1 TIME-DEPENDENT REDUCTION IN O 2 SUPPLY AND UTILIZATION IN THE DIAPHRAGM DURING MECHANICAL VENTILATION: ROLE OF VASCULAR DYSFUNCTION By ROBERT THOMAS DAVIS III A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2012
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TIME-DEPENDENT REDUCTION IN O2 SUPPLY AND UTILIZATION IN THE DIAPHRAGM DURING MECHANICAL VENTILATION:
ROLE OF VASCULAR DYSFUNCTION
By
ROBERT THOMAS DAVIS III
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2012
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© 2012 Robert Thomas Davis III
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I would like to dedicate this work to my parents, friends, and professors who have played a role in my educational growth. If it were not for your continued support and enthusiasm I wouldn’t be where I am today. Thank you for your love, understanding,
and encouragement throughout my graduate career.
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ACKNOWLEDGMENTS
I would like to acknowledge those who made the successful completion of this
project possible: my mentor, Dr. Brad Behnke; and my committee members, Dr. Scott
Powers, Dr. Thomas Clanton, Dr. Judy Delp, and Dr. Peter Adhihetty. Collectively and
individually, my committee provided me with invaluable guidance, instruction, and
patience. In addition, I would like to acknowledge Dr. Christian Bruells for his invaluable
contribution to this project. I would also like to give a special thanks to members of the
laboratory: Danielle J. McCullough, John N. Stabley, Payal Gosh and Bei Chen for their
enthusiastic participation assured the success of these experiments and also my
research achievements.
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TABLE OF CONTENTS page
ACKNOWLEDGMENTS .................................................................................................. 4
LIST OF TABLES ............................................................................................................ 7
LIST OF FIGURES .......................................................................................................... 8
LIST OF ABBREVIATIONS ........................................................................................... 10
ABSTRACT ................................................................................................................... 12
CHAPTER
1 INTRODUCTION .................................................................................................... 14
2 LITERATURE REVIEW .......................................................................................... 18
Brief History of Oxygen ........................................................................................... 18 Joseph Priestley (1733-1804) ........................................................................... 19 Antoine Laurent Lavoisier (1743-1794) ............................................................ 19 Carl William Scheele (1742-1786) .................................................................... 20
Oxygen Cascade: Atmosphere to Mitochondria ...................................................... 21 Oxygen Uptake: The Dynamic VO2 Response ....................................................... 22 Oxygen Transport and VO2; Does O2 Delivery Limit VO2? ..................................... 23
Diaphragmatic Microvascular Oxygenation (PO2m) .......................................... 26 Skeletal Muscle Resistance Vasculature ................................................................ 27 Mechanical Ventilation ............................................................................................ 30 History of MV .......................................................................................................... 31 Characteristics of the Diaphragm ............................................................................ 31 Mechanisms of MV-Induced Diaphragm Weakness ............................................... 32
Skeletal Muscle Atrophy ................................................................................... 32 Contractile Dysfunction .................................................................................... 34 ROS Production ............................................................................................... 35
Xanthine oxidase ....................................................................................... 35 NO synthase .............................................................................................. 35 NAD(P)H oxidase ....................................................................................... 36 Mitochondrial oxidants ............................................................................... 36
Blood Flow and Oxygen Delivery to the Diaphragm with MV .................................. 37 Summary ................................................................................................................ 37
3 METHODS .............................................................................................................. 39
Statistical Analysis .................................................................................................. 40 General Experimental Protocols and Analyses ....................................................... 40
Animals............................................................................................................. 40
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Mechanical Ventilation ..................................................................................... 41 Blood Flow ........................................................................................................ 41 Phosphorescence Quenching .......................................................................... 42
Calculation of diaphragm VO2 .................................................................... 43 Diaphragm contractions ............................................................................. 44
Isolated Microvessel Technique .............................................................................. 44 mRNA Analysis ....................................................................................................... 46
4 RESULTS ............................................................................................................... 48
Animals Weight, Hemodynamic Data, Arterial Blood Gases and pH ...................... 48 Diaphragm Blood Flow and Vascular Conductance are Diminished with
Mechanical Ventilation ......................................................................................... 48 Mechanical Ventilation Reduces Resting Diaphragm Microvascular PO2 (PO2m)
in a Time-Dependent Manner .............................................................................. 51 Resting Diaphragm O2 Uptake (VO2) ...................................................................... 51 Mechanical Ventilation Reduces the Ability to Augment Diaphragm Blood Flow,
Match O2 Delivery-to-Utilization, and Increase VO2 During Muscular Contractions ........................................................................................................ 52
Mechanical Ventilation Reduces NO-Mediated Vasodilation in Diaphragm Resistance Arterioles ........................................................................................... 53
Pressure-Diameter Relationship Is Altered in Diaphragm Arterioles Following Prolong Mechanical Ventilation ........................................................................... 54
5 DISCUSSION ......................................................................................................... 68
Mechanistic Basis for the Diminished Diaphragm Blood Flow Following Mechanical Ventilation ......................................................................................... 69
Implications from Altered PO2m Dynamics .............................................................. 72 Reduced O2 Delivery, Diaphragm VO2 and Cellular Energetics.............................. 73 Reduced PO2m and Cellular/Molecular Signals for Mitochondrial Dysfunction,
Atrophy, and Autophagy ...................................................................................... 74 QO2 and VO2 During Contractions: Ramifications on the Weaning Process .......... 75 Future Directions .................................................................................................... 76 Summary ................................................................................................................ 78
LIST OF REFERENCES ............................................................................................... 80
BIOGRAPHICAL SKETCH ............................................................................................ 92
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LIST OF TABLES
Table page 4-1 Body and diaphragm mass, blood basses and hematocrit. ................................ 55
4-2 Renal, respiratory and select hindlimb skeletal muscle blood flows. .................. 55
4-3 Microvascular PO2 dynamics across the rest-to-contractions transition after mechanical ventilation. ....................................................................................... 55
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LIST OF FIGURES
Figure Page 4-1 Resting diaphragm muscle blood flow (A) and vascular conductance (B)
measured during spontaneous breathing and after 30 min and 6 hr of MV.. ...... 56
4-2 Resting blood flow (A) and vascular conductance (B) to regionally delineated portions of the diaphragm muscle during spontaneous breathing and after 30 min and 6 hr of MV. ............................................................................................ 57
4-3 Resting diaphragm muscle blood flow (A) and vascular conductance (B) measured during spontaneous breathing and mechanically ventilated rats after 30 min and 6 hr.. ........................................................................................ 58
4-4 Comparison of blood flow (A) and vascular conductance (B) measured at rest between the diaphragm, red portion of the gastrocnemius muscle complex, soleus, and intercostal muscles during spontaneous breathing and after 30 min and 6 hr of MV. ............................................................................................ 59
4-5 Representative resting diaphragm microvascular PO2 profiles measured over time (A) and the average diaphragm microvascular PO2 (B) measured during spontaneous breathing and after 30 min and 6 hr of MV.. .................................. 60
4-6 Mean microvascular PO2m profiles including 95% CI (dashed lines) (A) and representative diaphragm microvascular PO2 profiles (B) in response to electrically stimulated muscle contractions after 30 min and 6 hr of MV.. ........... 61
4-7 Blood flow (A) and oxygen consumption (VO2) (B) at rest and during the steady-state of electrically stimulated contractions in the diaphragm muscle after 30 min and 6 hr of MV as compared to spontaneous breathing ................. 62
4-8 O2 delivery (A) and fractional O extraction (B) at rest and during electrically stimulated diaphragm muscle contractions after 30 min and 6 hr of MV as compared to spontaneous breathing. ................................................................. 63
4-9 Flow mediated vasodilation in diaphragm arterioles during spontaneous breathing and after 30 min and 6 hr of MV.. ....................................................... 64
4-10 Dose-responses to the endothelial-dependent vasodilation Acetylcholine (Ach) in the absence and presence of endothelial NO synthase inhibitor N-G-nitro-l-argine methyl ester (L-NAME) (A), Dose-responses to the endothelial-dependent vasodilation Acetylcholine (Ach) in the absence and presence of endothelial NO synthase inhibitor N-G-nitro-l-argine methyl ester (L-NAME) + COX inhibitor Indomethacin (INDO) (B), NO-dependent (endothelium-dependent) dilation (max dilationACh - max dilationACh + L-NAME) (C), Dose responses to the endothelium-independent vasodilator sodium
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nitroprusside (SNP) (D) in diaphragm arterioles during spontaneous breathing and after 30 min and 6 hr of MV. ........................................................ 65
4-11 eNOS mRNA expression in diaphragm arterioles during spontaneous breathing and after 30 min and 6 hr of MV.. ....................................................... 66
4-12 Active and passive myogenic responsiveness in arterioles during spontaneous breathing and following 30 min and 6 hr of MV……………………………………………………………………………….. ......... 67
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LIST OF ABBREVIATIONS
ADP ADENOSINE DIPHOSPHATE
ATP ADENOSINE TRIPHOSPHATE
cAMP CYCLIC ADENOSINE MONOPHOSPHATE
cGMP CYCLIC GUANOSINE MONOPHOSPHATE
COX CYCLOXYGENASE
EDRF ENDOTHELIAL-DERIVED RELAXATION FACTOR
EDHF ENDOTHELIAL-DERIVED HYPERPOLARIZING FACTOR
eNOS ENDOTHELIAL NITRIC OXIDE SYNTHASE
Gastred RED PORTION OF THE GASTROCNEMIUS
GTP GUANOSINE TRIPHOSPHATE
iNOS INDUCIBLE NITRIC OXIDE SYNTHASE
L-NAME L-NG-NITROARGININE METHYL ESTER
MAP MEAN ARTERIAL PRESSURE
MV MECHANICAL VENTILATION
NAD+ NICOTINAMIDE ADENINE DINUCLEOTIDE
NADH REDUCED FORM OF NAD+
NO NITRIC OXIDE
NOS NITIRIC OXIDE SYNTHASE
ONOO- PEROXYNITRITE
PCr PHOSPHOCREATINE
PGI2 PROSTACYCLIN
Pi INORGANIC PHOSPHATE
PO2m MICROVASCULAR OXYGENATION
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QO2 OXYGEN DELIVERY
Q CARDIAC OUTPUT
ROS REACTIVE OXYGEN SPECIES
SNP Sodium Nitroprusside
VO2 OXYGEN UPTAKE
VIDD VENTILATOR-INDUCED DIAPHRAGM DYSFUNCTION
VSMC VASCULAR SMOOTH MUSCLE CELL
XO XANTHINE OXIDASE
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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
TIME-DEPENDENT REDUCTION IN O2 SUPPLY AND UTILIZATION IN THE
DIAPHRAGM DURING MECHANICAL VENTILATION: ROLE OF VASCULAR DYSFUNCTION
By
Robert Thomas Davis III
December 2012
Chair: Brad Behnke Major: Health and Human Performance
Mechanical ventilation (MV) engenders several clinical complications associated
with the duration of MV. One consequence of MV is the difficulty to successfully wean a
large portion of patients from the ventilator. In skeletal muscle, a reduced O2 supply
results in contractile dysfunction and premature fatigue when performing external work.
However, whether MV induces an O2 supply-usage imbalance in the diaphragm, which
contributes to weaning difficulties, remains unknown. Here we demonstrate that MV
induces a time-dependent reduction in diaphragm blood flow which results in a greatly
diminished microvascular oxygenation. Furthermore, after as little as 6 hr of MV there is
a severely compromised ability to increase blood flow within the diaphragm during
contractions. Consistent with an O2 supply limitation, MV after 6 hr resulted in a ~80 %
reduction in O2 uptake during contractions compared to that achieved immediately after
the onset of MV, which would force reliance on non-oxidative energy sources and
hasten diaphragm fatigue. We also provide clear evidence for vascular dysfunction (i.e.
Nitric Oxide (NO)-mediated, structural alterations) as a potential mechanism that
contributes to the diminished diaphragm blood flow. These new and important findings
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reveal that prolonged MV results in a time-dependent decrease in the ability of the
diaphragm to augment blood flow to match O2 demand in response to contractile
activity. To our knowledge these are the first experiments that sought to determine the
effects of mechanical ventilation on diaphragm oxygenation and vasomotor control.
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CHAPTER 1 INTRODUCTION
Mechanical ventilation (MV) is used clinically as a life-saving intervention to
sustain adequate pulmonary gas exchange in patients that are incapable of maintaining
sufficient alveolar ventilation (e.g., patients in respiratory failure, coma, and drug
overdose). The removal of patients from MV is termed “weaning” and problems in
weaning are extremely common. The inability to wean patients from MV results in
increased risk of morbidity and mortality along with higher health care costs to patients,
insurance companies, and hospitals. Furthermore, long-term mechanical ventilation
results in perturbation in diaphragm function, collectively known as ventilator-induced
diaphragm dysfunction (VIDD; Vassilakopoulos et al., 2004). The physiological
mechanisms responsible for VIDD are unclear but likely multifaceted. Previous studies
have indicated increased reactive oxygen species (ROS) production, mitochondrial
damage, skeletal muscle atrophy, and muscle fiber remodeling as potential contributors
to weaning failure (Gayan-Rameriz et al., 2002, Kavazis et al., 2009, Sassoon et al.,
2002, Sieck et al., 2008). However, one aspect of VIDD that has not been investigated
is the impact of prolonged MV on diaphragmatic blood flow, oxygen delivery and
vasomotor control. In this regard, it is established that during acute MV (e.g. 30
minutes) blood flow to the diaphragm is decreased (Robertson et al., 1977, Uchiyama et
al., 2006, Virres et al., 1983, Hussain et al., 1985). Recently, our group has expanded
these observations and our data reveal that extending MV from 30 minutes to 6 hr
results in an additional decrease in diaphragmatic blood flow. However, at present,
there are no published reports regarding the impact of longer periods of MV (i.e., > 3
hrs) on blood flow, oxygen delivery to the diaphragm, or vasomotor control of the
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resistance vasculature (i.e., arterioles). Improving our knowledge of MV-induced
changes in blood flow and oxygen delivery to the diaphragm will advance our
understanding of how these variables contribute to VIDD and weaning difficulties.
Therefore, the overall objective of this project was to investigate the temporal pattern of
MV-induced alterations in diaphragmatic blood flow/oxygen delivery and investigate
mechanism(s) responsible for decrements in diaphragmatic blood flow and oxygen
delivery as well as putative mechanism(s) responsible for decrements in diaphragmatic
blood flow. Our central hypothesis is that prolonged MV results in a progressive decline
in diaphragmatic blood flow and oxygen delivery resulting from impaired vasomotor
function in resistance arteries due, in part, to decreased production/bioavailability of
nitric oxide (NO). Our central hypothesis was tested in four highly-integrated specific
aims using a well-established animal model of MV.
Specific Aim 1: To determine whether diaphragm blood flow decreases as a
function of time during MV.
Rationale: Previous work has demonstrated a significant reduction in diaphragm
blood flow immediately after the onset of MV due to the decreased metabolic activity of
the diaphragm (Robertson et al., 1977, Uchiyama et al., 2006, Virres et al., 1983,
Hussain et al., 1985). ROS generation in the diaphragm is significantly elevated after 6
hours of MV (i.e. prolonged MV) and an increased superoxide generation within the
diaphragm vasculature would likely reduce the bioavailability of NO (Kang et al., 2009).
NO has been demonstrated as a key regulator of resting arterial tone (Hirai et al., 1994,
Schrage et al., 2007) and a reduced bioavailability of NO after prolonged MV would
likely diminish resting diaphragm blood flow.
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Hypothesis: Diaphragm blood flow will be significantly reduced following 6 hr MV
versus blood flow measured immediately after the onset of MV.
Specific Aim 2: To determine the effect of prolonged MV on the matching of
diaphragmatic O2 delivery-to-O2 consumption (QO2-VO2) at rest and during
contractions; this will be accomplished through the measurement of
microvascular PO2 (PO2m).
Rationale: In order to sustain diaphragmatic metabolic and contractile function
there must be a tight coupling between the rates of O2 delivery (QO2) to that of O2
utilization (VO2) (Poole et al., 2001). An imbalance in either of these variables may force
an increased reliance on non-oxidative energy sources and, consequently, respiratory
muscle failure (for Rev see (Poole et al., 1997)). At present, we are unaware of any
other investigations determining whether the duration of MV affects diaphragm O2
delivery at rest, or induce alterations in the capacity to augment O2 delivery with
muscular contractions (e.g., during weaning).
Hypothesis: Following 6 hr MV there will be a diminished ability of the diaphragm
to augment O2 delivery rapidly during electrically-induced muscle contractions resulting
in a reduced PO2m.
Specific Aim 3: To determine how prolonged MV modifies resistance artery
function (i.e. vasomotor control).
Rationale: Previous work demonstrates that changes in shear-stress through
resistance arterioles can alter vasomotor control (e.g., flow-induced dilation) in as little
as 4 hours (Woodman et al., 2005). Therefore, in tissues that normally sustain a high
blood flow, a reduced shear-stress associated with inactivity may rapidly alter vascular
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control (e.g. blunted endothelial-dependent vasodilation). With a prolonged diminished
blood flow (and thus shear-stress) to the diaphragm during MV (versus that of
spontaneous breathing) we would expect a down-regulation of vasodilatory pathways
similar to that observed in other skeletal muscle models of disuse (McCurdy et al.,
2000). In addition, we wanted to test whether prolonged MV results in structural
alterations that have been demonstrated in other models of inactivity (i.e. bed rest,
Demiot et al., 2007).
Hypothesis: In diaphragm resistance arteries (i.e. arterial branch which provides
highest vascular resistance to blood flow) endothelium-dependent vasodilation, and
passive diameter responsiveness will be diminished after prolonged MV.
Specific Aim 4: To determine whether eNOS mRNA expression is reduced with
time of MV.
Rationale: Prolonged MV will result in a diminished blood flow (and thus shear-
stress) and stimulate a reduction in mRNA expression of eNOS following 6 hr MV
compared to that of spontaneous breathing.
Hypothesis: There will be a time-dependent reduction in eNOS mRNA and
protein expression in diaphragm resistance arterioles.
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CHAPTER 2 LITERATURE REVIEW
Being the primary muscle of ventilation, normal diaphragm function is requisite
for breathing. In this regard, it is clear that any perturbation to diaphragm muscle
function will have a negative impact on the overall health of the individual. Because MV
has been associated with deleterious effects on respiratory muscle function, elucidating
the mechanisms of VIDD is crucial. One aspect of VIDD that remains unknown is the
effect of MV on diaphragm muscle oxygen supply. It follows that obtaining new insights
regarding diaphragm oxygenation during prolong MV is important and will help us
develop therapeutic strategies to combat VIDD. Hence, this forms the rationale for the
experimental design within this proposal.
In this literature review, the discovery of oxygen will first be described, followed
by our current understanding of O2 transport and the pathways involved therein. To
study diaphragm O2 exchange we will measure blood flow, muscle oxygenation, and
vascular properties of diaphragm arterioles, all of which will be discussed. After the
foundation of these measures is established, the unique characteristic of the diaphragm
and the effects of MV will be discussed followed by a short summary.
Brief History of Oxygen
To many chemists and physiologists oxygen is regarded as the “elixir of life”
being the essential element for all living things. In Scandinavia, students are taught that
Swedish apothecary Carl William Scheele discovered oxygen in 1772, although his
publication is dated 1777. In America, it is taught that English Unitarian minister and
chemist Joseph Priestly discovered oxygen on August 1st, 1774 and personally
informed the French chemist Antoine Lavoisier in Paris in September of the same year.
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Lavoisier realized this gas was a new element which he termed “oxygen” (Roach et al.,
2003). Despite the contentious history behind the identification of oxygen, it is regarded
as the most important discovery in chemistry (Roach et al., 2003). This section will
provide a brief background of each of the three scientists and their contribution to the
discovery of this vital element.
Joseph Priestley (1733-1804)
Joseph Priestly was born near Leeds, England in 1733 and was the oldest of six
children. Initially he was a successful cloth dresser (Schofield, 1997) and later became
a self-taught chemist and teacher at Warrington Academy (Severinghaus, 2002).
Priestly was known to be very outspoken and a ferocious, free-thinking philosopher
(Schofield, 1997). On August 1st, 1774, he discovered a gas was liberated when he
heated the mineral red mercuric oxide in a sealed glass chamber. In the presence of
this gas, he demonstrated that a candle would burn more brightly and a mouse could
live longer in this chamber compared to a similarly sealed volume of air. He stated that
this gas is “much better than common air” (Comroe, 1977). Interestingly, Priestley also
discovered photosynthesis by demonstrating that a mint leaf left in the air in which the
mouse had died regenerated the substance needed to keep a mouse alive. In
September 1774 Priestly was invited to Paris by Antoine Laurent Lavoisier to present
his findings to a group of distinguished French scientists. As a consequence of this
meeting, Lavoisier began his own experiments and it is thought that this visit was the
critical catalyst of Lavoisier’s revolution of modern chemistry (Roach et al., 2003).
Antoine Laurent Lavoisier (1743-1794)
Antoine Lavoisier was born in Paris, France. His parents had a strong
educational background in science, philosophy, literature, and law. Lavoisier was a man
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of many professions. He was primarily a chemist but also a statesman, financier,
economist, manufacturer, and landowner (Poirier, 1993). Following Priestley’s visit,
Lavoisier repeated Priestley’s experiments using more elaborate techniques. During
this time, it is speculated that Carl William Scheele had also written a letter to Lavoisier
asking him to repeat his studies (completed in 1771) using his more elaborate burning
lens (Roach et al., 2003). Lavoisier later published his “discovery” and termed this
‘eminently breathable’ air oxygen in 1775 (West, 1980; West, 1996). Collectively,
Lavoisier’s work and “re-interpretation” of previous findings revolutionized the scientific
world.
Carl William Scheele (1742-1786)
Although Priestley and Lavoisier are usually credited with the discovery oxygen,
Carl William Scheele, a Swedish apothecary, may have generated this element as early
as 1771 (Severinghaus, 2002). Scheele was one of eleven children and received very
little formal education. In 1770, he became a lab assistant at Uppsala University in
Sweden. During his tenure there Scheele discovered oxygen by heating MnO2 with
H2SO4 (Oseen, 1942). Scheele wrote a book “On Air and Fire” describing his findings
but unfortunately it was not published until 1777. A couple of reasons for his delay in
the publication of his book were: 1) at this time he failed to fully understand how
important this discovery was and 2) he wanted to publish a book containing all his
discoveries collectively versus publishing independent papers (Severinghaus, 2002).
Undoubtedly, the debate regarding who discovered oxygen will always remain.
What is important to understand is that each individual scientist contributed in their own
way to this indispensable discovery. Whether it was the first person to isolate and
collect pure O2, differentiate it from other species of gases, or demonstrate that it is
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requisite for combustion; the discovery of oxygen was dependent on all three of these
remarkable scientists.
Oxygen Cascade: Atmosphere to Mitochondria
As mentioned previously, an adequate supply of oxygen is needed to sustain
normal cellular functions. The processes involved in the transport of oxygen from the air
to the cellular mitochondria are well defined (Weibel, 1984). Briefly, the major steps
include the convective movement of atmospheric oxygen (via the act of breathing) down
the airways to the alveoli. Thereafter, the oxygen undergoes alveolar gas mixing via
diffusion and convective forces from the lungs. Following this process, oxygen diffuses
out of the alveolar gas and into the pulmonary capillaries. This step is passive and
dependent on the magnitude of the diffusive gradient of O2 and the physicochemical
properties of the alveolar membrane. The fourth step in this cascade is the convective
transport of oxygen to the peripheral tissues and organs. Finally, oxygen diffuses from
the microvasculature into the tissue and ultimately the mitochondria. This oxygen
cascade is dependent on a sufficiently high PO2 gradient to sustain diffusion from the
atmosphere to the mitochondria.
Currently, the mechanism(s) responsible for skeletal muscle fatigue are complex
and not completely understood. There is compelling evidence demonstrating each step
in the oxygen cascade can modify oxygen transport and can cause impaired muscle
function (Roca et al., 1989; Pugh et al., 1964; Powers et al., 1989; Buick et al., 1980;
Saltin, 1985). As a result, the current notion is that all steps act in an integrated fashion,
and a disturbance to any of the steps in the cascade will result in a reduction of total O2
transport and skeletal muscle fatigue (Wagner, 1995). The following section will very
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briefly summarize the evidence that supports the notion that skeletal muscle oxygen
delivery is a major factor in determining exercise tolerance.
Oxygen Uptake: The Dynamic VO2 Response
In the conscious human, we are rarely in a resting metabolic steady-state; rather
we are continuously cycling between different metabolic demands. Therefore, a greater
understanding of the metabolic control and dynamics of oxygen exchange is of great
benefit to physiologists and clinicians alike. The speed of VO2 response (i.e. kinetics)
provides unique insight into skeletal muscle energetics and substrate utilization. For
example, faster VO2 kinetics across the rest-to exercise transition limit reliance on short-
term energy sources (e.g. glycolysis and phosocreatine (PCr) which can ultimately
impair skeletal muscle contractile function (e.g. excess proton formation from
accelerated glycolysis). On the contrary, slower VO2 kinetics will mandate a greater
disturbance of the intracellular milieu (e.g. H+, PCr, ADPfree and inorganic phosphate)
and deplete finite glycogen stores causing impaired contractile function and muscle
fatigue.
Historically, oxygen uptake (VO2) has been predominantly measured at the level
of the mouth, allowing for the characterization of pulmonary VO2 kinetics. However, in
order to investigate how the muscle responds to contractions, mitochondria located
within the muscle must be studied. However, in vivo measurements of mitochondria
function are not currently technically feasible.
At exercise onset, ATP demand increases in a square wave fashion whereas the
pulmonary VO2 response displays a finite kinetic response (Barstow et al., 1994). There
are three distinct phases of the pulmonary VO2 response in moderate intensity exercise.
Phase I constitutes an increase in cardiac output (Q; Casaburi et al., 1989) and
23
augmented perfusion through the pulmonary vasculature. This phase is dependent
upon the cardiopulmonary system (Wasserman et al., 1973) and is not indicative of the
muscle VO2 response (Grassi et al., 1996). Phase II represents the arrival of
desaturated venous blood from the exercising muscle, and reflects temporally the
increase in oxygen consumption at the level of the muscle (Whipp & Mahler, 1980;
Barstow et al., 1990, Grassi et al., 1996). The final phase (phase 3) represents the
steady-state in oxygen consumption.
Oxygen Transport and VO2; Does O2 Delivery Limit VO2?
The regulation of VO2 kinetics (i.e. the dynamic transition of VO2 upon the
initiation of exercise) during muscular contractions has been an area of debate since
A.V. Hills work in the early 1900s. The two main hypotheses for the regulation of VO2
are that, 1) there is an inherent inertia within the metabolic machinery of the muscle
(Whipp and Mahler, 1980., Grassi et al., 1996, Behnke et al., 2001) and 2) O2 delivery
regulates the speed at which VO2 can increase (Hughson et al., 1991, 1993). More
recently, it has become apparent that these two hypotheses are not mutually exclusive
and the regulation of VO2 kinetics is highly dependent on the health status of the
individual and the paradigm with which VO2 is measured. As our central hypothesis is
that the diaphragm displays a greatly reduced O2 delivery after MV, the evidence of an
O2 delivery limitation to VO2 will be discussed.
The notion of an O2 transport “limitation” to VO2 kinetics has been pioneered by
Dr. Richard L. Hughson (Hughson et al., 1991, Hughson et al., 1993, Hughson et al.,
1997) and others (Karlsson et al., 1975, Convertino et al., 1984, Hortsman et al., 1976).
Peripheral tissue oxygen delivery can be calculated as the product of cardiac output (Q)
and arterial oxygen content (CaO2) and is an indicator of the rate of convective transport
24
of oxygen to the tissues. In addition, there is a strong linear relationship between VO2
(i.e. oxygen consumption) and O2 delivery (Wagner, 1995). An example of how an
alteration in VO2 kinetics and thus muscle function might be related to a change in O2
transport is provided by examining the effects of supine exercise and heavy intensity
“priming” exercise. In the supine position, the hydrostatic gradient for muscle perfusion
due to gravity is reduced and muscle O2 availability may be compromised (Hughson et
al., 1993, Hughson et al., 1991, MacDonald et al., 1998). Indeed, it has been
demonstrated in the submaximal domain, pulmonary VO2 kinetics at exercise onset are
slowed in the supine position (i.e. reduction in the pressure head for muscle O2 delivery
during supine exercise in which the “gravitational assist” to muscle blood flow is absent)
(Hughson et al., 1991, Macdonald et al., 1998, Jones et al., 2006) and when performing
rhythmic forearm exercise with the arm elevated above the heart compared to when the
arm is places below the hear (Hughson et al., 1997). Under such conditions, “priming”
exercise accelerated blood flow dynamics during a subsequent bout of exercise and
removed possible constrainments of O2 delivery to VO2 and sped VO2 kinetics (Jones et
al., 2006). Similar to priming exercise, lower body negative pressure, which increases
the perfusion gradient for blood flow from the heart to the contracting muscle, returns
VO2 kinetics toward that of upright position (Hughson et al., 1993). Furthermore, Jones
and colleagues (2006) demonstrated that prior heavy exercise resulted in a speeding of
VO2 kinetics in the supine position.
It is noteworthy to mention that slower VO2 kinetics (seen with supine exercise)
can arise via: 1) slower dynamics of O2 utilization at the level of the myocyte (i.e.
inherent inertia in the metabolic contractile machinery), or 2) a reduction in O2 transport.
25
In this regard, it has been demonstrated that the activity of the sympathetic nervous
system (SNS) is attenuated in the supine position (Saul et al., 1991). This diminished
SNS may lead to an increased perfusion of non-exercising tissue (i.e. cutaneous
circulation, splanchnic regions) and affect the dynamic matching of O2 delivery to
consumption in the active tissue. As mentioned above, VO2 kinetics are slower during
forearm exercise performed above the heart, as compared to below or at the level of the
heart. In this position blood flow may be compromised at exercise onset, and therefore
this exercise paradigm is analogous to supine exercise (Hughson et al., 1993).
Oxygen delivery/supply regulates muscle function in numerous conditions such
as during strenuous exercise in the healthy individual. All levels of the respiratory
system and oxygen cascade present some inherent level of limitation (e.g. convective
oxygen transport to muscle, diffusive O2 conductance, and rate of O2 consumption in
the myocytes). Therefore, in order to fully understand the mechanism(s) responsible for
skeletal muscle fatigue, it is necessary to assess cellular metabolism as close to the site
of O2 utilization as possible (i.e. within the microcirculation).
Microvascular Oxygenation (PO2m)
As arterial blood transits from the central circulation to the periphery, oxygen
exits through the capillary wall by an oxygen diffusive gradient that extends from the red
blood cell all the way to the mitochondria (Tsai et al., 2007). There have been many
different techniques utilized to measure skeletal muscle oxygenation (e.g.,
microelectrodes, near-infrared spectroscopy). However, these techniques have had
several limitations (e.g. electrodes cause damage to the microvascular environment and
muscle fibers). In the late 1980s, David Wilson and colleagues (Vanderkooi et al., 1987,
Rumsey et al., 1988) developed a technique that allows the indirect measurement of
26
microvascular PO2m (which is indicative of these driving forces). This non-invasive
technique, known as phosphorescence quenching, utilizes palladium-porphyrin
compounds injected into the circulation. Following excitation of these compounds, the
only molecule that can quench phosphorescence is oxygen (Rumsey et al., 1991). Once
the lifetime decay has been determined, a simple manipulation of the Stern-Volmer
relationship allows for the calculation of microvascular PO2 (Rumsey et al., 1991, Wilson
et al., 1991). This technique has since been revised to examine muscle microvascular
PO2 in vivo and is detailed in the methods section.
Diaphragmatic Microvascular Oxygenation (PO2m)
The diaphragm is a unique skeletal muscle in that it is continuously active and
has a higher oxidative capacity than most other skeletal muscle (for exception see red
gastrocnemius; Hoppeler et al., 1981, Poole et al., 2000, Poole et al., 1992, Delp and
Duan., 1996, Powers et al., 1996). However, similar to other skeletal muscle, the
diaphragm can become fatigued during exercise (Johnson et al., 1993) and with chronic
diseases (e.g. emphysema) (Poole et al., 2001). This fatigue ultimately leads to
respiratory muscle failure and exercise intolerance. David Poole and colleagues (1995)
were the first to demonstrate that phosphorescence quenching is a feasible tool to
measure PO2m in the diaphragm. Since this seminal study, there have been other
investigations that sought to determine the PO2m in the diaphragm in healthy and
diseased conditions (e.g. emphysema; Poole et al., 2001). Geer and colleagues (2002)
demonstrated that the diaphragm has a higher resting PO2m (i.e. driving pressure for
oxygen) than other skeletal muscle (e.g. spinotrapezius). In addition, at the onset of
muscular contractions, the kinetics of PO2 in the diaphragm are more rapid than other
skeletal muscle (i.e. faster oxygen exchange dynamics). A higher PO2 in the diaphragm
27
is beneficial as it would reduce the degree of intracellular perturbations necessary to
achieve a given mitochondrial ATP flux (Wilson et al., 1977), and conserve limited
glycogen stores.
Skeletal Muscle Resistance Vasculature
As PO2m is dictated by the balance of QO2-to-VO2, mechanisms which regulate
the former, primarily the resistance vasculature, will be discussed. The most
mechanistic way to acutely regulate changes in peripheral skeletal muscle blood flow is
through changes in the vasomotor tone of the resistance vasculature. Vascular tone is
controlled by a balance between cellular signaling pathways (e.g. neural, humoral, and
metabolic) that mediate either vasoconstriction or vasodilation and ultimately the
internal diameter of the blood vessel. This section will focus on the structural
conformation of the resistance arterioles and how each component interacts functionally
to control vascular diameter and regulate blood flow.
Arterioles can be defined as the primary resistance vessels that enter an organ to
distribute blood flow into capillary beds and provide in excess of 80% of the resistance
to blood flow in the body (Martinez-Lemus, 2011). Consequently, they are vital in the
regulation of hemodynamics, regional distribution of blood flow (Christensen et al.,
2001, Meininger et al., 1984) and the maintenance of arterial pressure. Anatomically,
the vessel wall of arterioles consists of three structurally distinct layers, namely the
tunica intima, media, and adventitial layer (Rhodin et al., 1967).
The intima layer of resistance arterioles is predominantly composed of
endothelial cells. These endothelial cells play a role in the control of vascular tone by
the production and release of vasoactive factors that exert their action on neighboring
smooth muscle cells (Martinez-Lemus, 2011). Endothelial cells are arranged
28
longitudinally and in the direction of flow (Rhodin, 1980), and a large amount of
evidence indicates that these cells have the capacity to sense and transduce
mechanical forces and produce vasoactive compounds (Lui et al., 2008, Su et al., 2002,
Brum et al., 2005, Loufrani et al., 2008). The contribution of endothelium-derived
products in regulating blood vessel tone is well recognized [e.g. nitric oxide (NO),
endothelium-derived hyperpolarizing factor (EDHF), prostaglandins (PGI2), endothelin-
1, reactive oxygen species (ROS), Stankevičius et al., 2003]. One of the main
vasoactive compound released from the endothelium is NO. NO is a relatively stable
gas, with the ability to easily diffuse through the cell membrane and interact with various
substances in the cell (Nathan et al., 1994). NO stimulates guanylate cyclase in the
same cell or in a target cell, which converts guanosine triphosphate (GTP) to cyclic
guanosine monophosphate (cGMP) causing the concentration of cGMP to rise; and
lead to vascular smooth muscle relaxation (Vane et al., 1990). Another important
vasoactive compound that relaxes the vascular smooth muscle is EDHF. However, at
present, this factor has not been fully identified. On the contrary, there are at least
three vasoconstrictor substances that are released by the endothelium including
endothelin, prostanoids and thomboxane (Rubanyi et al., 1985). Endothelin-1 is the
most potent of these vasoconstrictors and was discovered in 1988 (Yanagisawa et al.,
1998). Its release can be initiated by numerous substances such as thrombin,
epinephrine, and interleukin-1 (Yanagisawa et al., 1988). Endothelin-1 is the most active
pressor substance discovered, with potency 10 times that of angiotensin II (Vane et al.,
1990).
29
The tunica media, or medial layer of arterioles, is primarily composed of vascular
smooth muscle cells (VSMC) and an internal elastic lamina (Martinez-Lemus, 2011).
VSMC’s activity are primarily regulated by changes in intracellular calcium
concentrations, activation of myosin light chain kinase, and increase in the
phosphorylation of the regulatory light chains (Kamm and Stull, 1985, Sommerville and
Hartshorne, 1986, Hai and Murphy, 1989). In addition, it has been established that an
increase in transmural pressure produces membrane depolarization of VSMC and
ultimately vasoconstriction (Harder et al., 1984). As stated previously, intracellular
calcium levels play a key role in regulating vascular tone (Missiaen et al., 1992).
Calcium influx occurs predominantly through voltage-gated and receptor-operated
calcium channels (Hurtwitz et al., 1986), and calcium efflux occurs via the sarcolemmal
calcium pump and Na+/Ca2+ exchanger (Missiaen et al., 1992). An increase in cAMP or
cGMP will induce VSMC relaxation by decreasing intracellular calcium via activation of
potassium channels (Nelson et al., 1990) or via the sarcolemmal calcium pump and
calcium uptake by the sarcoplasmic reticulum (Missiaen et al., 1992).
The adventitial layer consists of fibroblasts embedded in an extracellular matrix
made of thick bundles of collagen (Rhodin, 1967, Sangiorgi et al., 2006) and elastic
fibers which allow the resistance arterioles to elongate and recoil (Martinez-Lemus,
2011). Traditionally, it has been thought that the role of the adventitial layer was to
provide structural support only. However, current evidence suggests that adventitial
fibroblast are capable of producing ROS that can modulate the activity of smooth
muscle cells and initiate vascular remodeling (Haurani et al, 2007). In addition,
adventitial fibroblast can produce other growth factors and vasoactive compounds (e.g.
30
TGF-β, endothelin-1) and regulate vascular tone (Di Wang, et al., 2010). Therefore, at
present, it appears that the adventitial layer has a higher level of plasticity than
previously thought.
Mechanical Ventilation
The overall objective of the research herein is to elucidate the mechanism(s) of
prolonged MV induced alterations in diaphragmatic oxygen delivery and utilization. We
also want to learn how these variables may contribute to diaphragm muscle weakness
and the diminished ability to successfully wean patients from the ventilator. We will
utilize a highly integrative approach (e.g. blood flow, oxygen transport, microvascular
oxygenation, vasomotor control) to investigate events occurring within the diaphragm
during prolonged MV.
MV is an intervention utilized to sustain adequate alveolar ventilation in patients
who are incapable of doing so on their own (e.g. spinal cord injury, drug overdose,
surgery, and unconsciousness). The process of removing patients from MV is termed
“weaning”. Unfortunately, there are many problems associated “weaning” and this can
account for more than 40% of the total time spent on the ventilator (Esteban et al.,
1994). At present, the precise mechanism responsible for weaning difficulties is not
known, however, there are numerous studies that suggest MV-induced diaphragmatic
weakness is due, in part, to muscle atrophy, contractile dysfunction, and increased
reactive oxygen species (ROS) production (Gayan-Rameriz et al., 2002, Kavazis et al.,
2009, Sassoon et al., 2002, Sieck et al., 2008). In order to focus the scope of this
section, the remainder will provide a brief discussion of the history of MV and also
discuss our current knowledge and understanding of MV-induced diaphragmatic
weakness. Furthermore, it will provide a brief overview of new evidence collected by
31
our laboratory supporting the notion that a reduction in diaphragmatic oxygen delivery is
one possible mechanism responsible for respiratory muscle weakness and weaning
difficulties.
History of MV
Claudius Galenus (129 AD–circa 200 AD), better known as Galen, was a
prominent Roman physician. It is thought that he is one of the first to describe the
artificial ventilation of an animal (Galen, 1954). In 1543, Andreas Vesalius, also
described the importance of artificial ventilation by stating “that life may be restored to
the animal; an opening must be attempted in the trachea, into which a tube or reed can
be put; you will then blow into this, so that the lungs may rise again and the animal take
in air” (Chamberlain, 2003, Vesalius, 1543). Beginning in the 1800s, there was the
development and use of negative pressure ventilation (as opposed to positive-pressure
ventilation). This gave rise to the first use of an “Iron Lung”, which occurred in 1928
(Colice, et al., 1994). This type of ventilation was responsible for saving numerous lives
during the poliomyelitis epidemics in the 1940s (Colice, et al., 1994). Collectively, it is
easy to comprehend how the use of MV can have profound importance in the health
field and be an indispensable life-saving tool in certain clinical populations.
Unfortunately, the deleterious effects of MV often arise and increase patient morbidity
and mortality.
Characteristics of the Diaphragm
The largest muscle involved in mammalian ventilation is the diaphragm. It
accounts for up to 75% of the work of breathing during normal respiration and unlike
other skeletal muscle is continuously active throughout life. The diaphragm is a highly
plastic tissue, and therefore undergoes distinct adaptations to meet physiologic or
32
pathophysiologic demands (Poole et al., 1997). The diaphragm is composed of three
distinct regions, including the central tendon (non-contractile segment), and the costal
and crural regions (muscular portions). The fibers of the peripheral muscular regions
radiate towards the central tendon. In addition, the costal and crural segments have
different embryologic origins, segmental innervation, and functional attributes (De troyer
et al., 1988, Duron et al., 1979).
The metabolic characteristics of the diaphragm reflect its high tonic contractile
activity. In the rat, the diaphragm is composed of four different major muscle fiber types
(i.e. I, IIa, IId/x, and IIb) which are heterogenously distributed (Delp and Duan, 1996,
Powers et al., 1996). In addition, the oxidative capacity of the muscle fibers in the
diaphragm is higher than most other hindlimb skeletal muscle (Delp and Duan, 1996,
Powers et al., 1992, Powers et al., 1990, Sexton et al., 1995, Sieck et al., 1987).The
diaphragm also expresses a regional distribution of blood flow, with the medial and
dorsal costal regions accounting for most of its blood flow (Sexton et al., 1995). Other
unique characteristics of the diaphragm include; differential vasomotor regulation (Aaker
and Laughlin, 2002), prevalence of counter-current capillary flow (Kindig & Poole,
1998), and higher capillarity compared to other hindlimb skeletal muscle (Hoppeler et
al., 1981, Poole et al., 1992, Reid et al., 1992).
Mechanisms of MV-Induced Diaphragm Weakness
Skeletal Muscle Atrophy
MV is a method of reducing or removing the “work” of the diaphragm. It is one of
several experimental models and clinical conditions which result in skeletal muscle
atrophy (e.g. hindlimb unloading, immobilization, prolonged bed rest). Atrophy of the
diaphragm has been observed in both animal and human experiments following MV
33
(Anzueto et al., 1997, Bernard et al., 2003, Gayan-Ramirez et al., 2003, Knisely et al.,
1988, Le Bourdelles et al., 1994, Levine et al., 2008, Shanley et al., 2002, Yang et al.,
2002). MV-induced diaphragmatic atrophy is unique in that it occurs more rapidly (i.e.
within 12 hrs) when compared to disuse atrophy seen in other hindlimb skeletal
musculature (Le Bourdelles et al., 1994, McClung et al., 2007, Yang et al., 2002). The
rate of skeletal muscle atrophy is dependent on a decrease in protein synthesis (Ku et
al., 1995); an increase in protein degradation (Bodine et al., 2001); or due to a
combination of both of these variables. Work from Dr. Scott Powers’ laboratory has
demonstrated a reduction in protein synthesis in as little as 6 hr MV (Shanley et al.,
2004). In addition, reductions in insulin-like growth factor and myosin heavy chain (i.e.
type I and IIx) are evident after 12-24 hrs of MV (Gayan-Ramirez et al., 2003, Shanley
et al., 2004). However, at present, it is believed that that rapid onset of diaphragmatic
atrophy is primarily due to an increase in proteolysis (DeRuisseau et al., 2005, McClung
et al., 2007, Shanley et al., 2002).
The three primary pathways involved in skeletal muscle proteolysis are: 1)
lysosomal proteases (i.e. cathespins), 2) calcium actives proteases (i.e. calpains), 3)
and the protesome pathway. In regards to protease activation in the diaphragm
following MV; it appears lysosomal proteases do not play a dominant role in the MV-
induced skeletal muscle degeneration. Contrarily, calpains/caspases and the
proteasome pathways have been shown to contribute to the muscle protein breakdown
seen with MV (Shanely et al., 2002, DeRuissea et al., 2005). Specifically, elevated
levels of intracellular calcium (seen with inactivity) can activate both calpain and
caspases, which are responsible for sarcomeric protein release (Communal et al., 2002,
34
Koh et al., 2000, Purintrapiban et al., 2003). This is thought to be the initial step in
muscle protein loss during MV-induced diaphragmatic atrophy. The ubiquitin
proteasome pathway is the major proteolytic pathway responsible for skeletal muscle
protein breakdown and muscle atrophy following release for myofibrallar proteins. The
total proteasome complex (26S) consists of a core subunit (20S) coupled with a
regulatory complex (19S) at each end of the core subunit (Grune et al., 2003,
Hasselgren et al., 1997). The coordinated action of this three-enzyme system is
requisite for skeletal muscle protein breakdown (DeRuissea et al., 2005). Furthermore,
evidence indicates there is an increase in 20S proteasome activity following MV
(Shanley, et al., 2002, DeRuissea, et al., 2005).
Contractile Dysfunction
Utilizing a variety of animals models (i.e. rats, rabbits pigs, baboons), it has been
demonstrated that MV results in contractile dysfunction (Anzueto et al 1997, Sassoon et
al., 2002, Zhu et al., 2005, Shanely et al., 2003, Powers et al., 2002, Criswell et al.,
2003) which occurs in a time-dependent manner (Powers et al., 2002). For example, it
has been demonstrated that there is a significant reduction (~20%) in diaphragmatic
specific force in as little as 12 hrs of MV (Powers et al., 2002, Criswell et al., 2003). It is
noteworthy that decrements in force cannot be attributed to atrophy alone because the
force of the diaphragm was normalized to the cross-sectional area. In addition, it
appears the effects of MV are confined to the diaphragm, as there were no changes in
soleus skeletal muscle mass (Powers et al., 2002). Interestingly, Gayan-Ramirez and
colleagues (2002) demonstrated that intermittent spontaneous breathing during MV can
retard contractile dysfunction. These results provide supporting evidence that
diaphragm inactivity plays a fundamental role in promoting weaning failure.
35
ROS Production
It is well established that ROS are produced in inactive skeletal muscle thanks to
the seminal studies by Kondo et al. (1991, 1992, and 1993). Given the diaphragm is
inactive during controlled MV (Powers et al., 2002, Sassoon et al., 2004), the potential
for ROS production clearly exists. When cellular oxidant production exceeds the
capacity of intracellular antioxidants to scavenge these oxidants, oxidative-stress-
induced cellular injury can occur. In this regard, it has been previously demonstrated
that there is oxidative injury in the diaphragm during MV (within as little as 6 hr after the
onset of MV) (Falk et al., 2006, Shanely et al., 2002, Zergeroglu et al., 2003). At
present, numerous ROS producing pathways exist in the cell and may contribute
independently or cooperatively to myocyte damage including the following: 1) xanthine
oxidase; 2) production of NO via nitric oxide synthase (NOS); 3) NADPH oxidase; and
4) mitochondrial production of oxygen radicals.
Xanthine oxidase
Xanthine oxidase (XO) is produced via sulfhydrydryl oxidation or proteolysis of
xanthine dehydrogenase by calcium activated proteases (i.e. calpain) (Halliwell, et al
1999). XO catalyzes the formation of superoxide radicals and uric acid in the presence
of oxygen and purine substrates (i.e. hypoxanthine, xanthine). Superoxide radicals can
then lead to the formation of other damaging reactive species (e.g. peroxynitrite
(ONOO-)) (Halliwell et al., 1999). Nonetheless, it is unclear whether if XO-induced
production of oxidants in the diaphragm contributes to muscle dysfunction.
NO synthase
The free radical NO is produced via the enzyme NO synthase (NOS), and can
lead to the formation of other damaging reactive nitrogen species (e.g. ONOO-). There
36
are three isoforms of NOS that exists (Kaminski et al., 2001, Stamler et al., 2001) which
include: 1) type 1 or neuronal (i.e. nNOS), which is calcium activated, 2) type II or
inducible (i.e. iNOS), which is calcium independent, and 3) type III or endothelial (i.e.
eNOS), which is also calcium activated. Both nNOS and eNOS are expressed in the
diaphragm (Stamler et al., 2001, Van Gammeren et al., 2007). The formation of nitrogen
species is associated with cellular injury including mitochondrial dysfunction, lipid
peroxidation, and nitrosylation of proteins (Barreiro et al., 2002, Nin, et al, 2004, Stamler
et al., 2001, Supinski et al 1999). At present it appears that NO may not play a role in
MV-induced diaphragmatic dysfunction (Van Gammeren et al., 2007), however, further
studies are warranted.
NAD(P)H oxidase
NAD(P)H oxidase is a membrane-associated enzyme that catalyzes the one-
electron reduction of molecular oxygen into superoxide using NADPH or NADH as the
electron donor (Javesghani et al., 2002). It has been shown that numerous factors such
as the calcium-sensitive PKC-ERK1/2 pathway can increase NAD(P)H oxidase activity
in cells (Hazan et al.,1997). Given that skeletal muscle inactivity (e.g. MV) causes an
increase in calcium, one could assume that NAD(P)H activity would increase and be a
possible source of oxidant production.
Mitochondrial oxidants
The primary function of the mitochondria is to produce ATP; however, it’s also
well known that electron leakage from the electron transport chain (via complex I and III)
can result in formation of superoxide and subsequently hydrogen peroxide (Andreyev et
al., 2005; Cadenas et al., 2000; Powers et al., 2005). Clearly, the mitochondria have to
ability to alter cellular redox balance. Previously, it was thought that there was minimal
37
mitochondrial damage following prolonged MV (Fredriksson et al., 2005), however,
recently Powers and colleagues (2011) demonstrated the mitochondrial target
antioxidants help protect the diaphragm from VIDD.
Blood Flow and Oxygen Delivery to the Diaphragm with MV
In order to maintain contractile function and prevent muscle fatigue, the
diaphragm must be able to tightly regulate the rates of O2 delivery to those of O2
utilization (Poole et al., 2001, Poole et al., 2012). In this regard, it has been
demonstrated at the onset of MV there is a reduction in diaphragm blood flow
(Uchiyama et al., 2006) due to the reduced recruitment (and thus metabolic load) which
allows for redistribution of cardiac output to other vital organs (Viires et al., 1983;
Hussain et al., 1985). However, it is unknown whether prolonged MV results in a
reduction in diaphragmatic blood flow or has a negative impact on the diaphragms
ability to increase O2 delivery during muscular contractions (e.g. weaning). Clearly, a
substantial fall in diaphragm blood flow following prolonged MV could result in
impairment in the ability of the diaphragm muscle to contract (e.g. weaning) and
contribute to VIDD. In this regard our laboratory has demonstrated MV induces a time
dependent reduction in diaphragm oxygenation (Davis et al., 2012). These data are the
first to demonstrate there is a reduced oxygen supply following prolonged MV, which
may also contribute mechanism to weaning difficulties.
Summary
Failure to wean patients from MV is an important clinical problem and respiratory
muscle weakness is a major contributor to weaning difficulties. Improving our
knowledge of how changes in blood flow and O2 delivery to the diaphragm (induced by
prolonged MV) will help us design modalities to successfully wean patients off MV.
38
Data from this project supports the notion that a time-dependent reduction in oxygen
transport is one mechanism contributing, in part, to VIDD. Our long-term goal is to
develop methods for the prevention of MV-induced diaphragmatic weakness and
weaning difficulties.
39
CHAPTER 3 METHODS
This section will be divided into two segments. The first segment will contain the
experimental design used to test each of our specific aims (1-4), which were intended to
determine the effects of MV on diaphragmatic oxygenation and resistance artery
function. The subsequent section will provide detailed methods associated with each
experimental technique.
Experiment 1: In this experiment we measured diaphragm blood flow during
spontaneous breathing and after 30 min and 6 hr of ventilation using radioactive
microspheres in female Sprague-Dawley (SD) rats. Arterial O2 concentration and mean
arterial pressure were measured at these time points to calculate O2 delivery and
vascular conductance, respectively.
Experiment 2: Utilizing the phosphorescence quenching techniques (Geer et al.,
2002, Wilson et al., 1994) we determined if microvascular PO2m is reduced from the
spontaneous breathing condition to 30 min of MV, and if there will be a further reduction
following six hours of MV. In addition, we determined whether the duration of MV affects
the matching of QO2-to-VO2 across the rest-to-contraction transition after both acute
and prolonged MV.
Experiment 3: The purpose this experiment was to determine whether, and
through which signaling pathways (e.g. endothelium-dependent or independent,
structural/mechanical alterations) prolonged MV alter diaphragm arteriolar function.
Experiment 4: This experiment investigated whether there is a down-regulation
of eNOS mRNA in diaphragmatic arterioles following prolonged MV. This experiment
40
provides further mechanistic insight into potential signaling pathways responsible for a
reduction in vasomotor control with MV.
Statistical Analysis
Group and sample sizes were calculated using a power analysis of preliminary
data from our laboratory. Comparisons between blood flow, vascular conductance, O2
delivery, and VO2, percent maximal dilation, mRNA were analyzed with one-way
ANOVA. Resting, Steady-State contracting PO2m, blood pressure, arterial blood gases,
and pH across time were analyzed with repeated measure ANOVA. Individual
differences will be examined post hoc using a Tukey’s test. All data are presented as
means ± SE. Significance was established at P≤0.05.
General Experimental Protocols and Analyses
Animals
Adult (4-6 month old, ~250 g) female Sprague-Dawley obtained from Charles
River Laboratories (Boston, MA, USA) were used for this investigation (Spontaneous
Breathing: n=45; 30 min MV: n=41; 6 hr MV: n= 42) . The Sprague-Dawley rat was
chosen due to the similar properties (e.g. anatomical and physiological) with the human
diaphragm (Metzger et al., 1985, Mizuno et al., 1991, Poole et al., 1997, Powers et al.,
1997). Upon arrival, all rats were housed at the University of Florida Animal Care
Services Center. All procedures were approved by the University of Florida Institutional
Animal Care and Use Committee. Animals were maintained in a temperature-controlled
(23 ± 2°C) room with a 12:12-h light-dark cycle. Water and rat chow were provided ad
libitum through the experimental period.
41
Mechanical Ventilation
All surgical procedures performed used aseptic techniques. Animals in the MV
groups were anesthetized with sodium pentobarbital (50 mg/kg, i.p.), tracheostomized
and connected to a volume-cycled ventilator (Harvard Apparatus; Cambridge, MA). A
catheter was implanted in the carotid artery for measurement of blood pressure and
periodic blood sampling (every 3 hr) for analyses of blood gases (GemPremier 3000,
Instrumentation Laboratory, Bedford, MA). Arterial O2 saturation was monitored
continuously by using a Mouse OX (Asbury Park, PA) placed around the rats foot.
Expired PCO2 was measured continuously using a microcapnograph (Model Columbia
Instruments, Columbus, Ohio). Arterial PO2, PCO2, and pH were maintained within the
normal range (80 to 110 mm Hg, 30 to 40 mm Hg, and 7.35 to 7.45, respectively) by
minor adjustments to minute volume. A catheter was placed in the jugular vein for
infusion of sodium pentobarbital (10 mg/kg/hr) and fluids, when necessary. Body
temperature was monitored and maintained at 37 ± 1°C (via rectal thermometer) by use
of a recirculating heating blanket. Body fluid homeostasis was maintained by
administrating electrolyte solution (i.v., 2 mL/kg/hr). Operative care during the MV
period included expressing the bladder, removal of airway mucus, lubricating the eyes
and rotating the animal. The ventilator was maintained at an average breathing
frequency of 70 ± 10 breaths/min and tidal volume of 2.2 ± 0.5 mL/breath.
Blood Flow
Tissue blood flow was measured using radiolabeled microspheres (15 µm
diameter; 46Scandium, 85Strontium, 57Cobalt; random order) according to the methods of
Flaim et al., 1984). Microspheres (i.e. 2.5 X 105 of each label) were injected at three
time points; 1) during spontaneous breathing, 2) after 30 min (i.e. acute) and 3)
42
following 6 hr (i.e. prolonged) MV. After the final microsphere infusion the animal was
euthanized via an overdose of sodium pentonbarbital (≥ 100mg/kg i.a.) and the
following muscles were harvested for blood flow determination: diaphragm (dissected
into appropriate anatomical sections, i.e., crural, central tendon, dorsal costal, mid
costal, and ventral costal), soleus, red portion of the gastrocnemius, internal and
external intercostal, sternocleidomastoid, and scalene. The kidneys were also be
harvested to determine adequate distribution of the microspheres (i.e., <15% difference
in blood flow between the left and right kidney). Blood flow was expressed as
mL/min/100 g of tissue and vascular conductance was expressed as mL/min/100
g/mmHg to normalize for any possible changes in arterial pressure.
Phosphorescence Quenching
As stated previously, phosphorescence quenching was utilized to measure
diaphragm muscle PO2m during spontaneous breathing and after 30 min and 6 hr of MV.
Briefly, rats were anesthetized with pentobarbital sodium (50 mg/kg i.p., supplemented
as needed) and the right carotid artery was isolated and cannulated with a fluid-filled
catheter (PE-50) to monitor arterial blood pressure (Digi-Med BPA Model 400a; Micro
Med, Inc.; Louisville, KY). This catheter was also be used for the infusion of the
phosphorescent probe R2. The diaphragm was exposed via laparotomy and the liver
reflected gently downward, permitting access to the inferior surface of the medial and
ventral regions of the diaphragm. The phosphorimeter light guide was positioned 2-5
mm from the surface of the medial costal diaphragm, and exposed surfaces were
covered with Saran Wrap to prevent moisture and heat loss. During the experimental
procedure the abdominal cavity was kept moist via superfusion of a Krebs-Henseleit
bicarbonate-buffered solution equilibrated with 5% CO2/95% N2 at 37°C. A temperature
43
probe was placed between the liver and the diaphragm to monitor the temperature of
the abdominal cavity. The phosphor, palladium meso-tetra-(4-carboxyphenyl)-porphyrin
dendrimer (R2; Oxygen Enterprises Ltd., Philadelphia, PA), was infused ~10 min before
each experiment at a dose of 15 mg/kg and PO2m measurements were recorded every
2 s for 60 s to provide an average steady-state PO2m during spontaneous breathing and
after 30 min and 6 hr MV. All PO2m measurements were performed in a dark room to
avoid contamination of the signal with ambient light. Upon completion of the experiment
each rat was euthanized with an overdose of anesthesia (pentobarbital sodium, >100
mg/kg, i.a.) and a thoracotomy was performed to visually verify cardiac arrest.
Calculation of diaphragm VO2
Muscle oxygen uptake (VO2) of the medial costal diaphragm (i.e., region of PO2m
measures) was calculated from blood flow and PO2m measurements in the diaphragm
during the resting and contracting conditions as described by Behnke et al., (2002).
Specifically, VO2 is calculated using the Fick equation using PO2m as an analogue for
venous PO2 (Behnke et al., 2002, Wagner, 1991) and from the O2 dissociation curve
and microvascular blood O2 content (CmO2). As no discernible change in blood pH or
muscle temperature was observed, we expect there will be no significant shift in O2
dissociation curve. Therefore, diaphragm VO2 was calculated from data collected from
arterial blood samples, PO2m and muscle blood flow (Qm) utilizing the following
equation:
VO2 = Qm ˟ (CaO2-CmO2),
where VO2 is the oxygen uptake of the diaphragm muscle, Qm is diaphragm muscle
blood flow, and CaO2 and CmO2 are the oxygen contents of the arterial and
44
microvascular blood, respectively. Muscle VO2 was expressed as mL/min/per 100 g
tissue.
Diaphragm contractions
The diaphragm was exposed as described above and stainless steel electrodes
were sutured (6-0 silk; Ethicon, Somerville, NJ) to the right ventral costal (cathode) and
the right dorsal costal (anode) diaphragm according to the methods of Geer et al.
(2002). Electrically-stimulated twitch contractions (1 Hz, 3-6 V, 2-ms pulse duration)
were induced with a Grass S88 stimulator (Quincy, MA) for 3 min. This contraction
protocol elicits contractions at a similar frequency as with spontaneous breathing.
Following 30 min and 6 hr MV the stimulation protocol and PO2m was measured at 2 s
intervals across the rest-to-contractions transition. Blood flow determination was made
after 180 s of stimulation to assess the contracting steady-state muscle hyperemia.
VO2 was then calculated as described above for resting and steady-state contracting
conditions.
Isolated Microvessel Technique
To determine vasomotor control resistance arterioles (<200 μm intraluminal
diameter) were isolated from the diaphragm and studied in vitro to remove potentially
confounding metabolic, humoral, and neural influences. Resistance arterioles will be
harvested from three groups of animals which include; 1) spontaneously breathing
animals, 2) following acute, and 3) following prolonged MV. With the aid of a dissecting
microscope (Olympus SVH10), first-order (1A) arterioles from the diaphragm muscle
were isolated and removed from the surrounding muscle tissue as previously described
(Aaker & Laughlign, 2002, Muller-Delp et al., 2002, Spier et al., 2004). The arterioles
(length, 0.5–1.0 mm; inner diameter, 90–175 μm) were transferred to a Lucite chamber
45
containing PSS equilibrated with room air. Each end of the arteriole will be cannulated
with micropipettes and secured with nylon suture. Following cannulation, the
microvessel chamber will then be transferred to the stage of an inverted microscope
(Olympus IX70) equipped with a video camera (Panasonic BP310), video caliper
(Colorado Video 307A) and data-acquisition system (PowerLab) for on-line recording of
intraluminal diameter. Arterioles were initially pressurized to 75 cmH2O with two
independent hydrostatic pressure reservoirs. Leaks were detected by pressurizing the
vessel, and then closing the valves to the reservoirs and verifying that intraluminal
diameter remained constant. Arterioles that exhibit leaks were discarded. Arterioles that
were free from leaks were warmed to 37◦C and allowed to develop initial spontaneous
tone during a 30–60 min equilibration period. Upon displaying a steady level of
spontaneous tone we evaluated vasodilator and pressure responses in these resistance
arterioles. To determine endothelial function of diaphragm resistance arterioles, dose
responses to acetylcholine (Ach), which mediates smooth muscle relaxation indirectly
by binding to endothelial cell M2-receptors and stimulates the release of endothelium-
derived relaxing factor(s) (EDRF), will be tested. EDRFs typically released from
vascular endothelial cells are nitric oxide (NO) and prostacyclin (PGI2), and to a lesser
extent, endothelium-derived hyperpolarizing factor (EDHF). Therefore, the contribution
of the nitric oxide synthase (NOS) and cyclooxygenase (COX) signaling pathways to
ACh-induced vasodilation in the diaphragm resistance arterioles will be determined
using the NOS inhibitor AP-nitro-L-arginine methyl ester (L-NAME) and the COX
inhibitor indomethacin. Flow-induced vasodilation (endothelium-dependent),
endothelium-independent (via sodium nitroprusside, SNP), active (myogenic) and
46
passive diameter responses were assessed in the isolated vessels. The latter was
accomplished by altering the heights of the independent fluid reservoirs in equal and
opposite directions so that a pressure difference is created across the vessel without
altering mean intraluminal pressure. Diameter measurements were then determined in
response to incremental pressure differences of 4, 10, 20, 40 and 60 cmH2O.
Volumetric flow (Q) can be calculated according to the following equation (Davis, 1987;
Kuo et al. 1990; Muller-Delp et al. 2002):
Q = π(Vrbc/1.6)(d/2)2
where (d) is inner diameter and (Vrbc) represents mean red blood cell velocity, which will
be determined in a subset of arterioles at each of the pressure gradients mentioned
above. Passive myogenic will be assessed by allowing the vessels to equilibrate at 37◦C
and 75 cmH2O for 60 minutes. After equilibration, intraluminal pressure will be
increased in 20-cmH2O increments from 0 to 140 cmH2O. Diameter was recorded for 3
min at each pressure step and these pressure changes will occur in the absence of
flow.
mRNA Analysis
Diaphragm resistance arterioles were snap frozen and stored at 80°C. Arterioles
were then pulverized in lysis buffer, and total RNA extracted using a RNAqueous
isolation kit (Ambion). cDNA was made using the High-Capacity cDNA Archive kit
(Applied Biosystems). Real-Time PCR was performed as previously described (Spier et
al., 2004). Briefly, real-time PCR was performed in triplicate, with two no-template
control samples and two reverse transcriptase negative samples (GeneAmp 96-well
optical reaction plates). eNOS mRNA expression was quantified with predesigned
TaqMan primers (Applied Biosystems) using the ABI Prism 7900HT fast real-time PCR
47
system. Quantification of relative gene expression was performed using the
comparative threshold cycle method.
48
CHAPTER 4 RESULTS
Animals Weight, Hemodynamic Data, Arterial Blood Gases and pH
Body and diaphragm weight were not different between groups (Table 4-1).
Mean arterial pressure (MAP), blood gases and pH are summarized in Table 1 for
spontaneous breathing and during MV. There was a reduction in MAP after 6 hours of
MV, however, it remained above levels that affect PO2m dynamics (Behnke et al., 2006).
Indeed, we did not observe any relation between MAP and PO2m dynamics (i.e., mean
response time; r2=0.007, P=0.72). Contrary to that reported by Poole et al. (1995), we
did not observe a relation between MAP and PO2m (r2=0.11, P=0.29), which may be due
to circulatory volumetric changes induced to manipulate MAP in the previous study.
Arterial PO2 decreased slightly over time but no relations were observed between
arterial PaO2 and VO2 (r2=0.09, P=0.66) or PO2m (r2=0.07, P=0.17). Based on the
rightward-shifted O2 dissociation curve of the rat (P50= 38 mmHg) small changes in
PaO2 above 80 mmHg would have very little impact on O2 content as these PO2 values
would fall on the flat portion of the dissociation curve.
Diaphragm Blood Flow and Vascular Conductance are Diminished with Mechanical Ventilation
To address the changes in diaphragm blood flow that may be a contributing
mechanism to diaphragm muscle weakness with MV we measured diaphragm blood
flow as a function of time on the ventilator. During spontaneous breathing, diaphragm
blood flow averaged 36 ± 5 mL/min/100 g, which is consistent with what has been
reported for the rat diaphragm under anesthesia (Boegehold et al., 1991). Mechanical
ventilation was then initiated and set at a rate sufficient to suppress contractions within
the diaphragm (i.e., passive ventilation) and blood flow was measured again after 30
49
min of MV. Passive MV elicited a reduction of ~25% in diaphragm blood flow after 30
min to ~26 mL/min/100 g (Figure 4-1A). To assess whether diaphragm blood flow
decreased in a time-dependent manner with passive MV, diaphragm blood flow was
again measured after 6 hr MV. There was an additional ~75% reduction in diaphragm
blood flow versus that measured at 30 min MV, resulting in an average flow of 7
mL/min/100 g (Figure 4-1A). For comparison, the blood flow to the diaphragm
measured herein after 6 hr MV (Figure 4-1A) is considerably less than that measured in
the highly glycolytic white portion of the gastrocnemius of ~ 10-15 mL/min/100 g (Hirai
et al., 2011, Behnke et al., 2011). To normalize for changes in blood flow that may be
due to alterations in blood pressure we calculated vascular conductance (i.e., blood flow
÷ MAP) at the different time points. As demonstrated in Figure 4-1B, reductions in
vascular conductance paralleled changes in blood flow with the duration MV, resulting in
a significantly lower vascular conductance after 6 hr versus 30 min of MV.
Consistent with what others have found (Brancatisano et al., 1991, Sexton et al.,
1995) there was a marked heterogeneity in regional distribution of blood flow within the
diaphragm (Figure 4-2). During spontaneous breathing blood flow to the medial costal
portion of the diaphragm was the highest and showed the greatest reduction after the
onset of MV. After 6 hr of MV all regions of the diaphragm demonstrated significant
reductions in blood flow and vascular conductance versus both spontaneous breathing
and 30 min of MV (Figure 4-2A & B). In order to rule out the effects of anesthesia
causing the reduction in diaphragm blood flow we measured blood following 6 hr of
anesthesia alone (Figure 4-3). Blood flow and vascular conductance to the intercostal
50
muscles, which are subjected to similar alterations in intrathoracic pressures as the
diaphragm, did not change across the measurement period (Figure 4-4A & 4-4B).
The net reduction in blood flow to the diaphragm between the conscious standing
(Sexton et al., 1995, Poole et al., 2000) and the inactive condition (i.e., during MV) is
similar to that observed in the soleus muscle between the standing and anesthetized
condition (Behnke et al., 2011). Furthermore, the diaphragm has a high daily duty cycle
(i.e., ratio of active to inactive times) and the soleus muscle has the highest daily duty
cycle of the hindlimb locomotory muscles (Hensbergen et al., 1997). Therefore, we
wanted to measure blood flow with inactivity in the soleus muscle (which is inactive
during the entire protocol after the onset of anesthesia) to determine if similar reductions
in blood flow as observed in the diaphragm also occur in this muscle over time (Table 4-
2). Unlike the diaphragm, blood flow and vascular conductance remained unchanged in
the soleus muscle at every time point measured herein (i.e, up to 6 hr; Figure 4-4A & B).
Given the high oxidative capacity of the diaphragm, we also wanted to compare blood
flow over time in skeletal muscle which displays a similar oxidative capacity and fiber
type composition. Therefore, we measured blood flow to the red portion of the
gastrocnemius muscle complex (GastRed) as it displays a similar oxidative capacity
(citrate synthase activity; diaphragm ~ 39 vs. GastRed ~ 36 µmol/min/g) and oxidative
fiber type profile (type I fibers: diaphragm ~44% vs. GastRed ~ 51%; type IIa fibers,
diaphragm ~23% vs. GastRed ~ 35%; (Delp and Duan, 1996) as the diaphragm. Similar
to that of the soleus, we did not observe any change in blood flow (Figure 4-4A) or
vascular conductance (Figure 4-4B) in the GastRed across the 6 hr experimental
protocol.
51
Mechanical Ventilation Reduces Resting Diaphragm Microvascular PO2 (PO2m) in a Time-Dependent Manner
Figure 4-5A shows representative resting diaphragm PO2m responses from an
individual animal during the measurement periods. During spontaneous breathing (SB)
diaphragm PO2m was ~ 53 mmHg and was significantly reduced to ~37 mmHg after 30
min of MV (Figure 4-5B). PO2m decreased by an additional ~50% after 6 hr of MV
versus that after 30 min of MV to 18 ± 5 mmHg (Figure 4-5B). As PO2m reflects the
QO2/VO2 ratio in the diaphragm (Poole et al., 1995), the lower PO2m observed after 6 hr
could result from the reduced blood flow, an increased VO2, or a reduction in both
variables but with a great proportional decrease in O2 delivery. Therefore, we also
calculated diaphragm VO2.
Resting Diaphragm O2 Uptake (VO2)
Relative to spontaneous breathing, 30 minutes of MV significantly decreased
diaphragm VO2 (spontaneous breathing 1.44 ± 0.10; 30 min MV, 1.19 ± 0.08
mL/min/100 g; P<0.05). As the MV rate utilized was sufficient to suppress diaphragm
activity, the reduction in VO2 after the onset of MV is due to the quiescence of the
diaphragm motor units during passive ventilation. Specifically, as ~12-20% of maximal
diaphragm force is generated during normal, quiet breathing (Sieck et al., 1989, Mantilla
et al., 2010), a fall of ~17% in diaphragm VO2 can be expected with inactivity induced by
passive MV. However, after 6 hr of MV there was a trend (P=0.056) for a further
reduction in diaphragm VO2 (1.02 ± 0.06 mL/min/100 g) compared to that calculated
after 30 min of MV.
52
Mechanical Ventilation Reduces the Ability to Augment Diaphragm Blood Flow, Match O2 Delivery-to-Utilization, and Increase VO2 During Muscular Contractions
To determine whether the capacity to increase blood flow and VO2 during a
metabolic challenge in the diaphragm is negatively affected after 6 hr of MV, we elicited
twitch contractions and quantified the resultant blood flow, PO2m and VO2 responses in
the diaphragm. Average and representative diaphragm PO2m profiles across the rest-
to-contractions transition are demonstrated in Figure 4-6A and 6B, respectively, after 30
min and 6 hr of MV. Upon initiation of contractions, the diaphragm PO2m after 30 min of
MV decreased by ~16 mmHg from the pre-contracting value to a low PO2m value of ~21
mmHg (Table 4-3), which is similar to that reported by Geer et al. during contractions
after acute MV (Geer et al., 2002). After 6 hr of MV the decrease in PO2m with
contractions was significantly less (i.e., ~9 mmHg or roughly 55% of that observed after
30 min of MV; Table 4-3) resulting in a low PO2m value of 8 ± 1 mmHg. When looking at
PO2m dynamics the time delay before a statistically significant fall in PO2m (versus pre-
contracting values) was shorter in the 6 hr MV group versus that after 30 min of MV
(Table 4-3). The shorter time delay suggests that the relative increase O2 delivery was
slower than that of O2 uptake (Behnke et al., 2002) during contractions, resulting in a
more rapid exponential decrease (i.e., faster time constant, tau) in PO2m after 6 hr
versus 30 min of MV (Table 4-3). In comparison to 30 min of MV, 6 hr of MV elicited
significantly faster overall kinetics, with a mean response time of (i.e., TD + Tau) of ~15
s versus ~22 s after 30 min of MV (Table 4-3).
When quantifying the hyperemic response elicited by muscular contractions,
there was a 2.8 fold increase in diaphragm blood flow compared to the resting value
after 30 min of MV versus a change of only 1.3 fold after 6 hr of MV (Figure 4-7A). The
53
resultant change in diaphragm blood flow from rest-to-contractions (i.e., steady-state
contracting value minus pre-contracting baseline value) was ~90% greater after 30 min
of MV (40 ± 6 mL/min/100 g) versus 6 hr of MV (4 ± 1 mL/min/100 g). In fact, after 6 hr
of MV diaphragm blood flow during contractions was only ~ 80% of that measured in the
resting condition after 30 min MV.
We also wanted to explore the possibility that the severely reduced ability to
augment flow after 6 hr of MV could not be offset by an increase in fractional O2
extraction such that diaphragm VO2 would be reduced during contractions. After 30 min
of MV, contractions elicited a marked increase in diaphragm VO2 from 1.4 ± 0.1 at rest
to 7.0 ± 0.4 mL/min/100 g during the contracting steady-state (Figure 4-7B). The
increase in VO2 was significantly blunted after 6 hr versus 30 min of MV with a change
in VO2 from a resting value of 1.0 ± 0.1 to 1.6 ± 0.1 mL/min/100 g during the steady-
state of contractions (Figure 4-7B). The increase in VO2 during contractions after 6 hr
of MV was only ~ 22% of that observed after 30 min of MV during contractions. In
addition, there was a reduced diaphragm O2 delivery (i.e., arterial O2 content ˟ blood
flow; Figure 8A) and increased O2 extraction (i.e., O2 delivery ÷ VO2; Figure 8B) both at
rest and during contractions after 6 hr versus 30 min of MV.
Mechanical Ventilation Reduces NO-Mediated Vasodilation in Diaphragm Resistance Arterioles
In order to investigate the role of MV on resistance vascular function both
endothelium-dependent and independent mechanisms in diaphragm resistance
arterioles were assessed. The development of spontaneous tone did not differ between
arterioles from spontaneous breathing (42 ± 4%), 30 min (40 ± 6%), or 6 hr MV (38 ±
5%) rats (P > 0.05). In addition, the maximum arteriolar diameter was not different
54
between groups (Spontaneous Breathing, 201 ± 7 μm; 30 min MV, 202± 9 μm ; 6 hr
MV, 195 ± 6 μm; P > 0.05). Following 6 hr of MV, there was a 40% reduction in flow-
induced vasodilation in diaphragm arterioles (Figure 4-9A). These arterioles also
exhibited a significantly lower endothelium-dependent vasodilation to Ach (Figure 4-10
C). There was also a significant reduction in arteriolar vasodilator responsiveness to the
exogenous NO donor SNP (Figure 4-10D) and eNOS mRNA expression (Figure 4-11A)
in diaphragm arterioles following 6 hr of MV. These data demonstrate following
prolonged MV, vascular dysfunction occurs through endothelium-
dependent/endothelium-independent mechanisms. To our knowledge, this is the first
study that provides evidence for a reduction in NO bioavailability and/or NO “mis”-
handling in diaphragm arterioles following prolonged MV.
Pressure-Diameter Relationship Is Altered in Diaphragm Arterioles Following Prolong Mechanical Ventilation
Figure 4-12 illustrates the active and passive pressure-diameter relationship as
intraluminal pressure was increased stepwise from 10 to 130 cm H2O. There were no
changes in the active (myogenic) response, however, the passive-pressure response
was reduced following 6 hr of MV indicating structural alterations within the resistance
vasculature.
55
Table 4-1. Body and diaphragm mass, blood basses and hematocrit. Spontaneous breathing 30 min MV 6 hr MV Body mass (g) 274 ± 3 268 ± 3 271 ± 2 Diaphragm wt (mg) 873 ± 11 843 ± 30 855 ± 13 MAP (mmHg) 100 ± 4 105 ± 4 81 ± 4 * pH 7.4 ± 0.1 7.4 ± 0.1 7.4 ± 1 Arterial PO2 (mmHg) 96 ± 0.4 90 ± 6 80 ± 5 * Arterial PCO2 (mmHg) 47 ± 1 38 ± 4 30 ± 3 * Hematocrit (%) 44 ± 0.3 40 ± 1 37 ± 1 * MAP, mean arterial pressure. [Hb], arterial hemoglobin concentration. *P<0.05 versus both spontaneous breathing and 30 min MB. Values are means ± SE. Table 4-2. Renal, respiratory and select hindlimb skeletal muscle blood flows. Spontaneous breathing 30 min MV 6 hr MV Kidney 346 ± 43 319 ± 50 284 ± 39 Intercostal 13 ± 3 12 ± 2 8 ± 1 Rectus abdominus 5 ± 1 4 ± 1 4 ± 1 Soleus 33 ± 10 29 ± 8 28 ± 6 Red gastrocnemius 13 ± 1 15 ± 2 16 ± 1 ml/min/100g tissue. Values are means ± SE. Table 4-3. Microvascular PO2 dynamics across the rest-to-contractions transition after mechanical ventilation. 30 min MV 6 hr MV Baseline PO2 (mmHg) 37.2 ± 2.4 18.8 ± 1.5* Low PO2 (mmHg) 20.5 ± 1.9 7.7 ± 0.7 * Time delay (s) 10.8 ± 1.1 7.8 ± 1.4 * Time constant (s) 11.3 ± 2.1 6.8 ± 1.2 * Delta PO2 (mmHg) 15.9 ± 3.0 8.7 ± 1.0 * mmhg. Values are means ± SE.
56
0.0
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Dia
phra
gm B
lood
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00 g
)D
iaph
ragm
Vas
cula
r Con
duct
ance
(mL/
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/100
g/m
mH
g)
SpontaneousBreathing
30 min MV 6 hr MV
SpontaneousBreathing 30 min MV 6 hr MV
*
* †
*
* †
A
B
Figure 4-1. Resting diaphragm muscle blood flow (A) and vascular conductance (B) measured during spontaneous breathing (n=7) and after 30 min (n=8) and 6 hr of MV (n=5). (mean ± SE) *P<0.05 versus spontaneous breathing; †P<0.05 versus 30 min MV.
57
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0.6Medial CostalVentralDorsalCrural
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60Medial CostalVentralDorsalCrural
Bloo
d Fl
ow(m
L/m
in/1
00 g
)
SpontaneousBreathing
30 min MV 6 hrs MV
* †
A
B
Vasc
ular
Con
duct
ance
(mL/
min
/100
g/m
mH
g)
SpontaneousBreathing
30 min MV 6 hrs MV
* †
*
*
Figure 4-2. Resting blood flow (A) and vascular conductance (B) to regionally delineated portions of the diaphragm muscle during spontaneous breathing (n=7) and after 30 min (n=8) and 6 hr of MV (n=8). *P<0.05 versus spontaneous breathing; †P<0.05 versus 30 min MV.
58
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lood
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)D
iaph
ragm
Vas
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r Con
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ance
(mL/
min
/100
g/m
mH
g)
30 Min
*
* †
* †
A
B
Spontaneous BreathingMechanical Ventilation
Spontaneous BreathingMechanical Ventilation
6 Hrs
*
30 Min 6 Hrs
Figure 4-3. Resting diaphragm muscle blood flow (A) and vascular conductance (B) measured during spontaneous breathing (n=8) and mechanically ventilated rats (n=7) after 30 min and 6 hr. (mean ± SE) *P<0.05 versus spontaneous breathing; †P<0.05 versus 30 min MV.
59
0.0
0.1
0.2
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0.5 DiaphragmRed GastrocnemiusSoleus Intercostal
0
10
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Bloo
d Fl
ow(m
L/m
in/1
00 g
)
SpontaneousBreathing
30 min MV 6 hrs MV
*n.s
* †
n.s
Vasc
ular
Con
duct
ance
(mL/
min
/100
g/m
mH
g)
SpontaneousBreathing
30 min MV 6 hrs MV
n.s
* †
n.s*
A
B
n.s
n.s
Figure 4-4. Comparison of blood flow (A) and vascular conductance (B) measured at rest between the diaphragm, red portion of the gastrocnemius muscle complex, soleus, and intercostal muscles during spontaneous breathing (n=7) and after 30 min (n=8) and 6 hr of MV (n=8). *P<0.05 versus spontaneous breathing; †P<0.05 versus 30 min MV.
60
0
10
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0 5 10 15 20 25 300
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Spontaneous Breathing 30 min MV 6 hr MV
Mic
rova
scul
ar P
O2
(mm
Hg)
Time (s)
Mic
rova
scul
ar P
O2
(mm
Hg)
SpontaneousBreathing
30 min MV 6 hr MV
*†
*
A
B
Figure 4-5. Representative resting diaphragm microvascular PO2 profiles measured over time (A) and the average diaphragm microvascular PO2 (B) measured during spontaneous breathing and after 30 min and 6 hr of MV(n=11). *P<0.05 versus spontaneous breathing; †P<0.05 versus 30 min MV.
61
Figure 4-6. Mean microvascular PO2m profiles including 95% CI (dashed lines) (A) and representative diaphragm microvascular PO2 profiles (B) in response to electrically stimulated muscle contractions after 30 min and 6 hr of MV (n=5). Contractions were initiated at time zero.
Figure 4-6. Mean microvascular PO2m profiles including 95% CI (dashed lines) (A) and representative diaphragm microvascular PO2 profiles (B) in response to electrically stimulated muscle contractions after 30 min and 6 hr of MV (n=5). Contractions were initiated at time zero.
62
0
20
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60
806 hr Spontaneous Breathing30 min MV6 hr MV
0
2
4
6
830 min MV6 hr MV
Rest Contractions
*†*†‡
*
‡Bl
ood
Flow
(mL/
min
/100
g)
Rest ContractionsSpontaneous Breathing
VO2
(ml/m
in/1
00 g
)
*
*
#*†‡•
*‡A
‡6 hrs Spontaneous Breathing30 min MV6 hrs MV
Figure 4-7. Blood flow (A) and oxygen consumption (VO2) (B) at rest and during the steady-state of electrically stimulated contractions in the diaphragm muscle after 30 min (n=7) and 6 hr of MV (n=8) as compared to spontaneous breathing (n=7). *P<0.05 versus spontaneous breathing; †P<0.05 between 30 min and 6 hr of MV; ‡P<0.05 versus rest in the same condition; #P=0.056 between 30 min and 6 hr of MV.
63
0.0
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30 min MV6 hr MV
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Rest ContractionsSpontaneous Breathing
O2
Del
iver
y (m
L O
2/min
/100
g)
*†
*
B
*†‡
*‡
Rest ContractionsSpontaneous Breathing
Frac
tiona
l O2
Ext
ract
ion
*
*†*†‡
*‡
C
Figure 4-8. O2 delivery (A) and fractional O extraction (B) at rest and during electrically
stimulated diaphragm muscle contractions after 30 min (n=7) and 6 hr of MV (n=8) as compared to spontaneous breathing (n=7). *P<0.05 versus spontaneous breathing; †P<0.05 between 30 min and 6 hr of MV; ‡P<0.05 versus rest in the same condition.
64
Flow (nl/sec)
-10 0 10 20 30 40 50 60
Rel
axat
ion
(%)
0
10
20
30
40
50Spontaneous Breathing30 min MV6 hr MV
*ƚ
asdasdasdasd
Figure 4-9. Flow mediated vasodilation (A) in diaphragm arterioles during spontaneous breathing (n=7) and after 30 min (n=8) and 6 hr of MV (n=7).* P<0.05 versus spontaneous breathing; ƚP<0.05 versus 30 min MV.
A
65
**ƚ
sds
Spontaneous Breathing
30 min MV 6 hr MV
** ƚ
DSDSD
**ƚ
DSDSDS
A B
CD
Figure 4-10. Dose-responses to the endothelial-dependent vasodilation Acetylcholine (Ach) in the absence and presence of endothelial NO synthase inhibitor N-G-nitro-l-argine methyl ester (L-NAME) (A), Dose-responses to the endothelial-dependent vasodilation Acetylcholine (Ach) in the absence and presence of endothelial NO synthase inhibitor N-G-nitro-l-argine methyl ester (L-NAME) + COX inhibitor Indomethacin (INDO) (B), NO-dependent (endothelium-dependent) dilation (max dilationACh - max dilationACh + L-NAME) (C), Dose responses to the endothelium-independent vasodilator sodium nitroprusside (SNP) (D) in diaphragm arterioles during spontaneous breathing (n=19) and after 30 min (n=21) and 6 hr of MV (n=23). *P<0.05 versus spontaneous breathing. ƚP<0.05 versus 30 min MV.
66
Spontaneous Breathing
30 min MV 6 hr MV
*
Figure 4-11. eNOS mRNA expression in diaphragm arterioles during spontaneous
breathing (n=15) and after 30 min ( n=12) and 6 hr of MV (n=13). *P<0.056 versus spontaneous breathing.
67
Figure 4-12. Active and passive myogenic responsiveness in arterioles during spontaneous breathing (n=11) and following 30 min (n=10) and 6 hr of MV (n=8). *P<0.05 versus spontaneous breathing. ƚP<0.05 versus 30 min MV.
Pressure (cmH20)
0 20 40 60 80 100 120 140 160
Norm
alize
d D
iam
eter
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
Spontaneous Breathing Passive30 min Passive6 hr PassiveSpontaneous Breathing Active 30 min Active 6 hr Active
*ƚ
68
CHAPTER 5 DISCUSSION
These experiments provide several new and clinically relevant findings regarding
the regulation of O2 supply-demand and diaphragm contractile function with mechanical
ventilation (MV). Specifically, this is the first investigation to demonstrate: 1) there is a
time-dependent reduction in diaphragm blood flow and O2 delivery (QO2) with MV that is
not apparent in other skeletal muscle (during the time frame used herein), 2) diaphragm
microvascular PO2 (PO2m), which represents the sole driving force for capillary-to-
myocyte O2 diffusion, is reduced as a function of time of MV, 3) the ability to increase
blood flow in the diaphragm with muscular contractions is severely reduced after 6 hr of
MV, and 4) diaphragm O2 uptake (VO2) during muscular contractions is reduced by
~80% after 6 hr versus the onset of MV, 5) NO-mediated vasodilation is diminished
following prolonged MV, 5) there are also structural alterations to the diaphragm
resistance vasculature following prolonged MV. Overall, our findings demonstrate an O2
supply-demand imbalance in the diaphragm after mechanical ventilation that occurs
within hours after the onset of MV. The functional consequence of this is profound in
that the O2 supply-demand imbalance is exacerbated during muscular contractions,
resulting in a reduced aerobic metabolism in the diaphragm.
The time-dependent reduction in microvascular O2 supply (blood flow and
ultimately O2 pressure) in the diaphragm with MV present several potential
consequences, including 1) local areas of hypoxia and/or anoxia that may promote ROS
generation within diaphragm mitochondria and activate apoptotic pathways, and 2) an
O2 delivery limitation and exacerbated fall in intramyocyte PO2 during elevated
69
respiratory muscle O2 demand (e.g., during the weaning process), hastening the onset
of respiratory muscle fatigue. The precise role of a reduced QO2-to-VO2 ratio (i.e.,
diminished PO2m measured herein) in concert with the mitochondrial dysfunction
apparent after prolonged MV (Kavasiz et al., 2009), and their relative contributions to
VIDD, require additional investigations.
Mechanistic Basis for the Diminished Diaphragm Blood Flow Following Mechanical Ventilation
To our knowledge this is the first investigation examining the effect of MV over
time on diaphragm vasomotor function. Given the large vasodilator reserve evident in
the diaphragm even at maximal exercise (Poole et al., 2000), the finding of an relative
inability to increase blood flow during contractions following 6 hr of MV (Figure 4-6A)
was quite surprising and suggests a reduced vasodilation (possible due to increased
oxidative stress/antioxidant imbalances and NO bioavailability (Kang et al., 2009))
and/or structural modifications (e.g., short-term remodeling) associated with an
increased tonic vasoconstriction (Martinez-Lemus et al., 2011, Martinez-Lemus et al.,
2009) when the diaphragm is inactive. The following section will discuss the evidence
for endothelial dysfunction, vascular smooth muscle dysfunction and structural
modifications in the resistance vasculature following prolonged MV.
Endothelial Dysfunction
We have demonstrated prolonged MV results in a reduction in flow-mediated
(Figure 4-9A) vasodilation and, therefore, a reduction in shear stress along the apical
surface of the endothelial cell. Furthermore, it is known that a reduction in shear stress
(steady and oscillatory) alters the production of vasoregulating agents such as NO
(Chang et al., 2000; Kuchan et al., 1994; Rubanyi et al., 1986). The structure that
70
resides on the luminal surface (i.e. interface between blood flow and the endothelial
cell) and is in direct contact with the flowing blood is known as the endothelial
glycocalyx. These membrane-bound macromolecules are responsible for “sensing” fluid
mechanical shear stress. The physical displacement of these proteoglycans can be
transmitted to the cellular surface and provoke an intracellular response, such as
production of NO (Davies et al., 1995). Ach-induced vasodilation was also blunted
following prolonged MV (Figure 4-10 A) and was further reduced following treatment
with L-NAME or combined blockade with indomethacin and L-NAME. The inhibitory
effect of L-NAME on Ach-induced vasodilation and reduction in flow-mediated
vasodilation indicate a significant role for NO in mediating vascular dysfunction with MV.
Furthermore, given the drastic decline in diaphragm blood flow, it is also likely that there
is a reduced shear-stimulus or sensitivity to shear following prolonged MV (evidenced
by an reduction in eNOS mRNA expression) following prolonged MV. Although further
studies are warranted, we believe endothelial glycocalyx disruption (and ultimately
reduced NO production) may be one key instigator of the endothelial dysfunction
observed in this study. In this regard, it has also been demonstrated that hypoxia (i.e.
alterations in PO2, Ischemia/Reperfusion Injury) can severely damage the glycocalyx
(Ward et al., 1993; Czarnowska et al., 1995, Rubio-Gayosso et al., 2006 ). Moreover,
theese authors also concluded that the damage of the endothelial glycocalyx is
associated with the appearance/production of ROS, which is also elevated during MV
(Kavasis et al., 2009)
VSMC Responsiveness
Endothelial-independent vasodilation (i.e. smooth muscle vasodilation) was also
diminished following prolonged MV. The finding of an emaciated vasodilation to
71
exogenous NO in the prolonged MV group suggests there is NO “mis”-handling (as
compared to a reduction NO bioavailabilty) and a reduction smooth muscle
responsiveness to NO and cGMP-mediated relaxation following prolonged MV.
Vascular smooth muscle dysfunction has been reported by others (Behnke et al., 2010,
Delp et al., 1995, Miyata et al., 1992) and it is thought that this is possibly due to
reductions in the net intracellular accumulation of cGMP (due to increased cGMP-
phosphodiesterase activity) in smooth muscle (Moritoki et al., 1988), and/or a reduced
scavenging, or increased production of superoxide (Csiszar et al., 2002, Sindler et al.,
2009), causing a reduction in the bioavailabilty of NO (Gryglewski et al., 1986, Kang et
al., 2009).
Structural Modifications
Another important finding from this study was the reduction in the passive
pressure-diameter relationship. This indicates there maybe structural alterations in the
resistance vasculature with MV. Pistea and collegues (2008) have demonstrated there
maybe inward eutrophic remodeling of arterioles when exposed to chronic
vasoconstriction or inhibition of NO-mediated vasodilation (for review see Martinez-
lemus et al. 2009). In this regard, it has been demonstrated that there is an increased
in angiotensin II during prolonged MV (unpublished observations) which could be a key
contributor to the inward eutrophic remodeling. In addition, Li-Fu and colleges (2011)
recently demonstrated that there is an increase in collagen deposition in the diaphragm
following prolonged MV. This would likely cause vascular stiffness in diaphragm
arterioles and lead the reduction in passive-pressure response demonstrated herein
(Figure 4-12). These data yet represent another potential vascular mechanism for the
reduction in the hyperemic response following prolonged MV. It is important to note that
72
structural alterations can potentially affect vasodilation, however, the vessel data
presented herein was normalized to its maximal diameter, and therefore, we are sure
the changes found are not due to changes in the structure of the vessel.
It is unclear why the diaphragm displays a time-dependent reduction in blood
flow which was not apparent in other skeletal muscles that were also inactive (i.e.,
soleus and gastrocnemius; Figure 4-3). There are several unique properties of the
diaphragm in relation to other skeletal muscle (for review see (Mantilla et al., 2003))
which, when coupled with its remarkable activation history, may expedite the time-
course of vasomotor dysfunction with disuse in this muscle. Given the vasomotor
dysfunction observed after inactivity (e.g., after bed rest; (Demiot et al., 2007)), we
cannot preclude that similar reductions in blood flow to other skeletal muscle do not
occur over longer time periods than utilized in our protocol. In fact, reductions in blood
flow during disuse are thought to be a key signaling mechanism for structural and
functional adaptations within the resistance vasculature of some skeletal muscle (Delp
et al., 2009). Nonetheless, the reduced blood flow and O2 delivery to the diaphragm at
rest and during contractions following MV (Figures 4-6A and 4-7A, respectively) can
have profound ramifications on intracellular processes that contribute to ATP production
and molecular signaling mechanisms for mitochondrial dysfunction, atrophy, and
autophagy as discussed below.
Implications from Altered PO2m Dynamics
To investigate whether the rapidity with which QO2 and VO2 can increase is
affected with MV we quantified the PO2m profile (which is representative of the dynamic
QO2-to-VO2 ratio; (McDonough et al., 2011) across the rest-to-contractions transition.
Under healthy conditions, the evidence in vivo (Radedran et al., 1998, Bangsbo et al.,
73
2000) suggests that that QO2 dynamics are faster relative to VO2, such that the mean
venous PO2 (Grassi et al., 1996) or PO2m (Behnke et al., 2001) is maintained or
increased during the initial onset of contractions. This is advantageous as it maintains a
large capillary-to-intramyocyte PO2 gradient to facilitate blood-tissue O2 exchange.
However, with aging (Behnke et al., 2005) and pathological diseases (e.g., CHF
(Behnke et al., 2007,Diederch et al., 2002) and type II diabetes (Padilla et al., 2007)),
there is a shorter delay before PO2m decreases, as well as a more precipitous fall in
PO2m, after the onset of contractions in skeletal muscle. The faster PO2m kinetics in
these conditions are attributed, in part, to a slower increase in muscle blood flow (Copp
et al., 2009) and blunted arteriolar vasodilation dynamics (Behnke et al., 2010). In the
present study we observed faster PO2m dynamics (i.e., shorter time delay, faster MRT;
Table 3) during contractions after 6 hr relative to 30 min of MV in the diaphragm.
Whereas we did not measure QO2 or VO2 dynamics in separatum, the faster PO2m
dynamics after 6 hr of mechanical ventilation are consistent with a sluggish increase in
QO2 relative to VO2, which would force a greater reliance on non-oxidative energy
sources during the critical transition to muscular work.
Reduced O2 Delivery, Diaphragm VO2 and Cellular Energetics
Based upon Fick’s law of diffusion [VO2=DmO2 (PO2 capillary-PO2 intramyocyte)],
at a given VO2 and DO2 a significantly higher PO2 in the microvasculature would raise
the intracellular PO2. The latter of which would be advantageous in mitigating the
degree of intracellular perturbations (e.g. H+, lactate, phosphocreatine (PCr) and
inorganic phosphate Pi,) required to sustain a given mitochondrial ATP flux. However,
even a small reduction in O2 supply during the steady-state of submaximal exercise can
manifest large changes in cellular homeostasis (i.e., PCr degradation) (Haseler et al.,
74
1988, Hogan et al., 1999). This notion can be further conceptualized by considering the
equation for oxidative phosphorylation:
5 ADP + 5 Pi + O2 + 2NADH + 2H+ → 5 ATP + 2NAD+ + 2H2O
Under conditions of altered O2 supply, compensatory changes in the regulatory
parameters of mitochondrial respiration (i.e. cytosolic [ATP]/ [ADP][Pi] and mitochondrial
[NAD+]/[NADH]) occur to maintain a given rate of ATP production (and thus VO2)
(Hogan et al., 1992 ,Wilson et al., 1985). In the current study, 6 hr of MV lowered
contracting PO2m (Figure 5), thereby reducing intracellular PO2, and the maintenance of
a given rate of oxidative ATP production would mandate lowered intracellular energy
levels (i.e., reduced [ATP]/[ADP][Pi], [NAD+]/[NADH], and [PCr]) (Wilson et al., 1977).
Reduced PO2m and Cellular/Molecular Signals for Mitochondrial Dysfunction, Atrophy, and Autophagy
Whereas atrophy in locomotory skeletal muscle requires prolonged disuse
(Powers et al., 2007, Jackman et al., 2004, Lawler et al. 2003), the diaphragm displays
a rapid atrophic response with MV (Powers et al., 2005, Powers et al., 2007).
Furthermore, oxidative stress associated with diaphragm inactivity can occur within 3-6
hr after the onset of MV (Zergeroglu et al., 2003) and represents a molecular
mechanism for diaphragm atrophy (McClung et al., 2007, Betters et al., 2004) and
mitochondrial dysfunction (Kavasiz et al., 2009). However, the signaling mechanisms
for the enhance ROS production evident with MV remains unclear. The PO2m
measurements made in the current investigation reflect the composite PO2 of all the
plasma within the sampled region. It is likely that some areas of the diaphragm
vasculature would have considerably lower PO2’s then the aggregate value reported
herein. Therefore, given the large reduction in PO2m following 6 hr of MV in the current
75
study, we believe there may be hypoxic and/or anoxic loci within many diaphragm
myocytes which could promote mitochondrial ROS generation, although this has yet to
be determined.
It has been demonstrated that antioxidant administration attenuates MV-induced
muscle atrophy (McClung et al., 2007) and mitochondrial dysfunction (Powers et al.,
2011). In addition, in aged animals that have an elevated ROS production, antioxidant
administration has been demonstrated to elevate skeletal muscle PO2m (Herspring et
al., 2004). Therefore, the beneficial effects of antioxidant administration on mitigating
diaphragm contractile dysfunction with prolonged MV may be due, in part, to elevating
the QO2-to-VO2 ratio within the diaphragm.
QO2 and VO2 During Contractions: Ramifications on the Weaning Process
During muscular work under healthy conditions there is a tight coupling between
the increase in VO2 and QO2 (i.e., metabolic demands are precisely met by O2 delivery).
However, after 6 hr of ventilation there is an uncoupling between these two variables,
resulting in an increased fractional O2 extraction at rest and during contractions (Figure
7A). In human patients who failed spontaneous breathing trials, there was an increased
fractional O2 extraction until failure, which was attributed to a reduced O2 supply (Jubran
et al., 1998). Further, it has been demonstrated that impairments in the mechanical-
chemical coupling in the diaphragm can result, in part, from a decreased O2 availability
(Pierce et al., 2001). Accordingly, both an impaired contractile function and limited O2
supply have the potential to reduce aerobic metabolism within the diaphragm after MV.
In the current study, we observed a ~80% reduction in VO2 during contractions after 6
hr of MV compared to that calculated after 30 min of MV. This large reduction in
oxidative metabolism would hasten the onset of diaphragm fatigue, and if this same
76
reduction in VO2 occurs in the human diaphragm after MV, would predispose the patient
to weaning failure. However, from the current study it is not possible to delineate the
contributions of a reduced O2 supply from mitochondrial and contractile dysfunction to
this impaired VO2 response after MV.
Future Directions
MV is associated with respiratory muscle dysfunction in animal and human
models, however, little is known about the pathophysiology and specific molecular
mechanisms behind VIDD. Recently, more investigators are developing and utilizing
murine experimental models of MV. The murine model is very beneficial due to the wide
variety of molecular techniques and assays available, as well as the ability to genetically
modified animals, allowing for proof-of-concept experiments and dissection of specific
molecular pathways. It is important that future investigations attempt to utilize a highly
integrative approach (e.g. examining human diaphragm samples as well as utilizing
genetically modified mice) in order to obtain the greatest amount of knowledge about
this phenomenon.
The results of the present study raise the question whether diaphragm oxygen
supply-demand imbalance plays a role in VIDD and weaning difficulties. Future studies
are needed to investigate the role of the endothelial glycocalyx in diaphragm arterioles
following prolonged MV. The diminished flow-mediated and Ach-induced vasodilation is
indicative of endothelial dysfunction. It would beneficial to determine if there is a
reduction in NO production following prolonged MV due to the disruption of the
glycolcalyx. Additionally, new therapeutic strategies (cationic co-polymers) have been
developed to target and repair the endothelial glycocalyx (Giantsos et al., 2009).
Therefore, further investigations are needed to determine whether these biomimetic
77
polymers can be a form of infusible therapy to restore endothelial function and ultimately
diaphragm oxygenation during prolonged MV. Moreover, given the large body of
evidence suggesting oxidative stress as a central component of VIDD, interventions
aimed to reduce ROS and oxidative damage (e.g. anti-oxidant supplementation,
exercise training) will also be valuable in further dissecting the molecular pathways
involved in VIDD. Furthermore, other potential vascular molecular pathways (e.g.
Angiotensin pathway, TGF-β signaling) need to be examined to gain better insight of
diaphragm vascular function following prolonged MV.
Another valuable tool that can significantly improve respiratory muscle function in
patients experiencing weaning difficulties is diaphragm pacing. One of the primary
triggers of ROS production in the diaphragm is due to the reduction in contractile activity
by the unloading of the diaphragm. The diaphragm, in contrast to other skeletal
muscles, is activated rhythmically on a continuous period. MV imposes a unique form of
muscle disuse as the diaphragm is simultaneously mechanically unloaded, intermittently
shortened, and electrically suppressed by the ventilator (Petrof et al., 2010). Intermittent
or continuous phrenic nerve stimulation (and therefore diaphragm muscle contraction)
may be beneficial in maintenance of the intracellular processes and molecular signaling
pathways responsible for ATP production. Moreover, it is not currently known whether
intermittent or continuous pacing will improve the ability of the diaphragm to regulate the
matching of O2 delivery-to-O2 uptake (in response to contractions), or alter diaphragm
vasomotor function following prolonged MV. Conversely, given the data presented in the
current investigation, it will also be very beneficial to further examine the effect of “flow”
to the diaphragm following prolonged MV. By the development and implementation of a
78
diaphragm perfusion system, one could easily gain further insight into the role of blood
flow to the diaphragm during prolonged MV. Additionally, one could observe whether
the maintenance of blood flow during prolonged MV will prevent the characteristics of
VIDD (e.g, contractile dysfunction, ROS production, Mitochondrial Dysfunction,
Calpain/Protease Activation). Finally, it is imperative that we obtained detailed
understanding of the current ventilation protocols and physiological parameters
accessed by clinicians and doctors. It is very plausible that these variables (e.g.
amount of saline infusion, type and amount of inhalant analgesics used) could have an
impact impact on the ability to be weaned from a ventilator. Moreover, multiple factors
other than MV affect diaphragm function (e.g., sepsis, corticosteroids, neuromuscular
blockade, antibiotics, nutritional deficiency; Laghi et al., 2003). The potential impact of
these mediators also needs to be considered when investigating the pathophysiology
behind the development of VIDD.
Summary
In summary, we have demonstrated that mechanical ventilation elicits a
significant reduction in the microvascular PO2 in the diaphragm in as little as 6 hr, and
this lowered PO2m is associated with a diminished diaphragm blood flow. Further, upon
initiation of muscular contractions, there is an inability to augment diaphragm blood flow
and O2 delivery which results in an inadequate matching of O2 delivery-to-O2 uptake.
Taken together, the diminished O2 delivery results in a ~80% reduction in the aerobic
metabolism of the diaphragm during contractions. So what is causing this mismatching
of O2 delivery-to O2 uptake? Although the precise mechanisms are likely multifaceted,
we provide clear evidence in favor of vascular dysfunction being a key instigator in the
79
oxygen supply/demand mismatch with prolonged mechanical ventilation and believe this
is one potential mechanism contributing, in part, to weaning difficulties.
Collectively, these data provide strong support that a diminished O2 supply (in
addition to mitochondrial dysfunction) contributes to mechanical ventilation-induced
diaphragm dysfunction. Moreover, the results from these experiments provide insight
into the functional and structural mechanisms responsible for MV-induced diaphragm
atrophy in the diaphragm, and also for broader topics such as skeletal muscle wasting
due to prolonged bed rest, immobilization, and disease states. Important questions to
address in future work is the role of the diminished O2 supply on mitochondrial function,
and what vasomotor pathways contribute to the reduce blood flow response after
mechanical ventilation.
80
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BIOGRAPHICAL SKETCH
Robert Thomas Davis III was born in Detroit, MI and spent most of his childhood
between Winfield, KS and Aurora, IL. He graduated from Kansas State University (KSU)
with a bachelor’s degree in Kinesiology in 2006. He decided continue on his graduate
work at KSU and received his master’s degree. In 2009 Robert moved to Gainesville,
FL to attend the University of Florida and begin work on his Doctor of Philosophy in
exercise physiology. Upon completion of his PhD, Robert plans to begin post-doctoral
training at University of Illinois at Chicago in the area of cardiac and skeletal muscle
contraction under the direction of Dr. John Solaro.
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