Metabolic response to an acute jump in cardiac workload: effects on malonyl-CoA mechanical efficiency and fatty acid oxidation

Zhou et al, H-00557-2007.R1

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Metabolic Response to an Acute Jump in Cardiac Workload:

Effects on Malonyl-CoA, Mechanical Efficiency, and Fatty

Acid Oxidation An Acute Jump in Cardiac Workload

Lufang Zhou1, Hazel Huang2, Celvie L. Yuan3, Wendy Keung4, Gary D. Lopaschuk4 and

William C. Stanley2,3,5

1Departments of Biomedical Engineering, 2Physiology & Biophysics, and 3Nutrition, Case

Western Reserve University, Cleveland, OH 44106; 4Department of Pediatrics, University of

Alberta, Edmonton, Canada, T6G 2S2; and 5Division of Cardiology, Department of Medicine,

University of Maryland, Baltimore, MD 21201

Short title: Cardiac Metabolism at High Workload

Address for correspondence: William C. Stanley, Ph.D.

Division of Cardiology Department of MedicineUniversity of Maryland-Baltimore20 Penn St., HSF2, Room S022Baltimore, MD 21201, USA410-706-3585 (Phone)410-706-3583 (Fax)e-mail: [email protected]

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Articles in PresS. Am J Physiol Heart Circ Physiol (December 14, 2007). doi:10.1152/ajpheart.00557.2007

Copyright © 2007 by the American Physiological Society.

Zhou et al, H-00557-2007.R1

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ABSTRACT

Inhibition of myocardial fatty acid oxidation can improve left ventricular (LV) mechanical

efficiency by increasing LV power for a given rate of myocardial energy expenditure. This

phenomenon has not been assessed at high workloads in non-ischemic myocardium, so we

therefore subjected in vivo pig hearts to a high workload for 5 min, and assessed whether

blocking mitochondrial fatty acid oxidation with the carnitine palmitoyltransferase-I (CPT-I)

inhibitor oxfenicine would improve LV mechanical efficiency. In addition, the cardiac content

of malonyl-CoA, (an endogenous inhibitor of CPT-I) and acetyl-CoA carboxylase activity

(which synthesizes malonyl-CoA) were assessed. Increased workload was induced by aorta

constriction and dobutamine infusion, and LV efficiency was calculated from the LV pressure-

volume loop and LV energy expenditure. In untreated pigs the increase in LV power resulted in

a 2.5-fold increase in both fatty acid oxidation and cardiac malonyl-CoA content, but did not

affect the activation state of acetyl-CoA carboxylase (ACC). The activation state of the ACC

inhibitory kinase, AMP activated protein kinase, decreased by 40% with increased cardiac

workload. Pretreatment with oxfenicine inhibited fatty acid oxidation by 75% and had no effect

on cardiac energy expenditure, but significantly increased LV power and LV efficiency (37+5%

vs. 26+5%, respectively, p<0.05) at high workload. In conclusion 1) myocardial fatty acid

oxidation increases with a short-term increase in cardiac workload despite an increase in

malonyl-CoA concentration, and 2) inhibition of fatty acid oxidation improves LV mechanical

efficiency by increasing LV power without effecting cardiac energy expenditure.

Key words: acetyl-CoA carboxylase, AMP activated protein kinase, exercise, fatty acids, heart,

mitochondria

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INTRODUCTION

One of the major determinants of myocardial oxygen consumption (MVO2) at a given

rate of left ventricular (LV) power generation is mitochondrial substrate selection (23; 44).

Under normal resting conditions, fatty acid oxidation is the predominant source of energy for

cardiac power generation (60~80%), however studies in humans(39), dogs(30; 31) and pigs(25)

in vivo, and in isolated perfused rat (3; 21) and mouse(20) hearts show that with high rates fatty

acid oxidation, the external power is reduced for a given MVO2 (3; 30; 39). In the failing heart,

or during acute ischemia and/or reperfusion, pharmacological treatment with agents that either

inhibit myocardial fatty acid oxidation (5; 6) or directly activate carbohydrate oxidation (2; 28;

41) increase LV function without affecting MVO2, and therefore improve the left ventricular

mechanical efficiency (defined as the ratio of external LV power/LV energy expenditure).

However, the effect of inhibiting fatty acid oxidation on cardiac function and MVO2 during high

cardiac workloads in the healthy heart is not known.

Under normal or ischemic conditions, fatty acid oxidation strongly inhibits the

mitochondrial enzyme pyruvate dehydrogenase, which inhibits oxidation of pyruvate, and thus

glucose and lactate uptake and oxidation(35). On the other hand, it has been shown that

inhibition of fatty acid oxidation increases pyruvate oxidation and glucose uptake and oxidation

at rest and during exercise(26; 41; 45). Fatty acid oxidation in the heart is regulated at the level

of the mitochondrial outer membrane by the activity of carnitine palmitoyltransferase I (CPTI),

which is inhibited by malonyl-CoA on the cytosolic side of the enzyme(22; 44). Several studies

have shown an inverse relationship between myocardial malonyl-CoA content and fatty acid

oxidation(9; 24; 37; 40; 43), and specifically that adrenergic stimulation corresponds with a

reciprocal increase in fatty acid oxidation and decrease in malonyl-CoA content(13-16; 24; 36).

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Malonyl-CoA is produced by acetyl-CoA carboxylase (ACC), which is inhibited when

phosphorylated at serine-79 by AMP activated protein kinase (AMPK), while AMPK is activated

by phosphorylation at threonine-172 (10). We previously found that the reduction in malonyl-

CoA content that occurred when MVO2 was increased by adrenergic stimulation was not

associated with reduced ACC activity or increased AMPK activity in pigs (15), however tissue

was sampled between 15 and 30 minutes after the initiation of stimulation. It is not known if

there are changes in malonyl-CoA content and activation of ACC and AMPK during the initial

minutes of the transition from a low to a high cardiac workload.

The present study evaluated what effect inhibition of fatty acid oxidation on myocardial

LV function and mechanical efficiency. We hypothesized that a switch in myocardial energy

substrate use from fatty acid to carbohydrates would increase LV power without affecting

oxygen consumption, and therefore improve myocardial energy efficiency. The second aim of

this study was to determine if there is a decrease in malonyl-CoA content with an abrupt short-

term increase in cardiac workload. We hypothesized that with five minutes of increased cardiac

workload the myocardial content of malonyl-CoA would decrease due to activation of AMPK

and inhibition of ACC, resulting in greater fatty acid oxidation, as previously observed with a

longer duration of adrenergic stimulation (13-16; 24; 36). Studies were performed in an

established open-chest pig model, with animals subjected to an abrupt five minute increase in

cardiac workload induced by simultaneous adrenergic stimulation, parasympathetic blockade and

aortic constriction treatment (24; 38; 48). Fatty acid oxidation was inhibited at the level of

transport into the mitochondria using oxfenicine, a CPT-I inhibitor. There are numerous

approaches to inhibiting fatty acid oxidation(29; 42). In this study oxfenicine was selected

because it is devoid of cardiovascular effects under normal conditions(5; 7; 38; 48), has a rapid

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onset of action(19), and consistently reduces fatty acid oxidation in the pig heart under

conditions of increased cardiac workload(24; 38; 48).

METHODS

Studies were performed in accordance with the Guide for the Care and Use of Laboratory

Animals (NIH Publication Number 85-23, revised 1996) and with prior approval of the

Institutional Animal Care and Use Committee at Case Western Reserve University. Twenty four

domestic pigs of either sex (mean weight, 35.1 ± 1.1 kg) were entered into the study. Data from

these studies have been reported separately on the regulation of pyruvate dehydrogenase activity,

NADH, glycogen concentration, and basic hemodynamics (48).

Surgical preparation

The surgical preparation has been previously described in detail (24; 38; 48). Briefly,

overnight fasted pigs were sedated with Telazol (6 mg/kg im), anesthetized with isoflurane by

mask (5%), ventilated with 100% O2,and maintained on isoflurane (0.75-1.5%) and ketamine (4

mg kg-1 min-1 iv) to keep PCO2 and pH in the normal range (PO2 >100 mmHg, PCO2 35-45

mmHg, and pH 7.35-7.45). A femoral artery and vein were catheterized for blood sampling and

infusion, respectively, and the animals were heparinized (200 U/kg bolus followed by 100 U kg-1

min-1i.v) to prevent clotting and thrombus formation. The heart was exposed via a midline

sternotomy, and the left atrium was catheterized for infusion of dobutamine and atropine. A

vascular occluder was placed around the ascending aorta and constricted during the dobutamine

treatment period. The cardiac anterior interventricular vein was catheterized for coronary venous

blood sampling, and a Doppler ultrasonic flow meter was placed around the proximal left

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anterior descending coronary artery to record blood flow continuously (Transonics Inc). Four

sonimicrometry crystals were placed at midmyocardial depth in the base, apex, and septum of the

lateral wall of left ventricle to continuously measure LV volume using an online commercial

system (Sonometrics). A high fidelity pressure transducer (Millar Instruments) was positioned in

the LV, and the signal was integrated with LV volume, and the LV pressure – LV volume loop

area was calculated for each beat.

Experimental protocol

Three groups of pigs were studied: 1) an untreated group subjected to increased cardiac

workload with dobutamine infusion (DOB, n=8), 2) an oxfenicine treated group that was

subjected to increased cardiac workload (DOB + OXF, n=8), and 3) a control (CON, n=8) group

with sham instrumentation and normal cardiac workload. At the beginning of the protocol,

[9,10-3H]oleate tracer was infused (40 µCi/hr i.v.) for the measurement of fatty acid oxidation,

and the oxfenicine treatment was initiated in the DOB + OXF group (30 mg/kg iv bolus

oxfenicine followed by an infusion at 30 mg kg-1 min-1). After a 50 minute equilibrium period,

animals in the DOB and DOB + OXF were subjected to a 5 minute period of increased cardiac

work induced by constricting the aortic cuff sufficient to maintain the peak left ventricular

systolic pressure at ~190 mmHg, while simultaneously infusing dobutamine (100 µg/kg as a

bolus followed by 40 µmol kg-1 min-1) and atropine (2 mg i.v. bolus) into the left atrium to

increase heart rate and contractility. Arterial and venous blood samples for measurement of

blood glucose and lactate concentrations were taken before (-5 and -1 minutes) and during

dobutamine treatment (at 20, 45, 75 seconds, 2, 3, 4, and 5 minutes), and samples were drawn for

plasma free fatty acid and 3H2O concentrations at -5 and -1 minutes, and at 3, 4, and 5 minutes of

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dobutamine infusion. After 5 minutes of increased workload a large punch biopsy (~3 g) was

rapidly excised from the LAD bed and immediately freeze-clamped on aluminum blocks pre-

cooled in liquid nitrogen and stored at –80°C for later analysis(40). The CON group received the

same infusion of [9,10-3H]oleate, and arterial and coronary venous blood samples were drawn at

50-60 minutes, followed immediately by a myocardial biopsy, as in the other two groups.

Analytic methods

Arterial and venous O2 saturation and hemoglobin were measured spectrophotometrically

with a hemoximeter (A-VOX System, San Antonio, TX), and pH, PCO2, and PO2 were measured

in a blood gas analyzer (Nova Biomedical, Waltham, MA). Subsequent biochemical analysis

was performed with the investigator blinded to treatment. Blood samples were analyzed for

concentrations of glucose and lactate, and plasma was assayed for free fatty acids, [3H] oleate

and 3H2O as previously described(24; 38). Malonyl-CoA and adenine nucleotide contents were

assayed by high-pressure liquid chromatography with UV detection as previously described (40).

All tissue concentrations were expressed per gram wet weight of tissue. The amount of total and

phosphorylated ACC and AMPK were assessed by western blot using specific antibodies for

phosphor-ACC at serine-79 and phospho-AMPK at threonine-172, as previously described (24;

27). AMPK activity was measured on myocardial homogenates as previously described (11).

Calculations

Myocardial blood flow was measured from the ultrasonic flow meter and normalized by

dividing by the weight of the heart being perfused by the LAD (34; 40). The net uptakes (µmol

kg-1 min-1) of glucose, lactate, free fatty acids and oxygen were calculated as the product of

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arterial - venous difference and blood flow. The rates of exogenous fatty acid oxidation (µmol g-

1 min-1) was calculated as the product of the release of 3H2O (dpm/ml) and myocardial blood

flow, divided by the arterial specific radioactivity of free fatty acids (dpm/µmol)(38).

Stroke volume was calculated as LV end diastolic volume – LV end systolic volume, and

cardiac output as the product of stroke volume and heart rate. LV stroke work (J) was calculated

as the LV pressure (Pascals) times volume, and LV power (watts) was calculated as the product

of LV stroke work and heart rate(4). LV energy expenditure was calculated from MVO2

assuming 20.2J/ml of O2 (46), and LV mechanical efficiency as LV power/LV energy

expenditure.

Statistical analysis

All hemodynamic variables; rates of free fatty acid, glucose, and lactate uptakes; rate of

fatty acid oxidation; and tissue metabolite concentrations were compared between resting

conditions and increased cardiac work and between DOB and DOB + OXF groups using a one-

or two-way ANOVA with the Bonferroni post-hoc test for multiple comparisons, as appropriate.

Significance was set at P<0.05, and values are reported as means ± SE.

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RESULTS

As recently published separately from these experiments, there was a significant increase

in heart rate, peak LV pressure, myocardial blood flow and MVO2 in the DOB and DOB + OXF

groups compared to CON, while there were no differences between the DOB and DOB +OXF

groups (see reference (48)). Cardiac output, stroke work and LV energy expenditure increased to

a similar extent in the DOB and DOB + OXF groups (Table 1). Treatment with oxfenicine

significantly increased LV power compared with the DOB group (1.72±0.35 vs. 1.28±0.13 at 45

seconds and 1.70±0.33 vs. 1.27±0.23 at 5 minutes, respectively) (Fig. 1) despite no effect on LV

energy expenditure (Table 1). As a result, a significant improvement in mechanical efficiency

was seen in the DOB + OXF group compared to the DOB group (Fig. 1).

The arterial concentrations of lactate, glucose, and fatty acids were unchanged over the

course of study and values were similar among the three experimental groups (data not shown).

Increased cardiac work significantly increased glucose and fatty acid uptake in the DOB group

(Fig. 2). Pharmacological inhibition of CPT-1 suppressed fatty acid uptake in the DOB + OXF

group under resting and high cardiac work conditions, and further enhanced glucose and lactate

uptakes compared to the DOB group (Fig. 2). Myocardial fatty acid oxidation was also

suppressed by oxfenicine treatment under resting conditions (14±9 compared to 49±14nmol g-1

min-1; p<0.05) and at a high cardiac workload (Fig. 3). There was a decreased lactate uptake in

the DOB and DOB + OXF groups in the first minute of increased cardiac energy expenditure

(Fig. 2B), with 4 out of 8 pigs in the DOB group and 2 out of 8 pigs in the DOB + OXF group

showing a net release of lactate at 20 and/or 45 seconds.

The rate of free fatty acid oxidation was greater in the DOB group compared with the

DOB + OXF group before (data not shown) and during the dobutamine treatment period (Fig. 3).

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Malonyl-CoA concentration was greater in the DOB and DOB+OXF groups compared with the

CON group (P<0.05), with no significant difference between these two groups (Fig. 3). The

amount of total ACC and phospho-ACC were not different among groups, nor was the phospho-

ACC/total ACC ratio (Table 2). While total AMPK was not different among groups, there was a

significant reduction in phospho-AMPK and the ratio of phospho-AMPK/total AMPK in the

DOB group compared to the CON group, while the DOB + OXF group was not different from

the other two groups.

The cardiac content of AMP and ADP were not different among the groups (Table 3),

although the ATP content was 20% lower in the DOB and DOB + OXF groups compared to the

CON group. Interestingly, we previous measured ATP and ADP content on these same samples

using a less precise luciferase assay, which showed approximately twice the variability, but again

no differences among the groups(48).

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DISCUSSION

The results of this study show that despite the expected increase in myocardial fatty acid

oxidation during an acute work jump there was a paradoxical increase in the tissue content of

malonyl-CoA, an established inhibitor of CPT-I and cardiac fatty acid oxidation. In addition, the

increase in malonyl-CoA was not due to activation of ACC, but did correspond to a decrease in

AMPK activation. Treatment with oxfenicine had little effect in malonyl-CoA content despite a

75% decrease in fatty acid oxidation, suggesting that the observed increase in malonyl-CoA was

largely independent from changes in fatty acid oxidation.

The second main finding of this study is that inhibition of fatty acid oxidation with

oxfenicine increases LV power without increasing cardiac energy expenditure. This

phenomenon has been observed under conditions of normal workload, during demand-induced

ischemia, and with post-ischemic reperfusion(41; 44). The present study extends this concept to

conditions of high workload similar to intense exercise in healthy people, and suggests the

provocative idea that CPT-I inhibition might improve exercise performance in short-term intense

aerobic athletic events. These observations are consistent with previous studies showing that

switching from fatty acid utilization to carbohydrate increases LV energy efficiency (2; 3; 20;

21; 25; 30; 32; 39). Compared with fatty acids, carbohydrates are more oxygen efficient (i.e., for

a given amount of ATP synthesis fatty acid oxidation requires 11% more oxygen consumption

than pyruvate), and high concentrations of fatty acids have also been shown to uncouple

oxidative phosphorylation and increase oxygen utilization in isolated mitochondria and cells(44).

Thus, the improved efficiency with oxfenicine is likely attributed to greater ATP synthesis per

oxygen consumption and/or more effective ATP use by the heart.

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We have previously observed that 15 to 30 minutes of intense adrenergic stimulation in

pigs results in a reciprocal decrease in malonyl-CoA content and an increase in fatty acid uptake

and oxidation (12; 26; 38), suggesting that less malonyl-CoA inhibition of CPT-I is a primary

mechanism for the increase in cardiac fatty acid oxidation observed with physiological stresses

such as an acute bout of exercise(15; 16; 24). In contrast, the present investigation found the rate

of fatty acid oxidation increased with dobutamine treatment despite a 2.5-fold increase in tissue

malonyl-CoA concentration (Fig. 3). Since malonyl-CoA exist in both cytosol and mitochondria

(18; 22; 47), it is possible that the cytosolic malonyl-CoA decreased while mitochondrial

malonyl-CoA increased. Malonyl-CoA inhibits CPT-I on the cytosolic side of the enzyme(22;

44), and is produced in both the cytosol and mitochondrial matrix from acetyl-CoA (44). The

supply of acetyl-CoA is a major regulator of malonyl-CoA formation (36; 37). The increase in

workload in the present experiment caused a 50% increase in the acetyl-CoA concentration (48),

presumably due to the rapid stimulation of acetyl-CoA formation by pyruvate dehydrogenase,

which may have triggered a selective increase of malonyl-CoA in the mitochondrial matrix.

Thus the increased tissue malonyl-CoA content may be due to a specific increase in

mitochondrial malonyl-CoA, as previously suggested (24). It is impossible to prove this based

on current experimental results, since malonyl-CoA was measured in whole tissue without

distinguishing cytosolic and mitochondrial compartments. Future studies should rapidly separate

the cytosol and mitochondria and measure these CoAs in these two compartments, although

accurate measurements of cytosolic and mitochondrial malonyl-CoA has yet to be made due to

technical difficulties with this approach. In any case, the results of the present study clearly

indicate that a fall in total tissue malonyl-CoA content is not essential for the increase in

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cardiac fatty acid oxidation that occurs in response to an acute increase in cardiac

workload.

Consistent with our previous studies in pigs (15; 24) there was no increase in the

activation of AMPK or ACC phosphorylation with increased cardiac workload. Studies in

working perfused rat hearts also found no increase AMPK activity at either 1 or 15 minutes

following a 2 to 3 fold increase in cardiac power(1). In addition, mice expressing a cardiac-

specific dominant-negative AMPKalpha2 subunit have normal ATP content and glycogen

depletion in response to acute exercise stress, stress echocardiography, and maximal exercise

capacity(33). On the other hand, 10 minutes of treadmill running in rats approximately doubled

AMPK activity and phospho-AMPK at threonine-172, and also doubled the amount of phospho-

ACC at serine-79(8). The results of the present study, and our previous work in pigs(15; 24),

consistently suggest that there is not activation of AMPK or inhibition of ACC in response to

high dose dobutanime and increased aortic pressure in pigs. While AMPK appears to play a

central role in the regulation of cardiac energy metabolism under many conditions(10), the

results of the present in vivo study shows that despite a significant decrease in phospho-AMPK

and AMPK activity (Fig 4) there is a stimulation of glucose uptake, glycogenolysis, and fatty

acid oxidation, (Table 1, Fig. 1-3)(48). Taken together, activation of AMPK is not an essential

regulatory component of metabolic response to a step increase in cardiac workload.

Cardiac ATP content, as measured by high pressure liquid chromatography, was

decreased by 20% under conditions of high workload, however AMP and ADP were not

increased, suggesting that there was a net loss of adenine nucleotides during the 5 minutes of

increased cardiac workload. This is consistent with our previous observation of an increased

adenosine production and efflux with dobutamine-induced work in pigs (17). In terms of

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metabolic regulation, since there was also no increase in AMP or phospho-AMPK, it appears that

the modest fall in ATP is not a major regulator of the energetic response to an acute jump in

workload.

In conclusion, the results of the present study show that inhibition of fatty acid oxidation

improves LV mechanical efficiency by increasing LV contractile power without affecting MVO2

during an acute bout of high workload. This suggests a novel approach to improving LV

mechanical efficiency at high cardiac workloads with drugs that optimize myocardial energy

metabolism, and presents the possibility that inhibition of CPT-I could potentially enhance

performance in athletic events that are limited by cardiac pump function. In addition, we

observed a paradoxical elevation in malonyl-CoA concentration and fatty acid oxidation at high

workloads. Lastly, a significant fall in AMPK activation was observed under conditions of high

energy demand, which further illustrates that activation of AMPK is not an essential component

of the metabolic response to the increase in cardiac workload.

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ACKNOWLEDGEMENTS

This work was support by NIH grants HL074237 and GM-66309, and by a grant from the

Canadian Institutes of Health Research. Gary Lopaschuk is a Medical Scientist of the Alberta

Heritage Foundation for Medical Research The authors thank Drs. Monika Duda, Isidore Okere,

and Naveen Sharma for assistance with the animal experiments.

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Table 1. Hemodynamic responses to short-term increased cardiac power with or without pretreatment with oxfenicine in pigs.

DOB (n=8) DOB + OXF (n=8)CON(n=8) Baseline 45 sec 5 min Baseline 45 sec 5 min

Stroke Work (J) 0.33+0.03 0.27+0.03 0.39+0.06* 0.35+0.06 0.26+0.03 0.49+0.07* 0.40+0.05

Cardiac Output (L/min)

2.8+0.3 2.5+0.3 3.7+0.5* 3.2+0.4 2.3+0.3 3.8+0.8* 3.7+0.8

Peak +dP/dt, (mmHg/sec)

1500+170 1420 +120 4720+540* 4920+670* 1320+150 5910+400* 6430+440*

LV energy expenditure

(watts)1.41+0.12 1.69+0.19 4.04+0.39* 4.81+0.40* 1.41+0.17 3.96+0.42* 5.21+0.54*

Values are means + SE. *: P<0.05 vs. CON

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Table 2. Summary of Western blot assessment of total and phosphorylated AMPK and ACC. Data expressed in arbitrary units.

CON DOB DOB+OXF

Total AMPK 247±9 237±21 281±32

Phospho-AMPK 154±3 88±17* 127±11

Total ACC 48±8 60±7 69±12

Phospho-ACC 29±2 33±5 41±3

Phospho/Total ACC 0.70±0.13 0.58±0.10 0.86±0.31

* P< 0.05 compared to CON; † P<0.002 compared to CON

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Table 3. Myocardial content of adenine nucleotides (µmol/g).

CON DOB DOB+OXF

ATP 4.64±0.11 3.64±0.15* 3.47±0.23*

ADP 1.08±0.02 1.12±0.05 1.13±0.03

AMP 0.159±0.004 0.180±0.019 0.181±0.011

* P<0.004 compared to CON

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Figure Legend.

Fig. 1. Effect of oxfenicine on myocardial left ventricular external power (top panel) and

mechanical efficiency (lower panel) under normal conditions and at 45 seconds and 5 minutes of

the period of increased cardiac workload. *p<0.05 vs. baseline, † p<0.05 vs DOB at the same

time point.

Fig. 2. Net myocardial glucose (top), lactate (middle) and fatty acid (lower) uptake as a function

of time for DOB and DOB + OXF groups under resting condition and during increased cardiac

energy expenditure.

Fig. 3. Myocardial malonyl-CoA content and the rate of fatty acid oxidation at the end of

protocol. * p<0.05 vs. control group (CON).

Fig 4. Myocardial phospho-AMPK/total AMPK as measured by western blot (top panel), and myocardial AMPK activity (lower panel) * p<0.05 vs. control group (CON).

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Fig. 1.

0

0.5

1.0

1.5

2.0

2.5

Pre 45 Sec 5 min Pre 45 Sec 5 min

LVPo

wer

(wat

t)

Untreated OXF

††

Untreated OXF

LVEf

fici

ency

(%)

0

20

40

60

Pre 45 Sec 5 min Pre 45 Sec 5 min

†

†

* *

* *

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Fig. 2.

0.0

0.2

0.4

0.6

0.8 DOBDOB + OXF

-0.2

0.0

0.2

0.4

0.6

0.8

Lac

tate

Up

take

(µm

ol/g

/min

)

0

40

80

120

160

Fre

e F

atty

Aci

d U

pta

ke(n

mo

l/g/m

in)

-6 0 1 2 3 4 5Dobutamine + Aortic Constriction

-6 0 1 2 3 4 5Dobutamine + Aortic Constriction

-6 0 1 2 3 4 5Dobutamine + Aortic Constriction

-6 0 1 2 3 4 5Dobutamine + Aortic Constriction

0 1 2 3 4 5Dobutamine + Aortic Constriction

-6 0 1 2 3 4 5Dobutamine + Aortic Constriction

-6 0 1 2 3 4 5Dobutamine + Aortic Constriction

-6 0 1 2 3 4 5Dobutamine + Aortic Constriction

Time (minutes)

P < 0.007 DOB vsDOB+OXF

P < 0.001 DOB vsDOB+OXF

P < 0.001 DOB vsDOB+OXF

Glu

cose

Up

take

(µm

ol/g

/min

)

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Fig. 3

0.0

0.5

1.0

1.5

2.0

2.5

CON DOB DOB + OXF

Myo

card

ial M

alo

nyl

-Co

A(µ

mo

l/g)

Myo

card

ial F

AO

(n

mo

l/g/m

in)

0

40

80

120

160

CON DOB DOB+ OXF

**

*

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Figure 4.

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0

90

180

270

CON DOB DOB + OXF

*

*

CON DOB DOB + OXF

P-AMPK/total AMPK

AMPK Activity

pmol

sm

g pr

otei

n-1

min

-1A

rbitr

iary

units

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