Influence of the caveolae on the stimulation of β2 ... · compounds as epinephrine, norepinephrine, and dopamine. These substances prepare the body These substances prepare the body

Influence of the caveolae on the stimulation of

β2 adrenergic receptors

Student: S.D. Dams Raportnummer: BMTE05.16

Supervisors: Jeffrey H. Omens, Ph.D Department of Bioengineering and Medicine University of California SanDiego Carlijn V.C. Bouten, Dr. Department of Tissue Engineering Technical University of Eindhoven

Sponsored by AMC Research BV

Contents 1 Introduction ..................................................................................................................... 4

2 Physiology......................................................................................................................... 5 2.1 The cardiac myocyte.................................................................................................. 5 2.2 The caveolae and caveolins....................................................................................... 5 2.3 Catecholamines and the β adrenergic receptors....................................................... 6 2.4 Mechanotransduction ................................................................................................ 7

3 Experiment....................................................................................................................... 9 3.1 Locolization of β-ARs and Caveolin-3 ...................................................................... 9

3.1.1 Hypothesis ......................................................................................................... 9 3.1.2 Materials and methods....................................................................................... 9

3.2 Function of caveolae ............................................................................................... 10 3.2.1 Hypothesis ....................................................................................................... 10 3.2.2 Materials and methods..................................................................................... 10

4 Results............................................................................................................................. 15 4.1 Colocalization ......................................................................................................... 15 4.2 Controll experiment................................................................................................. 16

4.2.1 In plating medium, addition fenoterol ............................................................. 16 4.2.2 Incubated in methyl-β-cyclodextrin, addition fenoterol .................................. 17 4.2.3 In plating medium, addition isoproternol ........................................................ 18 4.2.4 Incubated in methyl-β-cyclodextrin, addition isoprternol ............................... 18

4.3 Stretch experiment ................................................................................................... 19 4.3.1 Normal, 2% stretch and addition fenoterol...................................................... 19 4.3.2 Incubation methyl-β-cyclodextrin, 2% stretch and addition of fenoterol........ 20 4.3.3 Normal, 2% stretch and addition isoproternol................................................. 21 4.3.4 Incubation methyl-β-cyclodextrin, 2% stretch and addition of fenoterol........ 22

4.4 Flow experiment ...................................................................................................... 23

5 Conclusion...................................................................................................................... 25

6 References ...................................................................................................................... 26

7 Recommendations ......................................................................................................... 27

8 Acknowledgements ........................................................................................................ 29

9 Appendix ........................................................................................................................ 30 9.1 Culturing of cardiac myocytes................................................................................. 30

9.1.1 Protocol ........................................................................................................... 30 9.2 Immunohistochemistry............................................................................................. 31

9.2.1 Protocol ........................................................................................................... 31 9.2.2 More results of staining (controll groups) ....................................................... 32

9.3 Data reference experiment ...................................................................................... 36 9.3.1 Protocol reference experiment......................................................................... 36

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9.3.2 Protocol measuring the contractile rate ........................................................... 36 9.3.3 No flow, only medium..................................................................................... 36 9.3.4 No flow, 5 mM Fenoterol ................................................................................ 37 9.3.5 No flow, incubation in Methyl-β-cyclodextrin................................................ 38 9.3.6 No flow, incubation in Methyl-β-cyclodextrin plus 5 mM Fenoterol ............. 39

9.4 Data stretch experiment .......................................................................................... 41 9.4.1 Protocol preparing stretchers ........................................................................... 41 9.4.2 Protocol stretching........................................................................................... 41 9.4.3 No stretch, only medium ................................................................................. 41 9.4.4 No stretch, incubation methyl-β-cyclodextrin................................................. 43 9.4.5 2% Stretch, only medium ................................................................................ 44 9.4.6 2% Stretch, incubation MβCD ........................................................................ 45 9.4.7 2% Stretch, medium, addition 1 ml 5 mM Fenoterol ...................................... 46 9.4.8 2% Stretch, incubation MβCD, addition 1 ml 5 mM fenoterol ....................... 47

9.5 Data flow experiment .............................................................................................. 48 9.5.1 Protocol flowchamber ..................................................................................... 48 9.5.2 Flowchamber, no flow..................................................................................... 48 9.5.3 100 µl/min flow of maintainance medium ...................................................... 50

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1 Introduction Caveolae (= ‘cave-like’) are 50-100 nm diameter vesicles found throughout the various celltypes of the body [1]. In cellmembranes, depending on the local concentration of cholesterol, sphingolipids, and the phospholipid bilayer, more rigid patches of membrane can form.These biochemically distinct patches of membrane are termed “lipid rafts”. Caveolae are biochemically almost indistinguishable from lipid rafts. The primary difference between these two entities is the invaginated, vesicular morphology of caveolae. This difference arises due to the presence of a set of proteins unique to caveolae, but absent from lipid rafts. These proteins are called caveolins [2]. Catecholamines act through cardiac β-adrenergic receptors, βARs. β1AR and β2AR are the primary subtypes responsible for the cardiac response to catecholamines [3]. β-ARs play an important role in the rapid modulation of cardiomyocyte contractile function and the long term induction of the gene program that leads to cardiomyocyte hypertrophy and cardiac failure. The βAR complex is freely mobile in the plasma membrane [4]. Although β1AR and β2AR have very similar signaling properties. It has been shown that β2adrenergic receptors are localized within the caveolae, and that this localization is essential for the physiologic signaling of this receptor sub type [3]. Furthermore, it is thought that the β2ARs are colocalized with caveolin-3 [5]. In this study first the β2ARs and Caveolin-3 in cardiac myocytes will be visualized to quantify the amount of β2ARs and the amount of Caveolin-3 . Then, the function of the caveolae in the β2AR signaling pathway will be defined by stimulating the β2ARs with a β2AR selective agonist in cells with and without caveolae. After that, stretch and fluid-induced membrane shear will be applied on the cardiac myocyte with and without caveolae and with and without stimulation of the β2ARs. These experiments will help delineate the role of the caveolae and β2ARs inmechanical signaling pathways.

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2 Physiology This chapter containsan overview of the physiology of the cardiac myocyte, the function of the caveolae, the role of the caveolins and their interactions with β-ARs. There is significance for the role of caveolae and the β-ARs in cardiac hypertrophy. There is evidece that β-ARs play an important role in the rapid modulation of cardiomyocytes contractile function and the long-term induction of the gene program that leads to cardiac hypertrophy and eventually to cardiac failure [6]. Because mechanotransduction takes place on the membrane, it is likely that when disrupting this pathway it could lead to cardiac hypertrophy. Since defects in mechanotransduction can have implications for various remodeling diseases of the heart, it is important to understand how mechanotranduction takes place in myocytes and myocardium in order to understand the role of normal and abnormal loading on these cells and tissues.

2.1 The cardiac myocyte The cardiac myocyte is a specialized muscle cell that is approximately 25 µm in diameter and about 100 µm in length. The myocyte is composed of bundles of myofibrils that contain myofilaments (Figure 1).

Figure 1: Cardiac myocyte composedm of myofibrils, each of which contains myofilaments. The sarcomere lies between two Z-lines.

As a myocyte is stretched (as occurs with increased ventricular preload), the sarcomeres within the myofibrils are also stretched. In the cardiac muscle, maximal force generation is obtained at a sarcomere length of 2.2 µm. The heart normally operates at sarcomere lengths less than this optimal length (usually 1.8 – 2.0 µm) [7].

2.2 The caveolae and caveolins Caveolae are 50-100 nm, in diameter, vesicles found throughout the various celltypes of the body [1]. Caveolae (Figure 2) can only form on those places of the cellmembrane that contain cholesterol, sphingolipids and caveolins.

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Figure 2: Caveolae. [8]

The caveolin protein family is composed of three distinct proteins: caveolin-1, -2, -3. Caveolin-3 is exclusively expressend in skeletal and cardiac muscle cells (myocytes) [2]. Caveolin-3 can also be seen as a “marker” protein for the caveolae in cardia myocytes [9]. It is thought that the high affinity for cholesterol, the oligomerization, and the oligomer-oligomer interactions can together form an environment in the lipid bilayer conducive for the creation of 50-100 nm caveolar invaginations. Caveolae are seen as signalosomes, or entities in which signal transduction events can take place efficiently (Figure 3) [2].

Figure 3: Caveolae as a signalosome. [2]

Therefore it is very interesting to look further into influencing this signaling pathway. Most certainly, because these caveolae are situated on the membrane, they area where mechanotransduction takes place, and therefore could be influenced by mechanical loads as e.g. stretch and shear.

2.3 Catecholamines and the β adrenergic receptors Catecholamines are any of several compounds occuring naturally in the body that serve as hormone or as neurotransmitters in the sympathic nervous system.They include such compounds as epinephrine, norepinephrine, and dopamine. These substances prepare the body to meet emergencies as cold, fatigue and shock, or function as a neurotransmitter at nerve synapses, or can be an intermediate in the synthesis of another catecholamine. β-ARs mediate the catecholamine-induced activation af adenylyl cyclase through the action of G-proteins (Figure 4)[10].

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Figure 4: Beta activation at the cell membrane [11]

Three β-AR subtypes have been cloned, β1AR, β2AR and β3AR. β1AR and β2AR are the primary subtypes repsonsible for cardiac response to catecholamines. They are also pharmacologically more similar to each other than they are to the β3AR (Figure 5). Although β1AR and β2AR have very similar signaling properties when expressed in undifferentiated cell line, there is evidence that suggets that they have different signaling properties in regulating cardiac funtion. Membrane fractionations shows that β2ARs are found predominantly in a caveolin-enriched membrane fraction, therefore it is believed that in contrast to the β1ARs the β2ARs colocalize with the caveolae [3].

Figure 5: Beta receptor.

The contractile state of the heart is influenced by catecholamines whichact through cardiac β-adrenergic receptors (β-ARs) [4] and therefore too through the β2ARs. Because the β2ARs colocalize with the caveolae, it is an interesting topic to look further into. As mechanotransduction takes place on the membrane it should influence the caveolae and therefore the β2ARs too.

2.4 Mechanotransduction Mechanotransduction, in other words, how the cell senses and responds to changes in mechanical loads, is of great interest in several diseases states such as hypertrophy and heart failure. Myocytes are able to sense external stimuli and initiate responses. External mechanical loading of myocytes results in increased protein synthesis rates and cellular hypertrophy [12-14]. Many signaling pathways, e.g. mechanical signals that act on the nucleus through the cytoskeleton, have been implicated in this process and several putative mechanotransducers have been proposed, although the mechanisms of mechanotransduction in the cardiac myocyte remain largely unexplained. Because mechanotransduction has it’s origin at the same place as where the caveolae and β2ARs are located, it is likely that mechanotransduction can influence the signaling mechanism of the caveolae and the β2ARs. For instance stretch could result in breaking up the

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caveolae and therefore intrupt it’s cooperation with the β2ARs, which could result in extreme contraction of the cardiac myocyte and eventually could lead to cardiac hypertrophy. A similar dysregulation could happen, if fluid-induced shear is applied on the membrane.

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3 Experiment 3 Sets of experiments were conducted: The first for staining of particular structures on the cellmembrane, the second for stimulation of the β-adrenergic receptors with and without caveolae to asses effects on contractile rate, and the third to measure the role of stretch and shear on contractile rate in the presence of β stimulation.

3.1 Locolization of β-ARs and Caveolin-3 In this experiment the locolization of β1AR and β2AR with caveolin-3 will be established by staining the β adrenergic receptors seperately with caveolin-3, using immunohistochemistry technique.

3.1.1 Hypothesis It has been shown that caveolin-3 is the unique protein that makes the formation of the caveolae possible. Therefore it is believed that caveolin-3 is situated inside the caveolae. It has also been shown that the β2ARs colocalize with caveolin-3, therefore it can be assumed that the β2AR is also situated within the caveolae. In this experiment caveolin-3 as well as the β2ARs are labeled with flourescent antibodies, green and red respectively, for immunohistochemistry. Once the two proteins are labeled they can be visualized with a flourescent microscope to test colocalization, and they also can be visualized with a confocal lasers scanning microscope. It is expected in this experiment that if caveolin-3 and β2AR do colocalize, the overlap of the green and red fluorescent tags would give a yellowish color.

3.1.2 Materials and methods

3.1.2.1 Immunohistochemmistry Immunohistochemistry (IHC) combines anatomical, immunological and biochemical techniques for the identification of specific tissue components by means of a specific antigen/antibody reaction tagged with a visible label. IHC makes it possible to visualize the distribution and localization of specific cellular components within a cell or tissue. The term immunohistochemistry is often used interchangeably with immunocytochemistry and immunostaining. For immunohistochemical analysis, it is essential that the morphology of the tissues and cells be retained and that the antigenic sites be accessible. Tissue blocks or tissue sections are typically immersed into a fixative solution. Fixation is necessary to prevent artifactual diffusion of soluble tissue components, to arrest enzymatic activity, to avoid decomposition of the structure and to protect the tissue against the deleterious effects involved with the various stages of the immunohistochemical process. The best fixative differs from tissue to tissue and from antigen to antigen. Furthermore, blocking of the reactive sites in tissues is essential for the development of an immunohistochemical reaction. The principle is that non-immune serum from the host species of the secondary antibody is applied to the tissue at the beginning of the procedure and will adhere to protein-binding sites either by nonspecific adsorption or by binding of specific, but unwanted, serum antibodies to antigens in the tissue. If a direct technique is being performed, the blocking serum should be from the host species providing the primary antibody.

3.1.2.2 Staining of the myocytes For the staining of the rat neonatal myocytes, the cells must be cultured and left on the slide for at least 5 days. In this research the β1- and the β2 adrenergic receptors and caveolin-3 will be stained.

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β1AR and β2AR are of interest, because it is said that β2AR colocolizes with caveolae and β1AR does not. In cardiac myocytes the main expression of caveolin is caveolin-3. This protein is unique for the formation of caveolae, therefore staining caveolin-3 indicates presence of caveolae in myocytes. Therefore to determine this colocolization of β2AR with caveolae, β1AR and β2AR will be stained seperately with caveolin-3. The primairy antibodies of β1AR and β2AR are both rabbit polyclonal IgG (Santa Cruz) and the secondaries are (as well as β1AR and β2AR) goat anti rabbit IgG(H+L) Texas Red (Molecular Probes). The primary for caveolin-3 is a mouse monoclonal IgG1 (Santa Cruz) and the secondary is a goat anti mouse IgG (H+L) FITC (Alexa Fluor, Molecular Probes). Thus the β-ARs will be stained red and caveolin-3 will be green.

3.2 Function of caveolae In this experiment the function of the caveolae together with the β2AR will be investigated, using a selective agonist for the β2AR together with applying stretch and shear on the cardiac myocytes.

3.2.1 Hypothesis It has been shown that the signaling parthway of the β2ARs stimulate the contractile rate of the cardiac myocyte. If the β2adrenergic receptors do colocalize with the caveolae, the difference in contractile rate of the cardiac myocytes with disrupted caveolae and stimulated with fenoterol should be higher then the cardiac myocytes that do not have disrupted caveolae and are also stimulated with fenoterol. Because isoproternol does not stimulate the β2ARs, stimulating the cardiac myocytes with isoproternol should give no difference between the two. The second experiment stretch is applied. In other words mechanical forces are applied on the cellmembrane that will influence the signaling mechanism of the caveolae with the β2ARs. So if stretch opens up the caveolae, then the contractile rate of the cardiac myocytes with disrupted caveolae, stimulated with fenoterol and stretched should be higher then the cardiac myocytes that do not have disrupted caveolae, stimulated with fenoterol and also stretched. Because isoproternol does not stimulate the β2ARs, stimulating the cardiac myocytes with isoproternol should give no difference between the two. In the third experiment fluid-induced membrane shear is applied. Therefore, in this experiment mechanical forces are also applied on the membrane, which would lead to intervention of the signaling mechanism of the caveolae and β2ARs. Thus, if shear opens up the caveolae, then the contractile rate of the cardiac myocytes with disrupted caveolae, stimulated with fenoterol and stretched should be higher then the cardiac myocytes that do not have disrupted caveolae, stimulated with fenoterol and also sheared. Because isoproternol does not stimulate the β2ARs, stimulating the cardiac myocytes with isoproternol should give no difference between the two.

3.2.2 Materials and methods

3.2.2.1 Methyl-β-CycloDextrine and Fenoterol Methyl-β-CycloDextrine can be seen as a “carrier” molecule. It facilitates the dissolution of cholesterol. This cyclodextrine is a cyclic oligosaccharide consisting of 7 glucopyranose units, usually referred to as β-cyclodextrins. The structure these compounds have is a relatively rigid doughnut shape structure (Figure 6).

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Figure 6: The doughnut shaped structure of the beta-cyclodextrins.

Methyl-β-CycloDextrin will be used to extract cholesterol out of the membrane and therefore disrupt the caveolae. Fenoterol hydrobromide is a β2 adrenergic receptor agonist and will be used for the stimulation of these receptors. Isoproternol is an agonist for many receptors on the membrane (also β1ARs). Therefore it will be used to stimulate the whole cell.

3.2.2.2 The controll experiments There are a many factors that contribute to the increase of the contractile rate of the cardiac myocyte. Therefore to exclude these other, mostly unknown, factors a control experiment is needed. In this experiment the contractile rate of cardiac myocytes without any stretch or flow applied will be measured. There are 6 controll experiments:

1. in maintainance medium 2. addition of 1 ml of 5mM fenoterol 3. addition of 1 ml of 10 µM isoproternol 4. incubated in 10 µM methyl-β-cyclodextrin 5. incubates in 10 µM methyl-β-cyclodextrin and addition of 1 ml of 5 mM fenoterol 6. incubates in 10 µM methyl-β-cyclodextrin and addition of 1 ml of 10 µM

isoproternol. Of all these controll experiments, the contractile rate will be measured for 20 minutes at 37 degrees Celcius.

3.2.2.3 Stretch To apply stretch on neonatal rat cardiac myocytes stretchchambers, as shown in Figure 7 and Figure 8, were used. The cardiac myocytes are cultured on a stretchable collagen-coated silicone membrane, left piece Figure 7. The collagen is mounted with SureCoatTM so that it allows the cells to stick on the membrane. Once the cellss are properly adhered to the collagen different amounts of stretch can be applied by turning the top, right piece Figure 7, for different angles. These angles are also called turning angles and refer to a certain percentage of stretch.

Figure 7: Different parts of the stretch chamber.

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Figure 8: The stretcher put together.

To measure the amount of percentage stretch the stetcher must be calibrated. Figure 9 shows two calibration curves for the round stretchers. In this experiment the amount of stretch that was applied was one turn. This equals between the 2% and 2,5% stretch (red lines in Figure 9).

Figure 9: Calibration curve for round stretchers.

Another way to calibrate the amount of stretch is by marking the membrane with four spots, two in x-direction and two in y-direction, and maesure the distance between those spots as a reference. After turning the top measure the distance between those spots again. This will give the elongation of the cells. In this experiment the spots were 8 mm apart and after stretching they were 8,2 mm. So an increase of about 2 %. The spots are a help to locate individual cells. The contractile rate of 8 different situations will be measured:

1. maintainance medium, no stretch. 2. maintainance medium 2% stretch. 3. 2% stretch and addition of 5 mM fenoterol. 4. 2% stretch and addition of 10 µM isoproternol. 5. incubated in 10 µM methyl-β-cyclodextrin, no stretch 6. incubated in 10 µM methyl-β-cyclodextrin 2% stretch.

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7. incubated in 10 µM methyl-β-cyclodextrin 2% stretch and addition of 5 mM fenoterol.

8. incubated in 10 µM methyl-β-cyclodextrin 2% stretch and addition of 10 µM isoproternol.

Of all these controll experiments, the contractile rate will be measured for 20 minutes at 37 degrees Celcius.

3.2.2.4 Flow For the flowexperiments a flowchamber is used, see figure 10a: Figure 10a: Left, Flowchamber without the top. Middle, flowchamber put together. And right, the flowchamber from closeby. The cardiac myocytes are cultured on a glass slides and after 5 days the cells are ready to be used for the experiment. The slide goes upsidedown into the chamber with the medium underneath it. The flow that is applied to the cells is a laminar flow. The velocity of the flow is 100 µl/min. The flow over the cells is analogous to fluid-induces shear that may occur between cells in-vivo when the extracellulae fluid flows past the cells during the cardiac cycle. (Figure 10b). NOTE: It should be taken into account that when applying flow a lot of other factors, like different signaling pathways and diffusion into the cell, will be changed.

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Figure 10b: (A) Impression of the fluid-induced shear in the flowchamber. (B) An impression of the fluid-induces shear in-vivo.

The space where the extracellular fluid flows through in-vivo is about 10µm wide. This calculated from an intersection of a rat heart , Figure 10.

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Figure 10: Intersection of a rat heart.

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4 Results In this chapter the results of the different experiments will be shown.First the colocalizing of the β2 adrenergic receptorswill be discussed, than the contractile rate with and without caveolae and after that the contractile rate with and without caveolae with stretch, and lastly with and without caveolae with flow will be discussed.

4.1 Colocalization In the first experiment it was desirable to find colocalization of the β2 aderenergic receptors with caveolin-3. It was done by immunohistochemistry (see methods). To show the colocalization the samples where obseved underneath a fluorescent (Figure 11, Figure 12) and confocal microscope (Figure 13).

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Figure 11: Fluorescent microscope pictures. A: the myocytes by regular light, B: caveolin-3, C: β1AR.

In Figure 11 (A) shows the network of cardiac myocytes by regular light. (B) Shows the same myocytes, but only the part that is stained for caveolin-3. (C) Also shows the same network of cardiac myocytes, but now only for the β1ARs.

B C A

Figure 12: Fluorescent microscope pictures. A: the myocytes by regular light, B: caveolin-3, C: beta-2-AR.

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In Figure 12 (A) shows the network of cardiac myocytes by regular light. (B) Shows the same myocytes, but only the part that is stained for caveolin-3. (C) Also shows the same network of cardiac myocytes, but now only for the β2ARs. In Figure 11 and Figure 12 a difference can be seen in shape of the structure. Figure 11 clearly show a different structure in caveolin-3 and β1AR as in Figure 12 there seems to be a similar structure between caveolin-3 and β2AR. From this result it can be said that the β1ARs do not colocalize with caveolin-3, but the β2ARs might do.

Figure 13: Colocalization of beta-2-AR (red) and caveolin-3 (green).

In Figure 13 the green area is the area were there is only caveolin-3. The red area shows the area where there are only β2ARs. The yellowish area is the area where the β2ARs colocalize with the caveolin-3 proteins. From this figure it cannot be said that the β2ARs colocalise, because there is too much noise. So, from now on it is assumed that the β2ARs do colocalize with caveolin-3, because Xiang, Y. et al [3] has already proven it.

4.2 Controll experiment In this experiment the contractile rate of the cardiac myocytes was measured without stretching the cells or apply shear stress on them.

4.2.1 In plating medium, addition fenoterol Situation 1 is the mean contractile rate of cardiac myocytes in maintainance medium. Situation 2 is the contractile rate of the same cells in the same petridish with addition of 1 ml 5 mM Fenoterol. It can be seen that fenoterol has a significant effect on the contractile rate of the cardiac myocyte, because the mean contractile rate of the stimulated cardiac myocytes is higher.

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Controll group addition fenoterol

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Figure 14: This graph shows that the mean contractile rate of the cardiac myocytes stimulated by fenterol (2) is higher then the non-stimulated cardiac myocytes (1).

4.2.2 Incubated in methyl-β-cyclodextrin, addition fenoterol Situation 1 is the mean contractile rate of 5-day-old cardiac myocytes incubated in methyl-β-cyclodextrin. Situation 2 is the mean contractile rate of the same cardiac myocytes with addition of 1 ml 5 mM fenoterol. Methyl-β-cyclodextrin disrupts the caveolae as can be seen in Figure 15.

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Figure 15: This graph shows that the mean contractile rate of the cardiac myocytes with the disrupted caveolae decreases over time (1). It also shows that addition of fenoterol to these cardiac myocytes has a significant stimulating effect (2).

A possible explanation for that could be that the methyl-β-cyclodextrin disrupts the caveolae so badly that the total signaling pathway into the cardiac myocyte fades and the cell therefore almost stops contracting. Another explanation could be that the cultured cells were not

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contracting good enough in the beginning and therefore would almost stop contracting when incubated in methyl-β-cyclodextrin

4.2.3 In plating medium, addition isoproternol Situation 1 is the mean contractile rate of cardiac myocytes in maintainance medium. Situation 2 is the contractile rate of the same cardiac myocytes with addition of 1 ml 10 µM Isoproternol. It can be seen that isoproternol has a significant stimulating effect on the contractile rate of the cardiac myocyte, because the mean contractile rate of the stimulated cardiac myocytes (2) is higher.

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Figure 16: These results show that the mean contractile rate of the cardiac myocytes stimulated by isoproternol (2) is higher then the non-stimulated cardiac myocytes (1).

4.2.4 Incubated in methyl-β-cyclodextrin, addition isoprternol Situation 1 is the mean contractile rate of 5-day-old cardiac myocytes incubated in methyl-β-cyclodextrin. Situation 2 is the mean contractile rate of the same cardiac myocytes with addition of 1 ml 10 µM isoproternol. As seen in the results in Figure 16 isoproternol has also the effect of increasing the contractile rate of the cardiac myocytes.

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Controll group incubation methyl-beta-cyclodextrin and addition isoproternol

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Figure 17: These results show that the mean contractile rate of the cardiac myocytes with the disrupted caveolae is consistent over time (1). It also shows that addition of isoproternol to these cardiac myocytes has a significant stimulating effect (2).

As can be seen in Figure 17 the mean contractile rate of line 1 does not decrease with time. This could either mean that the cardiac myocytes were not incubated long enough in methyl-β-cyclodextrin

4.3 Stretch experiment In this experiment the contractile rate of the cardiac myocytes, with a 2% stretch applied, was measured.

4.3.1 Normal, 2% stretch and addition fenoterol Situation 1 is the mean contractile rate of 5-day-old cardiac myocytes in maintainance medium. Situation 2 is the mean contractile rate of the same cardiac myocytes with 2% stretch applied. Situation 3 is the mean contractile rate of the same cardiac myocytes with 2% stretch applied and addition of 1 ml 5 mM fenoterol.

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Figure 18: The results show that the mean contractile rate of 1 is higher then 2. It also shows that stretch and addition of fenoterol, 3, resulte is a very hig mean contractile rate.

These results show that applying stretch and addition of fenoterol (3) are of great influence on the mean contractile rate of the cardiac myocytes, for this contractile rate is much higher. This could mean that by stretching the membrane of the cardiac myocyte you open up the caveolae and therefore make the β2ARs more reachable.

4.3.2 Incubation methyl-β-cyclodextrin, 2% stretch and addition of fenoterol

Situation 1 is the mean contractile rate of 5-day-old cardiac myocytes incubated in methyl-β-cyclodextrin. Situation 2 is the mean contractile rate of the same cardiac myocytes with 2% stretch applied. Situation 3 is the mean contractile rate of the same cardiac myocytes with 2% stretch applied and addition of 1 ml 5 mM fenoterol.

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Figure 19: These results show that disrupting the caveolae (1) and applying 2% stretch on the same cardiac myocytes (2) does not make a significant difference in mean contractile rate. Applying 2% stretch and addition of fenoterol to the same cardiac myocytes (3) result in a higher mean contractile rate then 1 and 2.

These results show that applying stretch and addition of fenoterol (3) to the caveolae disrupted cardiac myocytes are of great influence on the mean contractile rate of the cardiac myocytes, for the contractile rate is extremely high. What might have caused the extremely high contractile rate (3) is that by disrupting the caveolae and after that stretching up the membrane, the β2ARs could be considered a 100% outside the caveolae an therefore even more reachable.

4.3.3 Normal, 2% stretch and addition isoproternol Situation 1 is the mean contractile rate of 5-day-old cardiac myocytes in maintainance medium. Situation 2 is the mean contractile rate of the same cardiac myocytes with 2% stretch applied. Situation 3 is the mean contractile rate of the same cardiac myocytes with 2% stretch applied and addition of 1 ml 10 µM isoproternol.

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Figure 20: This graph shows that the mean contractile rate of the cardiac myocytes in maintaince medium (1) and the appliance of 2% stretch same cardiac myaocytes (2) does not make a significant difference. Addition of isoproternol to the stretched myocytes (3) results in a steady, but higher mean contractile rate.

These results show that applying stretch and addition of isoproternol (3) are of significant influence on the mean contractile rate of the cardiac myocytes, for this contractile rate is much higher. But the relatively speaking, the contractile rate has not increased as much (3) as in the previous two experiments (Figure 18 and Figure 19). This indicates that isotproternol does not stimulate the β2ARs.

4.3.4 Incubation methyl-β-cyclodextrin, 2% stretch and addition of fenoterol

Situation 1 is the mean contractile rate of 5-day-old cardiac myocytes incubated in methyl-β-cyclodextrin. Situation 2 is the mean contractile rate of the same cardiac myocytes with 2% stretch applied. Situation 3 is the mean contractile rate of the same cardiac myocytes with 2% stretch applied and addition of 1 ml 5 mM fenoterol.

22

Stretch experiment incubation methyl-beta-cyclodextrin and addition isoproternol

0

5

10

15

20

25

0 2 4 6 8 10 12 14 16 18 20

Time [min]

Con

trat

ile ra

te1 2 3

Figure 21: This graph shows that disrupting the caveolae results in that the mean contractile rate of 1, 2, and 3 are consistent. Appliance of 2% stretch, 2, result in a significant higher contractile rate then in the same cardiac myocytes without the 2% stretch, 1. Addition of isoproternol results in a even higher mean contractile rate.

These results show that the mean contractile rate in all the situation is consistant and that applying 2 % stretch results in a significant higher mean contractile rate. Applying stretch and addition of fenoterol (3) to the caveolae disrupted cardiac myocytes are of great influence on the mean contractile rate of the cardiac myocytes, for it can be clearly seen that the contractile rate is much higher. But as in the previous experiment (Figure 20), the contractile rate (3) does not increase as far is in the first two experiments. This is another argument for that isoproternol does not stimulate the β2ARs and therefore it can be said that stretch does open up the caveolae which causes the β2ARs to be more reachable. In conclusion, it can be said that stretch can be seen as a mechanical factor that influences the membrane in such a way that it is causes the β2ARs to be more reachable. And therefore it can be concluded that the contractile rate of the cardiac myocytes with disrupted caveolae, stimulated with fenoterol and stretched is higher then the cardiac myocytes that do not have disrupted caveolae, stimulated with fenoterol and also stretched. Stimulating the cardiac myocytes with isoproternol shows no significant difference between the two.

4.4 Flow experiment In this experiment the contractile rate of the cardiac myocytes was measured in two situations:

1. Measuring the contractile rate of the cardiac myocytes in the flowchamber without applying flow on the cells

2. Measuring the contractile rate of the cardiac myocytes in the flowchamber, while applying a 100 µl/min flow of maintainance medium.

The results are show in Figure 22. They show that they are not consistent with the hypothesis, for the hypothesis was that the contractile rate of the cardiac myocytes should increase when flow would be applied. There could be a couple of reasons why the cardiac myocytes have a lower contractile rate when a flow of 100 µl/min is applied:

23

1. The fluid that flows over the cells is too cold, which would make the cells stop contracting.

2. The temperature of the flowchamber is to cold so that the fluid that flows over the cells is too cold, which would make the cells stop contracting.

3. The collagen coating is not good enough, so that the cells do not contract good enough to begin with.

4. The time it takes to put the flowchamber together takes too much time, so that the cells have cooled down so much that the stop contracting anyways.

5. The amount of fluid that is used when putting the chamber together is so little that the cells are dried out by the time you apply flow in them.

6. The flowchamber is leaking. This caused the fluid to flow out of the chamber and therefore does not cause a laminar flow.

Flowchamber main every 5 minutes

0

10

20

30

40

50

60

70

80

0 2 4 6 8 10

Time [min]

Con

trac

tile

rate

12

no flow, alleen chamber flowchamber

Figure 22: The graph shows that the cardiac myocytes without flow have a higher contractile rate then the cells with flow.

There could also be a physiological explanation and that is that when flow applied, the caveolae close up, so that no fluid can enter the caveolae and therefore cannot stimulate the β2ARs. This results in a lower contractile rate then usual. Though, this is still highly hypothetical, especially since the contractile rate of the cardiac myocytes is also extremely low when stretch is applied. And stretching out cardiac myocytes should open up the caveolae.

24

5 Conclusion In this study first the β2ARs and Caveolin-3 in cardiac myocytes were visualized to quantify the amount of β2ARs and the amount of Caveolin-3. Results show (Figure 13) that there is a certain amount of colocalisation, though it is hard to show that the β2ARs actually do colocalize with caveolin-3. A possible explanation for this could be that the antibodies were not specific enough and therefore cause a lot of noise, which makes it impossible to verify that the two specific proteins do colocalize. Therefore, the assumption is made that β2ARs do colocalize with caveolin-3, based on previous results from Steinberg et al. [5]had proven. When this assumption was made the signaling mechanism of the caveolae and the β2ARs was investigated by stimulating the β2ARs with a β2AR selective agonist in cells with and without caveolae. So the hypothesis was If the β2adrenergic receptors do colocalize with the caveolae, the difference in contractile rate of the cardiac myocytes with disrupted caveolae and stimulated with fenoterol should be higher than the cardiac myocytes that do not have disrupted caveolae and are also stimulated with fenoterol. Such a significant difference is not expected when stimulating the cardiac myocytes with isoproternol. These results show that disrupting the caveolae indeed has a significant influence on the contractile rate and therefore on the signaling pathway into the cell. When comparing the results of stimulation by fenoterol with stimulation by isoproternol the conclusion can be drawn that the difference in contractile rate of the cardiac myocytes with disrupted caveolae and stimulated with fenoterol is higher then the cardiac myocytes that do not have disrupted caveolae and are also stimulated with fenoterol. Stimulating the cardiac myocytes with isoproternol shows no significant difference between the two. After obtaining these results, stretchexperiments were done. In this experiment it was investigated if stretch, and therefore mechanotransduction, was of influence on the signaling mechanism of the caveolae and β2ARs. It was expected that if stretch opens up the caveolae, then the contractile rate of the cardiac myocytes with disrupted caveolae, stimulated with fenoterol and stretched should be higher than the cardiac myocytes that do not have disrupted caveolae, stimulated with fenoterol and also stretched. It was not expected that stimulating the cardiac myocytes with isoproternol would give such a significant difference. The results show that stretch does not appear to increase the baseline contractile rate. When comparing the results of stimulation by fenoterol with stimulation by isoproternol the conclusion can be drawn that the contractile rate of the cardiac myocytes with disrupted caveolae, stimulated with fenoterol and stretched is higher than the cardiac myocytes that do not have disrupted caveolae, stimulated with fenoterol and also stretched. Stimulating the cardiac myocytes with isoproternol shows no significant difference between the two. Therefore it could be possible that disrupting the caveolae, adding fenoterol and stretching the cells result in a total vulnarability of the β2ARs, which causes an extremely high contractile rate of the cardiac myocytes and therfore a cooperation between caveolae and β2ARs must exist.

25

6 References 1. Gratton, J.-P., P. Bernatchez, and W.C. Sessa, Caveolae and Caveolins in the

Cardiovascular System. J Circ Res, 2004. 94: p. 1408-1417. 2. Razani, B. and M. Lisanti, The Role of Caveolae and the Caveolins in Mammalian

Physiology. 2004. 3. Xiang, Y., et al., Caveolar Localization Dictates Physiologic Signaling of b2-

Adrenoceptors in Neonatal Cardiac Myocytes. J Bio Chem, 2002. 277(37): p. 34280–34286.

4. ?, The effect of caveolae associated with stretch on cardiomyocytes. ---, ? 5. Steinberg, S.F., b2-Adrenergic receptor signaling complexes in cardiomyocyte caveolae/lipid rafts. J Mol Cell Cardio, 2004. 37: p. 407–415. 6. Rybin, V.O., et al., Developmental Changes in b2-Adrenergic Receptor Signaling in

Ventricular Myocytes: the Role of Gi proteins and Caveolae Microdomains. Mol Pharmacol, 2003. 63: p. 1338-1348.

7. Richard, E. and P.D. Klabunde, Cardiovascular Physiology Concepts. www.oucom.ohiou.edu/ CVPhysiology/CF020.htm, 2004.

8. Gumbleton, D.M. 1992. 9. Williams, T.M. and M.P. Lisanti, The caveolin proteins. Gen Bio, 2004. 5(3): p. 214. 10. catecholamine and beta adrenergic receptors. us.expasy.org/cgi-

bin/niceprot.pl?P07550. 11. . 12. Sadoshima, J. and S. Izumo, Mechanotransduction in stretch-induced hypertrophy of

cardiac myocytes. J Recept Res, 1993. 13((1-4)): p. 777-794. 13. Grossman, W., Cardiac hypertrophy: useful adaptation or pathologic process? Am J

Med, 1980. 69: p. 576-583. 14. Mann, D.L., R.L. Kent, and G. Cooper, IV, Load regulation of the properties of adult

feline cardiocytes: Growth induction by cellular deformation. Circ Res, 1989. 64: p. 1079-1090.

26

http://www.oucom.ohiou.edu/

7 Recommendations Several recommendations can be done after doing these experiments. About the staining experiment the following recommendations can be made: • Use monoclonal primary antibodies. The primary antibody for the βARs was a

polyclonal, which means that it could attach to other proteins as well and therefore reduce specificity of the output.

• Use antibodies of alexa fluor. Namely, the staining of caveolin-3, which was an alexa flour green staining, was very good. The staining for the βARs on the other hand was not acceptable and could be improved.

• Instead of staining the proteins can also be labeled by using an adenovirus. The control experiments were succesful, though from time to time the cells were not always beating together. In order to get consistently beating cells the use of a good collagencaoting on the slides is nessecary. During my experiments I discovered that using SureCoatTM gave me better results than using the ‘home-made’ mixture of collagencoating. Though, it would be wise to take a closer look at both coatings. About the stretching experiments in the future I would recommend to do a lot more of these experiments, because only 5 experiments is not enough. Also the amount of stretch is fictional in this experiment, so in future work I would recommend to do research on the amount of stretch the cells in a rat heart undergo and then apply the same amount of stretch on the cultured cells with the stretcher. That way the data that will be obtained will be more ‘real’. Then there is the stretcher itself. In this experiment the round stretcher was used. This stretcher applied stretch in all directions and this is also contradictive with reality. So it might be considered to use the ellips-shaped stretcher, which are more close to reality. Evenso the commercial membrane, that is used, organizes the cell in random groups. This is also contradictive with reality, so therefore I would recommend the specially-made membrane where the cells are alligned, for this is closere to reality. About the flowchamber experiments. This experiment took a long time to work, because the cells would not beat in the flowchamber. There were several reasons why the chamber would not work and they are the following: • The fluid that flows over the cells is too cold, which would make the cells stop

contracting. • The temperature of the flowchamber is to cold so that the fluid that flows over the cells is

too cold, which would make the cells stop contracting. • The collagen coating is not good enough, so that the cells do not contract good enough to

begin with. • The time it takes to put the flowchamber together takes too much time, so that the cells

have cooled down so much that the stop contracting anyways. • The amount of fluid that is used when putting the chamber together is so little that the

cells are dried out by the time you apply flow in them. • The flowchamber is leaking. This caused the fluid to flow out of the chamber and

therefore does not cause a laminar flow. • The chamber was cleaned with lysol instead of ethanol. So for future work on fluid-induced membrane shear the following experiment is recommended: 1.In the flowchamber without applying flow on the cells 2.Apply a 100 ml/min flow of maintainance medium.

27

3.Apply a 100 ml/min flow of 5 mM Fenoterol. (Wash the fenoterol out) 1.Incubate the cells with methyl-beta-cyclodextrin 2.Apply a 100 ml/min flow of maintainance medium. 3.Apply a 100 ml/min flow of 5 mM Fenoterol

28

8 Acknowledgements This project was supported by the University of California of San Diego department of Bioengineering and Medicine and financial support was obtained from the department of cardiothoracic surgery of the Academic Medical Center at the University of Amsterdam.First of all I would like to thank AMC Research BV for giving me the financial support to go the San Diego. Then I would like to give my sincere thanks to Jeffrey H. Omens, Ph.D. who gave me the opportunity to do my internship at UCSD and who has supported me along the way. I am grateful for the freedom and support that was given to me. Along goes my thanks to Andrew D. McCulloch, Ph.D. for giving me the space to work. Furthermore, I would like to thank Professor Schmid-Schönbein and Hainsworth Shin for their inexhaustable input and help and I would like to thank Jerry Norwich for his help. Then, I would also like to thank Frans van de Vosse and Roy Kerckhoffs who were willing to use their contacts to arrange an internship for me at UCSD. Last, but not least, I would also like to thank all the other employees and students who were always there to help me and made me feel very welcome.

29

9 Appendix

9.1 Culturing of cardiac myocytes

9.1.1 Protocol The myocytes were cultured with the Neomyts kit of Cellutron. Product name: Neomyts kit Product #: nc-6031 Manufacturer’s information: Cellutron life technologies P.O. Box 1423, Highland park, NJ 08904 Tel: 1-800-326-3403 Fax: 1-800-327-2819 www.cellutron.com

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9.2 Immunohistochemistry

9.2.1 Protocol Materials: • β2 primary antibodies • β2 secondary antibodies • β1 primary antibodies and • β1 secondary antibodies • Caveolin-1 antibodies, with green fluorescent tail • Caveolin-3 primary antibodies • Caveolin-3 secondary antibodies • Blocking solution

- BSA • Fixative

- Formaldehyde • Permeabilizing solution

- Triton X-100 • PBS • Vectashield • DAPI Base Solutions: All base solutions should be filtered immediately before use Blocking solution: 3% BSA in PBS (500 ml) Fixative: 3.7% Formaldehyde (Polysciences) in PBS (50 ml) Permeabilizing Solution: 0.1% Triton X-100 in PBS (50 ml) 1. Grow cells on glass slides (slides must be covered with collagen to let the cells stick) 2. Put the PBS and the fixative in the 37◦C bath, so that they’ll warm up to 37◦C. (leave the

Permeabilizing solution at 4◦C and put the blocking solution at roomtemperature) 3. Wash cells with three quick rinses in PBS 4. Add fixative to each well and let sit for at least 10 minutes 5. Rinse three times in PBS 6. Incubate cells in Permeabilizing solution for 10 minutes 7. Rinse cells three times in blocking solution 8. Incubate in fresh blocking solution for at least 1 hour and up to overnight. 9. 1:200 dilution of caveolin-3 (or caveolin-1) primary antibody diluted in blocking solution

and incubate for at least 2 hours at room temperature. 10. Wash 3 times in blocking solution. 11. Add 1:100 dilution of βAR primary antibody in blocking solution for 4 hours up to

overnight (4◦C) at at room temperature. 12. Wash cells 3 times in blocking solution. 13. Incubate the cells in blocking solution and put them at and let them stay overnight. (NEXT DAY) 14. Add 1:1000 dilution of secondary antibody (green) in blocking solution and incubate at

room temperature for at least 2 hours.

31

15. Wash 3 times in blocking solution 16. Add 1:1000 dilution of secondary antibody (red) in blocking solution and incubate at

room temperature for at least 4 hours. 17. Wash 3 times in blocking solution 18. Mount chamber slides with thin glass cover slips using 1 drop of vectashield. Then seal

edges of cover slip with nail polish. (first corners) 19. Visualize and store samples at –20 ◦C.

9.2.2 More results of staining (controll groups) Pictures 20-24.

Table 1: The different controll groups of the β-1-adrenergic receptors and caveolin-3.

A B C D E slide antibody

β1AR primary

+ + + + -

β1AR secondary

+ + - + +

Cav-3 primary

+ + + - +

Cav-3 secondary

+ - + + +

A B C

Figure 23: (A) Normal light, (B) FITC, (C) TxRed (β1). Slide A

32

A B C

Figure 24: (A) Normal light, (B) FITC (C) TxRed (β1). Slide B

A B C

Figure 25: (A) Normal light, (B) FITC (C) TxRed (β1). Slide C

A B C

Figure 26: (A) Normal light, (B) FITC (C) TxRed (β1). Slide D

33

A B C

Figure 27: (A) Normal light, (B) FITC (C) TxRed (β1). Slide E

Pictures 25-27. Slide e is the same as slide E and slide b is the same as B, because the secondary of the β-AR is the same for β1 and β2.

Table 2: The different controll groups of the β-2adrenergic receptors and caveolin-3.

a b c d e slide antibody

β2AR primary

+ + + + -

β2AR secondary

+ + - + +

Cav-3 primary

+ + + - +

Cav-3 secondary

+ - + + +

Figure 28: (A) Normal light, (B) FITC (C) TxRed (β1). Slide a

34

Figure 29: (A) Normal light, (B) FITC (C) TxRed (β1). Slide c

Figure 30: (A) Normal light, (B) FITC (C) TxRed (β1). Slide d

35

9.3 Data reference experiment

9.3.1 Protocol reference experiment Use stocksolutions of 5 mM fenoterol and 10 µM MβCD.

1. Culture the cardiac myocytes on glass slides. 2. Let them grow for about 5 days. 3. Take slide 1 in the dish with medium to the microscope and measure the contractile

rate for approximately 20 minutes. 4. Gently lift the lit of the petridish (so that you can see the same cell) and add 1 ml 5

mM fenoterol to the medium in the dish. 5. Start measuring the contractile rate again for approximately 20 minutes after 1

minute. 6. Incubate slide 2 in 10 µM MβCD for at least 1 hour. 7. Take slide 2 in the dish with 5 mM MβCD to the microscope and measure the

contractile rate for approximately 20 minutes 8. Gently lift the lit of the petridish (so that you can see the same cell) and add 1 ml 5

mM fenoterol to the medium in the dish. 9. Start measuring the contractile rate again for approximately 20 minutes after 1

minute. 10. Repeat this procedure several times

9.3.2 Protocol measuring the contractile rate Once you have taped the beating cardiac myocytes for a certain amount of time the following procedure (and programm) can be used to measure the contractile rate:

1. Start up the DVD-player and the computer 2. start up the programm ‘Scion’ of ‘NIH image’. 3. Go to special in the menu 4. Click ‘load macros’ 5. Go to desktop/datafolder/Jeffrey Tsai/Jeffrey Tsai1/Hraccountmacro20030510.txt and

open this file 6. Put the DVD in the DVD-player and press play 7. Select region 8. make a 10 second movie (by pressing [Q] on the keyboard) or a 1 minute movie (by

pressing [M] on the keyboard). 9. make a M-mode of this movie by pressing [2] on the keyboard. 10. calculate the Contractile rate by pressing [3] on the keyboard and clicking on the

obtained window.

9.3.3 No flow, only medium Reference experiment: no flow, only medium 1 2 3 4 5 Time [min]

Contractile rate

Time [min]

Contractile rate

Time [min]

Contractile rate

Time [min]

Contractile rate

Time [min]

Contractile rate

1 148 1 60 1 86 1 129 1 672 121 2 84 2 89 2 135 2 673 165 3 129 3 86 3 122 3 614 170 4 107 4 86 4 129 4 675 165 5 104 5 86 5 135 5 676 159 6 104 6 86 6 129 6 617 184 7 127 7 86 7 122 7 618 165 8 110 8 92 8 135 8 55

36

9 141 9 0 9 86 9 135 9 6110 122 10 0 10 92 10 132 10 5511 110 11 0 11 92 11 132 11 6112 165 12 0 12 86 12 129 12 6113 120 13 184 13 86 13 122 13 6114 141 14 0 14 86 14 122 14 5515 43 15 0 15 86 15 116 15 6116 49 16 162 16 92 16 110 16 6717 104 17 0 17 86 17 116 17 6118 122 18 0 18 92 18 116 18 6119 80 19 0 19 86 19 122 19 5520 55 20 0 20 86 20 116 20 67

Main data reference experiment: no flow, only medium

Main of every 5 minutes of the main data: no flow, only medium

Time [min] Contractile rate Time [min] Contractile rate 1 98 1 982 99,2 5 106,63 112,6 10 1004 111,8 15 84,765 111,4 20 76,26 107,87 1168 111,49 84,6

10 80,211 7912 88,213 114,614 80,815 61,216 9617 73,418 78,219 68,620 64,8

Main contractile rate = 91,89

9.3.4 No flow, 5 mM Fenoterol Reference experiment: no flow, 5 mM Fenoterol 1 2 3 4 5 Time [min]

Contractile rate

Time [min]

Contractile rate

Time [min]

Contractile rate

Time [min]

Contractile rate

Time [min]

Contractile rate

1 116 1 110 1 129 1 141 1 902 129 2 116 2 120 2 159 2 1043 135 3 98 3 135 3 147 3 904 130 4 110 4 122 4 147 4 925 135 5 92 5 141 5 159 5 986 135 6 80 6 171 6 147 6 867 159 7 89 7 129 7 153 7 868 149 8 80 8 135 8 153 8 869 141 9 80 9 129 9 147 9 98

10 141 10 92 10 129 10 141 10 86

37

11 110 11 80 11 129 11 141 11 8612 110 12 72 12 135 12 141 12 10413 116 13 98 13 129 13 159 13 9214 122 14 110 14 129 14 147 14 8615 116 15 104 15 135 15 153 15 9016 122 16 98 16 141 16 147 16 5017 135 17 80 17 135 17 153 17 9618 129 18 80 18 129 18 141 18 9219 130 19 89 19 122 19 139 19 8620 130 20 80 20 129 20 147 20 98

Main data reference experiment: no flow, 5 mM Fenoterol

Main of every 5 minutes of the main data: no flow, 5 mM Fenoterol

Time [min] Contractile rate Time [min] Contractile rate 1 117,2 1 117,22 125,6 5 121,83 121 10 120,884 120,2 15 115,765 125 20 115,126 123,87 123,28 120,69 119

10 117,811 109,212 112,413 118,814 118,815 119,616 111,617 119,818 114,219 113,220 116,8


9.3.5 No flow, incubation in Methyl-β-cyclodextrin Reference experiment: no flow, incubation in Methyl-β-cyclodextrin 1 2 3 4 5

Time [min]

Contractile rate

Time [min]

Contractile rate

Time [min]

Contractile rate

Time [min]

Contractile rate

Time [min]

Contractile rate

1 174 1 159 1 239 1 190 1 1592 104 2 141 2 214 2 196 2 1103 0 3 178 3 110 3 202 3 984 160 4 189 4 86 4 141 4 1605 0 5 190 5 73 5 98 5 1596 190 6 190 6 0 6 86 6 1907 0 7 189 7 0 7 59 7 1168 178 8 190 8 0 8 110 8 1599 0 9 178 9 0 9 39 9 110

10 0 10 178 10 0 10 20 10 0

38

11 0 11 129 11 0 11 0 11 9812 0 12 110 12 0 12 0 12 013 0 13 129 13 0 13 0 13 014 0 14 130 14 0 14 0 14 015 0 15 141 15 0 15 0 15 016 0 16 159 16 0 16 0 16 017 0 17 141 17 0 17 0 17 018 0 18 129 18 0 18 0 18 019 0 19 141 19 0 19 0 19 020 0 20 159 20 0 20 0 20 0

Main data reference experiment: no flow, incubation in Methyl-β-cyclodextrin

Main of every 5 minutes of the main data: no flow, incubation in Methyl-β-cyclodextrin

Time [min] Contractile rate Time [min] Contractile rate 1 184,2 1 184,22 153 5 141,23 117,6 10 87,284 147,2 15 29,485 104 20 29,166 131,27 72,88 127,49 65,4

10 39,611 45,412 2213 25,814 2615 28,216 31,817 28,218 25,819 28,220 31,8


9.3.6 No flow, incubation in Methyl-β-cyclodextrin plus 5 mM Fenoterol

Reference experiment: no flow, incubation MβCD plus 5 mM Fenoterol 1 2 3 4 5 Time [min]

Contractile rate

Time [min]

Contractile rate

Time [min]

Contractile rate

Time [min]

Contractile rate

Time [min]

Contractile rate

1 160 1 220 1 214 1 178 1 1602 164 2 220 2 210 2 141 2 1643 159 3 233 3 239 3 135 3 1594 153 4 210 4 233 4 104 4 1535 156 5 233 5 239 5 139 5 1566 173 6 239 6 196 6 104 6 1737 152 7 220 7 210 7 116 7 1528 168 8 231 8 210 8 110 8 1689 147 9 251 9 233 9 122 9 147

39

10 135 10 233 10 239 10 133 10 13511 135 11 220 11 210 11 98 11 13512 152 12 210 12 202 12 104 12 15213 116 13 251 13 222 13 80 13 11614 147 14 233 14 216 14 141 14 14715 116 15 220 15 202 15 159 15 11616 116 16 233 16 214 16 139 16 11617 135 17 230 17 239 17 141 17 14718 135 18 233 18 211 18 141 18 13519 152 19 233 19 28 19 116 19 16820 116 20 259 20 222 20 159 20 152

Main data reference experiment: no flow, incubation in MβCD plus 5 mM Fenoterol

Main of every 5 minutes of the main data: no flow, incubation in MβCD plus 5 mM Fenoterol

Time [min] Contractile rate Time [min] Contractile rate 1 186,4 1 186,42 179,8 5 181,283 185 10 175,884 170,6 15 1645 184,6 20 145,286 1777 1708 177,49 180

10 17511 159,612 16413 15714 176,815 162,616 163,617 151,418 14419 10920 158,4


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9.4 Data stretch experiment

9.4.1 Protocol preparing stretchers 1. Wash stretcher in distal water and put them in a jar with 70% ethanol for 1 minute. 2. Wash 2 times in doubble dital water 3. Let stretchers dry in the hood with UV-light on. 4. Cut the commercial membrane in 9 pieces 5. Put the membranes on the stretchers (just before putting the membranes on, mark the

membrane on the outside with 2 dots 5 mm apart in x-direction and 2 dots 5 mm apart in y-direction)

6. Fill the stretchers with 70% ethanol 7. Check the membrane for leaks 8. Wash the stretchers 2 times with doubble distal water 9. Expose the stretchers for at least 20 minutes to UV-light in the hood 10. Put SureCoatTM on and let them dry overnight in incubator at 37 degrees Celcius 11. Remove SureCoatTM 12. Plant cells

9.4.2 Protocol stretching Use stocksolutions of 5 mM fenoterol and 10 µM MβCD. 1. Culture the cardiac myocytes on stretchers. 2. Let them grow for about 5 days. 3. Take stretcher 1 with medium to the microscope and measure the contractile rate for

approximately 20 minutes. 4. Gently turn the lit one turn (360 degrees) and measure the contractile rate for

approximately 20 minutes. 5. add 1 ml 5 mM fenoterol to the medium in the dish and go back to the same cell. (Tip: take a cell close to the dots, because that will be easier to find back) 6. Start measuring the contractile rate again for approximately 20 minutes after 1

minute. 7. Incubate stretcher 2 in 10 µM MβCD for at least 1 hour. 8. Take stretcher 2 to the microscope and measure the contractile rate for approximately

20 minutes 9. Gently turn the lit one turn (360 degrees) and measure the contractile rate for

approximately 20 minutes. 10. Add 1 ml 5 mM fenoterol to the medium in the dish and go back to the same cell. 11. Start measuring the contractile rate again for approximately 20 minutes after 1

minute. 12. Repeat this procedure several times

9.4.3 No stretch, only medium Stretch experiment: no stretch, only medium 1 2 3

Time [min] Contractile

rate Time [min]Contractile

rate Time [min] Contractile

rate1 14 1 67 1 102 15 2 62 2 213 11 3 67 3 234 13 4 60 4 22

41

5 9 5 61 5 216 12 6 68 6 237 14 7 65 7 218 11 8 63 8 229 8 9 67 9 23

10 9 10 62 10 2511 13 11 61 11 2112 10 12 53 12 2413 12 13 67 13 2214 9 14 56 14 2215 11 15 72 15 2216 8 16 61 16 1017 14 17 53 17 2118 15 18 31 18 2119 11 19 67 19 2320 11 20 98 20 24

Mean data stretch experiment: no stretch, only medium

Mean of every 5 minutes of the mean data: no stretch, only medium

Time [min] Contractile rate Time [min] Contractile rate 1 30,33333 1 30,32 32,66667 5 31,733333 33,66667 10 32,866674 31,66667 15 38,066675 30,33333 20 31,26 34,333337 33,333338 329 32,66667

10 3211 31,6666712 2913 33,6666714 2915 3516 26,3333317 29,3333318 22,3333319 33,6666720 44,33333


42

9.4.4 No stretch, incubation methyl-β-cyclodextrin Stretch experiment: no stretch, incubation MβCD 1 2

Time [min] Contractile rate Time [min] Contractile rate1 129 1 862 122 2 863 130 3 864 121 4 865 147 5 866 80 6 867 98 7 868 111 8 829 106 9 86

10 122 10 8611 130 11 8612 111 12 8613 129 13 8614 131 14 8615 141 15 8616 132 16 8617 122 17 8618 141 18 8619 129 19 8620 116 20 86

Mean data stretch experiment: no stretch, incubation MβCD

Mean of every 5 minutes of the mean data: no stretch, incubation MβCD

Time [min] Contractile rate Time [min] Contractile rate 1 107,5 1 107,52 104 5 107,93 108 10 94,34 103,5 15 107,25 116,5 20 1076 837 928 96,59 96

10 10411 10812 98,513 107,514 108,515 113,516 10917 10418 113,519 107,520 101


43

9.4.5 2% Stretch, only medium Stretch experiment: no stretch, only medium 1 2 3




rate1 10 1 23 1 102 11 2 21 2 213 13 3 25 3 224 9 4 22 4 215 11 5 26 5 226 12 6 21 6 107 14 7 23 7 218 13 8 23 8 229 15 9 24 9 21

10 15 10 22 10 2211 14 11 21 11 1012 15 12 22 12 2113 13 13 22 13 2214 18 14 22 14 2115 16 15 23 15 2216 17 16 21 16 1017 18 17 21 17 2118 19 18 24 18 2219 16 19 24 19 2120 15 20 21 20 22

Mean data stretch experiment: no stretch, only medium

Mean of every 5 minutes of the mean data: no stretch, only medium

Time [min] Contractile rate Time [min] Contractile rate 1 11,25 1 112 13,5 5 13,953 15,75 10 14,54 14 15 14,75 15,25 20 15,26 11,257 14,758 15,259 16

10 15,2511 11,7512 14,7513 1514 16,2515 15,7516 12,517 15,2518 1719 16,2520 15


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9.4.6 2% Stretch, incubation MβCD Stretch experiment: 2% stretch, incubation MβCD 1 2


10 147 10 8611 121 11 8612 130 12 8613 141 13 8614 122 14 8615 129 15 8616 106 16 8617 130 17 8618 129 18 8619 110 19 8620 122 20 86

Mean data stretch experiment: 2% stretch, incubation MβCD

Mean of every 5 minutes of the mean data: 2% stretch, incubation MβCD

Time [min] Contractile rate Time [min] Contractile rate 1 104 1 1042 111 5 104,83 103,5 10 1064 113,5 15 107,35 92 20 102,76 97,57 1088 1049 104

10 116,511 103,512 10813 113,514 10415 107,516 9617 10818 107,519 9820 104


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9.4.7 2% Stretch, medium, addition 1 ml 5 mM Fenoterol Stretch experiment: 2% stretch, only medium, fenoterol 1 2 3




rate1 140 1 98 1 1472 110 2 122 2 1413 92 3 121 3 1314 106 4 141 4 1475 90 5 159 5 1566 92 6 110 6 1687 83 7 110 7 1598 92 8 98 8 1689 91 9 92 9 147

10 91 10 106 10 15611 89 11 86 11 18012 92 12 126 12 15913 110 13 111 13 16714 98 14 106 14 16715 92 15 98 15 17916 91 16 129 16 18017 98 17 126 17 18018 92 18 110 18 18019 110 19 121 19 17920 92 20 120 20 180

Mean data stretch experiment: 2% stretch, only medium, fenoterol

Mean of every 5 minutes of the mean data: 2% stretch, only medium, fenoterol

Time [min] Contractile rate Time [min] Contractile rate 1 128,3333 1 128,32 124,3333 5 126,73333 114,6667 10 117,53334 131,3333 15 1245 135 20 132,53336 123,33337 117,33338 119,33339 110

10 117,666711 118,333312 125,666713 129,333314 123,666715 12316 133,333317 134,666718 127,333319 136,666720 130,6667


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9.4.8 2% Stretch, incubation MβCD, addition 1 ml 5 mM fenoterol Stretch experiment: 2% stretch, incubation MβCD, fenoterol 1 2


10 220 10 19011 241 11 19012 260 12 19013 235 13 18914 241 14 19015 211 15 19016 209 16 19017 210 17 19018 222 18 19019 201 19 19020 241 20 190

Mean data stretch experiment: 2% stretch, incubation MβCD

Mean of every 5 minutes of the mean data: 2% stretch, incubation MβCD

Time [min] Contractile rate Time [min] Contractile rate 1 223,5 1 223,52 215 5 219,73 211,5 10 215,24 225,5 15 213,75 223 20 203,36 218,57 213,58 223,59 215,5

10 20511 215,512 22513 21214 215,515 200,516 199,517 20018 20619 195,520 215,5


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9.5 Data flow experiment

9.5.1 Protocol flowchamber Make sure that the chamber is cleaned with ethanol before using it. 1. Culture the cardiac myocytes on glass slides. 2. Let them grow for about 5 days. 3. Heat the room of the microscope 4. Put the flowchamber, tubes and syringes (2 of 5 ml and 1 of 20 ml) in the incubator

and let it warm up to approximately 37 degrees Celcius. 5. Put the maintainance medium in a hot tub of 37 degrees Celcius 6. When chamber, tubes, syringes and medium are warmed up put them with the slide in

the heated room of the microscope. 7. Connect the flowchamber 8. Fill the two syringes (5 ml) with medium and connect them to the flowchamber. 9. Make sure that the midarea of the flowchamber is filled with warm medium 10. Take slide 1 from the dish and put it upsidedown in the chamber. 11. Take the top of the flowchamber and put it on top. 12. Tighten the top by first screwing the middle screws and than the cornersscrews

diagonally. 13. Wipe the chamber clean with a tissue and put it under the microscope 14. Start measuring the contractile rate. To put flow on it: 15. Fill the 20 ml syringe with warm maintainance medium. 16. Attach the small tube and press the fluid through it (all the way to the end). 17. Connect the tube to one side of the the chamber. 18. Connect an empty tube on the other side of the chamber. 19. Put the syringe in the syringe pump and make sure that the syringe is tightened. 20. Then open the channels of the flowchamber and press start on the syringepump (make

sure that the amount of flow is right) 21. Now you can start measuring the contractile rate. 22. Repeat this procedure several times (NOTE: make sure that there are no bubbles whatsoever)

9.5.2 Flowchamber, no flow Flow experiment: no flow, only in chamber 1 2


10 122 10 10411 89 11 10412 98 12 10413 110 13 10414 110 14 10415 98 15 104

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16 89 16 10417 80 17 10418 110 18 10419 89 19 10420 98 20 104

Mean data flow experiment: no flow, only in chamber

Mean of every 5 minutes of the mean data: no flow, only in chamber

Time [min] Contractile rate Time [min] Contractile rate 1 101 1 1012 107 5 1073 113 10 101,64 101 15 102,55 113 20 98,66 927 1018 1079 95

10 11311 96,512 10113 10714 10715 10116 96,517 9218 10719 96,520 101


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9.5.3 100 µl/min flow of maintainance medium Flow experiment: 100 µl/min flow maintainance medium 1 2


10 80 10 3711 80 11 3012 80 12 4213 80 13 5614 80 14 015 80 15 5516 80 16 4117 80 17 3018 80 18 3019 80 19 2920 80 20 22

Mean data flow experiment:100 µl/min flow maintainance medium

Mean of every 5 minutes of the mean data: 100 µl/min flow maintainance medium

Time [min] Contractile rate Time [min] Contractile rate 1 64,5 1 64,52 61 5 63,63 64,5 10 52,94 67 15 58,35 61 20 55,26 45,57 65,58 559 40

10 58,511 5512 6113 6814 4015 67,516 60,517 5518 5519 54,520 51


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Influence of the caveolae on the stimulation of β2 ... · compounds as epinephrine, norepinephrine, and dopamine. These substances prepare the body These substances prepare the body

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