EFFECTS OF AGING AND EXERCISE TRAINING ON THE MYOGENIC MECHANISM OF SKELETAL MUSCLE RESISTANCE ARTERIES

EFFECTS OF AGING AND EXERCISE TRAINING ON THE MYOGENIC MECHANISM OF SKELETAL MUSCLE RESISTANCE ARTERIES By FREDY RAFAEL MORA SOLIS A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2011 1

2011 Fredy Rafael Mora Solis 2

To my Mom for her unconditional love and support 3

ACKNOWLEDGMENTS I want to start by thanking the Fulbright scholarship program for giving me the opportunity to come to the United States and be able to get a world-class education. It was a long wait and a 2 years application process, but now at the end of my program I realized it was worth it and every step of the way has been an amazing experience. I want to thank Renee Han Burke, who was the person who interviewed me at the end of this long process and selected me as one of the grantees for the fall of 2009 and who has also been my advisor at LASPAU (Latin American Scholarship Program of American Universities). All this amazing experience would not have been possible without Dr Michael Delp, who took a chance by being my advisor and accepting me into his laboratory as one of his students. Thanks go out to Dr. Delp for assigning me this amazing project, from which I have learned so much and being always there to answer my questions. I would also like to thank Dr. Judy Muller-Delp for giving me space in her lab to develop my research, for doing the tough job of cannulating the vessels so I could run the experiments, for answering every question I had and for making me feel like I was part of her lab. I also thank Dr. Brad J Benhke, for being a member of my committee and for having his office door always open to help me out with anything I needed. Another person I need to thank is Dr. Alvaro N. Gurovich for helping me with the statistics, without his help I wouldn t have been able to do it by myself. I want to thank the department chair of the Physical Therapy program in Nicaragua, Josefa Conrado, PT, for being such a great friend, who has guided me in every aspect of my personal and professional career, she is the one who encouraged me to apply to the Fulbright program and without her support I would not be here. 4

Finally, I would like to thank Dr. Terese Chmielewski for accepting me as a PhD student in her lab. Because of her my academic life doesn t have an end with this masters but a new beginning under her mentorship. 5

TABLE OF CONTENTS page ACKNOWLEDGMENTS... 4 LIST OF TABLES... 8 LIST OF FIGURES... 9 LIST OF ABBREVIATIONS... 10 ABSTRACT... 12 CHAPTER 1 INTRODUCTION... 14 Background... 14 Establishment of Basal Vascular Tone... 15 Autoregulation of Blood Flow and Capillary Hydrostatic Pressure... 15 Blood Vessel Structure and Function:... 17 The Myogenic Mechanism and its Relationship with the Endothelium... 18 Intracellular Ca 2+ Concentration... 19 Depolarization of Vascular Smooth Muscle... 22 Membrane Depolarization Hypotheses... 23 Stretch-Activated Channels... 23 Calcium-Activated Potassium Channels... 23 Chloride Channels... 24 Calcium Channels... 25 The Myogenic Mechanism in the Systemic Circulation in Vivo... 25 Pulsatile Pressure... 26 Neurotransmitters... 26 Endothelium-Derived Factors... 26 Local Factors... 27 Vascular Adaptations to Chronic Exercise... 29 Hypotheses... 30 2 MATERIAL AND METHODS... 31 Exercise Training... 31 Microvessel Preparation... 31 Experimental Design... 32 Evaluation of Myogenic Mechanism... 33 Data Presentation... 34 Statistical Analysis... 34 6

3 RESULTS... 36 Animal Characteristics... 36 Vessel Characteristics... 36 Myogenic Response... 37 4 DISCUSSION... 44 Myogenic Mechanism in Endothelium-Intact and Denuded Vessels... 44 Effect of Exercise Training on the Myogenic Mechanism... 49 LIST OF REFERENCES... 52 BIOGRAPHICAL SKETCH... 56 7

LIST OF TABLES Table page 1-1 Animal characteristics... 36 1-2 Vessel characteristics from exercise trained animals... 37 1-3 Vessel characteristics from sedentary animals... 37 8

LIST OF FIGURES Figure page 2-1 Diameter changes in response to increasing intraluminal pressure in soleus with an intact endothelium.... 40 2-2 Diameter changes in response to increasing intraluminal pressure in soleus with endothelium removed.... 41 2-3 Diameter changes in response to increasing intraluminal pressure in superficial portion of the gastrocnemius with an intact endothelium.... 42 2-4 Diameter changes in response to increasing intraluminal pressure in denuded arterioles from the superficial portion of the gastrocnemius..... 43 9

LIST OF ABBREVIATIONS Ach ANOVA ATP Ca 2+ cmh 2 O EDCF EDTA ET ET-1 ID b ID max ID s Acetylcholine Analysis of Variance Adenosine triphosphate Calcium ion Centimeter of water Endothelium-derived contracting factors Ethylenediaminetetraacetic acid exercise trained Endothelin Steady-state baseline diameter Maximal inner diameter steady-state diameter after each incremental in pressure change K+ Potassium ion Kca KCl Kv MgSO 4 ml MLC MLCK mm MmHg MOPS Calcium activated potassium channels Potassium chloride voltage-dependent potassium channels Magnesium sulfate Milliliters Myosin light chain Myosin light chain kinase Millimoles Millimeter of mercury 3-(N-morpholino)propanesulfonic acid 10

NA Na+ NaCl NaH 2 PO 4 NE NO Pc PGI 2 PO 2 PSS SA SED SNP SR VDCC VSM YT Noradrenaline Sodium ion Sodium chloride Monosodium phosphate Nor epinephrine Nitric oxide Hydrostatic pressure Prostacyclin Partial pressure of oxygen Physiological saline solution Stretch-activated Sedentary Sodium nitroprusside Sarcoplasmatic reticulum Voltage-dependent calcium channels Vascular smooth muscle Young trained µl Microliter 4-AP 4-Aminopyridine 11

Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science EFFECTS OF AGING AND EXERCISE TRAINING ON THE MYOGENIC MECHANISM OF SKELETAL MUSCLE RESISTANCE ARTERIES Chair: Michael Delp Major: Applied Physiology and Kinesiology By Fredy Rafael Mora Solis August 2011 Aging is commonly associated with alterations in the cardiovascular system, which in turn results in changes in the cardiovascular physiology; one of these alterations is reduced blood flow capacity to exercising muscles. These age-related changes in which blood vessels walls become less tolerant of sudden increases in pressure have detrimental effects on the aerobic capacity and cardiovascular performance during exercise. Endurance exercise training protocols have been shown to improve the attenuated blood flow response in working muscles during exercise in the aged population. Therefore, the purpose of this study was to determine the effect of ageing and exercise training on the myogenic response in skeletal muscle resistance arterioles from two muscles composed of different fiber type (soleus and white portion of the gastrocnemius) and whether this effect takes place in the smooth muscle cells. For this to be accomplished, endothelium-intact and endothelium-removed resistance arterioles from each muscle from young (5-6 months) and old (24-25 months) male Fischer 344 rats were studied. Four experimental groups were used, young sedentary (YS), old sedentary (OS), young exercise-trained (YT) and old exercise-trained (OT). The exercise training protocol consisted of 10 to 12 weeks of treadmill running. Skeletal 12

muscle resistance arterioles were isolated from the muscles and studied in vitro. Responses to step increases in intraluminal pressure were recorded from 0 cmh 2 O to 140 cmh 2 O in the presence of extracellular Ca 2+. After each step change in intraluminal pressure, diameter was recorded continuously, all pressure changes occurred in the absence of intraluminal flow. The results from this study provide evidence that the myogenic response from isolated resistance arterioles from the soleus and superficial portion of the gastrocnemius muscles are impaired in senescent Fischer 344 rats, and that endurance exercise training induces development of a stronger and more robust vasoconstriction of skeletal muscle resistance arterioles as intraluminal pressure increases. This myogenic constriction is diminished in arterioles in which the endothelial cell layer has been removed, indicating that this change in vascular responsiveness in exercise trained animals is primarily mediated by changes in the endothelial cell lining and not in the smooth muscle cells. 13

CHAPTER 1 INTRODUCTION Background In 1902 Bayliss' reported that arterial smooth muscle contracted in response to augmented intraluminal pressure. Previous evidence has shown that this response, the myogenic response, is not mediated by nerves, vasoactive metabolites, or circulating vasoactive substances (28). Bayliss also found that the wall of denervated vessels reacts to diminished tension in the opposite direction to its reaction to increased tension, and that any fall of intravascular pressure, however produced, brings about a relaxation of the vascular wall (1). It is very well known that regulation of the contractile activity of vascular smooth muscle cells in the systemic circulation is dependent on a complex interplay of vasodilator and vasoconstrictor stimuli from circulating hormones, local metabolic by-products, neurotransmitters, endothelium-derived factors, and blood pressure (51). The myogenic response of small arteries and arterioles has been shown to contribute significantly to autoregulation of blood flow in different vascular beds. It is characterized by a decrease in vessel diameter after an increase of transmural pressure and by an increase in vessel diameter after a decrease of transmural pressure. The myogenic response has been found in a wide variety of small arteries and arterioles from different vascular beds and vessel wall tension has been identified as the stimulus for the myogenic response. This response is critically important for the development of resting vascular tone, upon which other control mechanisms exert vasodilator and vasoconstrictor influences (46). It may be considered an established fact that the myogenic response depends mainly on the reaction of the smooth muscle cells in the 14

vessel wall of small arteries and arterioles, and not on either the nerves or the endothelium (46). In the vascular system, the myogenic response has been proposed to participate in a number of physiologically important functions. The two most important of these are: (a) establishment of basal vascular tone and (b) autoregulation of blood flow and capillary hydrostatic pressure (5). Establishment of Basal Vascular Tone Basal vascular tone is a prerequisite for dilator influences. It establishes an underlying arteriolar constriction, a regional blood flow reserve (14), upon which other control mechanisms produce vasodilation or vasoconstriction. The myogenic response has also been postulated to play a central role in the maintenance of constant blood flow and capillary hydrostatic pressure (Pc) during variations in systemic arterial pressure (5). Autoregulation of Blood Flow and Capillary Hydrostatic Pressure This autoregulatory mechanism in which vessels contract or dilate in response to changes in transmural pressure may be impaired with advancing age. The magnitude of myogenic tone development declines with aging in both mesenteric (24) and skeletal muscle arterioles (37). Age-induced modifications of vascular smooth muscle contractile mechanisms may contribute to this reduced myogenic reactivity (37). In addition, previous work has demonstrated that both spontaneous tone development and myogenic activity differ in skeletal muscles of differing fiber type (glycolytic vs. oxidative). Muller-Delp et al. (37) found that active responses of arterioles from both the soleus and gastrocnemius muscles from aged rats were significantly less than those of arterioles from young rats and more closely resembled passive responses to increasing pressure. Furthermore, arterioles from the soleus muscle exhibited more 15

robust myogenic constriction than arterioles from the gastrocnemius in both young and old animals. In another study conducted by Delp (6), it was found that hindlimb unloading diminishes the contractile responsiveness of arterioles isolated from muscle composed of fast-twitch type IIB fibers, nonetheless, hindlimb unloading did not affect vasoconstrictor or myogenic responsiveness of arterioles from muscle composed of slow-twitch type I fibers. Rat hindlimb unloading model was initially developed to study musculoskeletal, cardiovascular, and metabolic changes caused by weightlessness, and it also has been useful for investigating responses of many other physiological systems to unloading and the recovery from unloading on Earth (36). Given the critical role of the myogenic response in maintaining local blood flow and total peripheral resistance, age-related declines in myogenic responsiveness may significantly alter physiological responses in aged individuals. Functions such as orthostatic tolerance and exercise capacity decline with aging and may be directly related to reductions in myogenic control mechanisms (24). The effects of diminished arteriolar contraction could also have other functional consequences, for example, a lowering of vasoconstrictor responsiveness and myogenic activity could result in a reduced ability to elevate vascular resistance in vivo. Alterations in the ability to control vascular resistance could subsequently diminish the precision in which arterial pressure and muscle blood flow are regulated (6). Carlson and colleagues (3) describe this arteriolar contraction to variations of intraluminal pressure as triphasic. First, at low pressures, arterioles dilate passively with increasing pressure, then increasing pressure from 20 to 120 mmhg elicits myogenic constriction and finally at very high pressures, above 140 mmhg, the vessel is dilated 16

despite the generation of nearly maximal VSM tone due to the myogenic response, and any additional increase in pressure causes further dilation. The strength of the myogenic response varies considerably depending on the diameter of small arteries and arterioles. Larger and very small vessels possess a relatively weak myogenic response, while vessels of intermediate diameter have the largest myogenic response (46). Differences in the strength of the myogenic response between vessels with similar diameter were also observed within one vascular bed; for instance, Kuo et al. (29) examined myogenic responses of isolated porcine subepicardial and subendocardial arterioles (80-100 micron in diameter) and found that subepicardial arterioles demonstrated greater myogenic constriction than subendocardial arterioles. This implies that myogenic autoregulation in subepicardial arterioles is better than that in the subendocardial arterioles at both low and high pressures. Blood Vessel Structure and Function: Classically, the vessel wall consists of three major cell elements, namely endothelial cells, smooth muscle cells and nerve endings in the adventitia, and all three cell elements are exposed to the influence of transmural pressure; however, not all of these seem to participate in the myogenic response (46). Evidence has demonstrated that after elimination of a tonic nervous influence on the vessel by blocking nerve endings in the adventitia, no alteration of the myogenic response has been observed (34). Harder (17) demonstrated that the pressure-mediated muscle cell depolarization occurring in the presence of neural blockade and inhibition of excitatory α-adrenergic receptors suggests that this response is myogenic in nature. However, given the vast body of literature demonstrating endothelial factors controlling arterial diameter, further 17

work needs to be done to demonstrate a purely myogenic mechanism. Likewise, the role of the endothelium was tested by observing the effects of its removal. Contradictory results were initially obtained; several studies showed that endothelium removal altered the myogenic response, while others demonstrated no influence of the endothelium on the myogenic response. Data supporting a role of the endothelium in the myogenic response were obtained after chemical removal of the endothelium, whereas with mechanical methods of removal a participation of the endothelium was not seen (46). Nevertheless, in almost all vessels investigated so far the endothelium is not involved in the myogenic response. In conclusion, it seems to be established that the myogenic response depends mainly on the reaction of the smooth muscle cells in the vessel wall of small arteries and arterioles, and not on either the nerves or the endothelium (46). This response is accompanied by a membrane depolarization and an increase of the intracellular Ca 2+ concentration, which depends largely on an influx of extracellular calcium via voltage-operated calcium channels. The Myogenic Mechanism and its Relationship with the Endothelium An important contribution of the endothelium is its role in maintaining vascular tone in large and small arteries by releasing contracting [e.g. endothelin-1 (ET-1)] and relaxing [e.g. nitric oxide (NO), prostacyclin (PGI 2 )] substances (50). Recently, the myogenic concept has been challenged by studies that show the response to be dependent on an intact, functional endothelium. Kuo et al. (28) demonstrated in their study that pressure-dependent responses occur in isolated coronary arterioles and that this response is not dependent on the endothelium. Therefore, pressure-induced changes in coronary arteriolar tone are a true myogenic response in that they originate from smooth muscle. MacPherson and collegues (32) found that that myogenic 18

response in rabbit ear artery is mediated independently of endothelial-derived factors. In human studies, Wallis et al. (50) demonstrated that human isolated cerebral resistance arteries display pressure-induced myogenic responses at pressures between 20 and 90 mmhg, and these responses may be independent of a functional endothelium, but because it is not known what pressure range vessels of this size experience in vivo, it is difficult to ascertain of the physiological role for this pressure-induced myogenic tone. On the other hand, Harder (18) examined the role of the endothelium in pressureinduced activation of isolated cat cerebral arteries. With an intact, undisturbed endothelium, isolated middle cerebral arteries exhibited membrane depolarization, action potential generation, and reduction in diameter. However, when the endothelium is disrupted this same pressure-dependent arterial muscle cell activation is no longer observed. Thus, these data suggest that the endothelial cell may serve as the transducer mediating changes in transmural pressure to activation of cerebral arterial muscle. These data suggest that an increase in transmural pressures creates a stress across the endothelial cell that activates the release of a chemical mediator. It is not possible at this point to determine the specificity of the type of mechanical stimulus. At present, it is believed that the myogenic response occurs as a result of mechanical stimuli that act directly on the vascular smooth muscle cells to generate constriction. The endothelium exerts its influence by releasing vasoactive factors, but the presence of an intact endothelium is not a prerequisite for myogenic reactivity in blood vessels (22). Intracellular Ca 2+ Concentration Myogenic tone is abolished by the removal of external Ca 2+ and by Ca 2+ channel blockers, which suggests that myogenic tone depends on Ca 2+ influx through voltage- 19

dependent Ca 2+ channels. Like all muscle cells, vascular smooth muscle uses Ca 2+ as the trigger for contraction. Calcium influx through channels in the plasma membrane and Ca 2+ release from intracellular stores are the major source of activator Ca 2+ (51). Knot and Nelson (26) studied the regulation of arterial wall Ca 2+ in intact cerebral arteries and its regulation of arterial diameter and demonstrated that a rise of intravascular pressure causes a graded membrane potential depolarization that leads to a graded increase in the steady open of voltage-dependent Ca 2+ channels. This elevates Ca 2+ entry, and thus steady-state intracellular Ca 2+. An elevation of arterial wall Ca 2+ leads to force development, cell shortening and vasoconstriction. Contraction in vascular smooth muscle can be initiated by mechanical, electrical, and chemical stimuli (5). Passive stretching of VSM can cause contraction that originates from the smooth muscle itself and is therefore termed a myogenic response. Electrical depolarization of the VSM cell membrane also elicits contraction, most likely by opening voltage dependent calcium channels which causes an increase in the intracellular concentration of calcium. Finally, a number of chemical stimuli such as norepinephrine, angiotensin II, vasopressin, endothelin-1, and thromboxane A 2 can cause contraction; each of these substances bind to specific receptors on the VSM cell (or to receptors on the endothelium adjacent to the VSM), which then leads to VSM contraction. The mechanism of contraction involves different signal transduction pathways, all of which converge to increase intracellular calcium (46). The mechanism by which an increase in intracellular calcium stimulates VSM contraction can result from either increased flux of calcium into the cell through calcium channels or by release of calcium from internal stores (e.g., sarcoplasmic reticulum; 20

SR). The free calcium binds to a special calcium binding protein called calmodulin. Calcium-calmodulin activates myosin light chain kinase (MLCK), an enzyme that is capable of phosphorylating myosin light chains (MLC) in the presence of ATP. Myosin light chains phosphorylation leads to cross-bridge formation between the myosin heads and the actin filaments, and hence, smooth muscle contraction (46). Compared with the vast number of animal studies performed, there are fewer human studies that have examined the myogenic response. In humans, isolated coronary artery preparations have been shown to display a myogenic response to changes in pressure. Miller et al. (35) examined responses of human coronary resistance vessels in vitro to changes in intraluminal pressure and found that arterioles from patients with hypertension demonstrated enhanced myogenic constriction compared with vessels from normotensive patients. The myogenic response is essential in regulating peripheral vascular resistance as well as tissue-specific blood flow. This autoregulatory mechanism, in which vessels contract or dilate in response to changes in transmural pressure, may be impaired with advancing age (24). Previous research has reported that in both muscle fiber types (soleus and white portion of the gastrocnemius) young animals exhibited a more robust myogenic constriction than old animals. Data from Muller-Delp et al. (37) suggest that arterioles from soleus muscle undergo some remodeling with age, but this does not impact the mechanical response of the vessels as pressure (stress) increases. Therefore the decrease in the myogenic tone in skeletal muscle arterioles is likely due to an impairment of a pressure-sensitive signaling mechanism (possibly linked to the 21

composition of the vascular wall) but not directly related to a structural impairment of the vessel wall. Depolarization of Vascular Smooth Muscle Typically, membrane depolarization elicits the influx of Ca 2+ through ion channels sensitive to changes in membrane potential, or voltage-dependent Ca 2+ channels (VDCC). The ensuing rise in intracellular Ca 2+ concentration precipitates the enzymatic cascade leading to muscle shortening or force production (4). It has been established that an increase in transmural pressure produces a membrane depolarization of the smooth muscle cells. This has been shown in cat cerebral arteries (17, 18), and other animal models. The pressure-induced membrane depolarization was always accompanied by a contraction of the vessel. At present, experimental evidence of the mechanism responsible for the pressure-induced membrane depolarization is not available. Nevertheless, several hypotheses based on indirect evidence have been suggested. Harder (17) demonstrated that elevation of transmural pressure in cat middle cerebral arteries results in muscle cell membrane depolarization. Pressure-mediated muscle cell depolarization occurring in the presence of neural blockade and inhibition of excitatory α-adrenergic receptors suggests that this response is myogenic in nature. However, given the amount of literature demonstrating endothelial factors controlling arterial diameter, further work needs to be done to demonstrate a purely myogenic mechanism. 22

Membrane Depolarization Hypotheses Stretch-Activated Channels The first hypothesis states that the membrane depolarization accompanying the myogenic response is caused by an activation of stretch-activated (SA) cation channels, which results in an increase of the inward current of the smooth muscle cells. Stretchactivated cation channels, which change their activity in response to an alteration of membrane stretch, have been found in vascular smooth muscle cells (47). Setogochi, M. et al. (47) have shown that SA channels exist in smooth muscle cells from mesenteric resistance arteries. The SA channels in this tissue share basic characteristics with other smooth muscle cells and non-muscle cells. The direct influx of Ca 2+ and Na + through SA channels as well as the concomitant membrane depolarization may contribute to the initiation of the cell responses induced by the membrane stretch. Thus, the SA channel may mediate signal transduction of membrane stretch to cell responses in vascular smooth muscle cells. Calcium-Activated Potassium Channels The second hypothesis states that the membrane depolarization accompanying the myogenic response is caused by an inhibition of calcium-activated potassium channels, which results in a decrease of the outward current of the smooth muscle cells. Stretch-induced depolarization could result from inhibition of any of the various K + currents identified in smooth muscle, provided the channel was active when the vessel had basal vascular tone. A direct role for a K + channel in initiating the myogenic response has not been shown, but there is evidence that K + currents can and do counteract myogenic tone (2). Of the five major types of K + currents identified in VSM, three appear to play no significant role in the myogenic response (46). 23

However, there is evidence that the other two types of channels, voltagedependent K + (Kv) channels and Ca 2+ activated K + (KCa) channels, can provide potentially powerful repolarizing mechanisms to counteract stimuli resulting from VSM stretch. The Kv channels exhibit exponential increases in open probability upon depolarization and likely serve an important role in the repolarization of excitable cells (23, 40). Knot and Nelson (27) tested the hypothesis that voltage-dependent K + channels are involved in the variations in diameter of small cerebral arteries in rabbit to changes in intravascular pressure by using 4-Aminopyridine (4-AP), a Kv channel inhibitor, and found that indeed these channels play in important role in the regulation of arterial diameter. Chloride Channels The third hypothesis states that the membrane depolarization accompanying the myogenic response is caused by an activation of chloride channels (23), which results in an increase of the inward current of the smooth muscle cells. This hypothesis is based on recent findings in rat cerebral small arteries, where some chloride channel blockers were shown to have no effect on vessel diameter and membrane potential at 20 mmhg, but a large effect at 80 mmhg. This may represent a fundamental mechanism of vasoconstriction of resistance arteries in the brain where myogenic tone is an important regulator of blood flow (39). Since smooth muscle cells are subjected to various physical forces such as altered transmural pressure, stretch, and wall tension associated with the development of myogenic tone, it is reasonable to suggest that the chloride channels involved in myogenic depolarization may be regulated by such cellular forces (39). The results of Nelson et al. (39) support the proposal that activation of chloride channels is a major 24

mechanism for pressure-induced myogenic tone in cerebral arteries. Activation of chloride channels in vascular smooth muscle cells leads to depolarization, increased entry of calcium through voltage-dependent calcium channels, and vasoconstriction. Calcium Channels It should also be considered that the membrane depolarization accompanying the myogenic response could be caused by an activation of calcium channels, which results in an increase of the inward current of the smooth muscle cells. This hypothesis is supported by patch-clamp experiments showing that in rat cerebral small arteries voltage-dependent calcium currents can be stimulated by membrane stretch (33). McCarron and colleagues (33) demonstrated that myogenic contraction in posterior cerebral arteries was dependent on Ca 2+ entry through dihydropyridine-sensitive Ca 2+ channels. In addition, the voltage dependent Ca 2+ current can be increased by membrane stretch. It is possible therefore, that stretch modulation of the voltagedependent Ca 2+ current plays a role in myogenic contraction. In summary, it is well established that the myogenic response is accompanied by a membrane depolarization. The mechanisms of the depolarization and its importance for the development of the myogenic response are still under debate. The Myogenic Mechanism in the Systemic Circulation in Vivo A clear understanding of the in vitro behavior of a vessel is the basis for the assessment of the role of the myogenic response in vivo. However, in vivo, small arteries and arterioles are not only under the influence of transmural pressure, which produces the myogenic response, but are also exposed to, for example, the pulsatility of the blood pressure, neurotransmitter and endothelial influences, conducted signals from neighboring vessel segments and local factors like the PO 2 (partial pressure of oxygen). 25

Consequently, the influence of these factors on the myogenic response should be understood (46). Pulsatile Pressure Small arteries and arterioles in vivo are not under the influence of a static transmural pressure, but of a pulsatile pressure (46). For instance, coronary arterioles show a myogenic response to a change in static transmural pressure. However, transmural pressure of in situ coronary arterioles is not static but varies strongly within the heart cycles, and vascular tone of the coronary resistance vessels may be modulated by this pulsation. Goto et al. (16) demonstrated that the myogenic responsiveness of isolated coronary small arteries remains present in a pulsatile pressure regime. Hence, his results are consistent with a role for myogenic responsiveness in coronary autoregulation in the beating heart (35). Neurotransmitters Transmitters released from nerve endings located in the adventitia of small arteries and arterioles are powerful modulators of vascular contractility in vivo. Adrenergic transmitters like noradrenaline play an important role in regulation of vessel diameter in vivo. Thus, neural influences, especially from adrenergic nerves, probably modulate the myogenic response by increasing its strength (46). Endothelium-Derived Factors The endothelium releases a variety of different factors induced by, for example, the influence of blood-flow-dependent shear stress on the endothelial cells, which are known to modulate vascular contractile responses in vivo. Apparently, there is a competitive interaction between the effect of transmural pressure and of blood flow on vessel diameter regulation, which makes interpretations of in vivo experiments more 26

complicated. At present, it is not established which of the known endothelium-derived factors are responsible for the blood-flow induced reduction of the strength of the myogenic response in small arteries and arterioles. Thus, factors released from the endothelium modulate the myogenic response by decreasing its strength (46). Local Factors It has been observed that contractile responses of blood vessels in vivo can be induced by signals conducted from neighboring vessel segments. Concerning the myogenic response, it was shown that a pressure change in an isolated vessel segment caused a myogenic response in a remote segment, which was insulated from the pressure changes as well as flow changes (45). The contractility of small arteries and arterioles in vivo is influenced by a variety of local factors, producing the metabolic control of vessel responses. Oxygen-dependent vascular control mechanisms also exist in some circulatory beds, and these may alter the effectiveness of myogenic mechanisms in regulating regional blood flow. Nonetheless, the exact nature of the interaction between myogenic and oxygen-dependent mechanisms remains unclear, especially at the level of the vascular smooth muscle (VSM) cell membrane (30). Hypoxia is a well-recognized vasodilatory stimulus. However, the influence of oxygen tension on myogenic reactivity has not been previously characterized. Findings by Earley and Walker (12) demonstrate that an endothelium-dependent influence is responsible for the associated blunted myogenic vasoconstriction and altered vessel-wall (Ca 2+ ) that follow chronic hypoxia. On the other hand, Toporsian and Ward (49) show in their study that in diaphragmatic arterioles from rats exposed to hypoxia for 48 hours, the response to increased transmural pressure is severely suppressed in both endothelium-intact and endothelium-removed vessel. These 27

conflicting effects of endothelium removal may be due to differences in the vascular beds that were studied. An important factor regulating local blood flow is PO 2. A decrease in PO 2 to hypoxic levels produced vessel dilation but no alteration of the myogenic response was observed in rat skeletal muscle small arteries (15). In contrast, in cat cerebral arteries a decrease in PO 2 reduced the membrane depolarization accompanying the myogenic response. Lombard et al. (30) employed small segments of cat middle cerebral artery to investigate the direct effect of reduced PO 2 upon cerebral VSM. He suggested that the inhibition of active VSM tone in middle cerebral artery by reduced PO 2 may be partially mediated by an inhibition of action potentials. A likely explanation for the direct relaxation of VSM by reduced PO 2 is that a decrease in PO 2 reduces the availability of the activator Ca 2+ which is necessary for the generation of contractile force by the muscle. Within the cardiovascular system, homeostatic mechanisms function to regulate peripheral vascular resistance to control mean arterial pressure. On a moment-tomoment basis, vascular resistance is determined by a complex interaction of multiple factors, among which are neural, humoral, and local autoregulatory influences. The extent to which these interactions are important in determining vascular resistance is beginning to become more clearly understood as a result of quantitative studies describing some of these interactive relations involving autoregulatory mechanisms (4). The reason for these contradictory results is not clear, but may be explained by the heterogeneous properties of arteries from different vascular beds. Hayashi et al. (20) studied segmental responses of the interlobular arteries to acute elevations in renal 28

arterial pressure from normotensive rats and spontaneously hypertensive rats and found that the myogenic reactivity of the interlobular arteries to pressure varies along the length of the vessel, with greater responses occurring in the more distal segments. Vascular Adaptations to Chronic Exercise It has been known for some time that chronic exposure to physical activity (i.e. exercise training) results in improved cardiovascular function as seen in increased maximal oxygen consumption, increased maximal cardiac output and increased blood flow capacity in skeletal and cardiac muscle (51). Vascular resistance is the main control mechanism for blood flow during exercise, and this resistance is controlled at the local vascular level of the muscle tissue by altering VSM contraction in the resistance arteries (8). There are complex interactions of vasodilating signals and vasoconstrictor signals in the VSM of the resistance arteries in active skeletal muscle. Evidence suggests that skeletal muscle hyperemia during bouts of exercise is influenced primarily by local control mechanisms such as metabolic control, endothelium-mediated control, propagated responses, myogenic control, and the muscle pump (8). Under resting conditions, within each tissue, the resistance arteries determine regional peripheral resistance in order to provide adequate blood supply to meet the metabolic demands of the body. Vascular resistance is determined by the caliber of the resistance arteries which is controlled by the level of contraction of the vascular smooth muscle (VSM) surrounding the arteries. These arteries have a level of basal tone (basal level of contraction of the VSM) and are also influenced by central control signals (sympathetic constriction) and local chemical and mechanical factors (51). 29

Skeletal muscle receives a greater blood supply following training. This is due to: increased number of capillaries, greater opening of existing capillaries, more effective blood redistribution, and increased blood volume. It is possible that based on these factors changes in the myogenic response with exercise training would occur on blood redistribution. Hypotheses The purpose of this study was to test the following hypotheses: The myogenic mechanism of skeletal muscle resistance arterioles from both the soleus muscle, which is composed mainly of slow-twitch fibers (type I), and the gastrocnemius muscle, composed mainly of fast-twitch fibers (type IIB), (7) would be stronger in young Fischer-344 rats. Exercise training would increase strength of the myogenic response of skeletal muscle resistance arterioles from soleus and gastrocnemius muscle in both experimental groups, young and old. The vasoconstrictor response would be greater in vessels where the endothelium had been removed as opposed to those where the endothelium was intact. 30

CHAPTER 2 MATERIAL AND METHODS Exercise Training All rats were habituated to treadmill exercise, during this habituation period each rat walked on a motor-driven treadmill at 5 m min 1 (0 deg incline), 5 min per day for 3 days. After habituation, young and old rats were randomly assigned to either a control sedentary (SED) group (young SED, n = 11, and old SED, n = 12) or an exercisetrained (ET) group (young ET, n = 10, and old ET, n = 8). ET rats performed treadmill running at 15 m min 1 (15 deg incline), 5 days per week, for 10 12 weeks. The duration of running was gradually increased in the first 4 weeks until a 60 min duration was reached. The rats continued to run 5 days per week for 60 min per day for the remainder of the 10 12 week training period as previously described (9, 24). Vascular responses were determined at least 48 h after the last exercise bout in ET rats. Microvessel Preparation Rats were anesthetized with pentobarbital sodium (100 mg/kg ip). The gastrocnemius-plantaris-soleus muscle group was carefully dissected free from both hindlimbs and placed in cold (4 C), filtered physiological saline solution (PSS) containing (in mm) 145 NaCl, 4.7 KCl, 2.0 CaCl2, 1.17 MgSO4, 1.2 NaH2PO4, 5.0 glucose, 2.0 pyruvate, 0.02 EDTA, 3.0 MOPS buffer, and 1% bovine serum albumin as previously described (37). With the aid of a dissecting microscope, first-order (1A) arterioles were isolated from the soleus, a predominantly slow-twitch muscle, and the superficial portion of the gastrocnemius, a predominantly fast-twitch muscle (7). In soleus muscle, 1A arterioles were defined as the first branch after the feed artery entered the muscle tissue. In gastrocnemius, 1A arterioles were defined as the first 31

branch off the feed artery that traverses the superficial portion of the muscle. The arterioles (80 to 225 µm inner diameter) were transferred to a Lucite chamber containing PSS equilibrated with room air. Arterioles were cannulated with micropipettes secured with suture (Alcon 11-0 nylon microfilament). Once the arteriole was cannulated at one end, the denuding process began by passing 5 ml of air though the lumen of the vessel in order to remove the endothelium, this procedure was performed only in the endothelium-removed vessels. After cannulation, the microvessel chamber was transferred to the stage of an inverted microscope equipped to measure and record arteriolar intraluminal diameter (24). Arterioles were pressurized to 70 cmh 2 O with two independent hydrostatic pressure reservoirs. Vessels were checked for leaks by pressurizing the vessel and then closing the lines to the fluids reservoirs to determine whether vessel diameter was maintained. Arterioles with leaks were discarded. Vessels determined to be free of leaks were warmed to 37 C and allowed to develop spontaneous tone during an initial equilibration period. The bathing solution was changed every 20 mins during equilibration. Recording of the myogenic response were not initiated until arterioles developed a minimum of 20% spontaneous tone that remained steady for at least 10 min. Arterioles that failed to achieve spontaneous tone were also discarded. Experimental Design To determine whether the myogenic mechanism is affected by age and exercise training one arteriole from the soleus muscle and one arteriole from the superficial gastrocnemius muscle were studied from each animal. During the experiment, spontaneous tone development, active myogenic response, and maximal inner arteriolar diameters were determined. 32

Evaluation of Myogenic Mechanism The micropipettes cannulating the arterioles were connected to independent reservoir systems. Intraluminal pressure was measured through sidearms of the two reservoir lines by low-volume-displacement strain-gauge transducers as previously described by Delp (6). The myogenic responses were recorded in the presence of 2 mm extracellular Ca 2+. The isolated vessels were initially pressurized at 70 cmh 2 O by setting both reservoirs at the same hydrostatic level. Vessels were equilibrated at 37ºC and 70 cm H 2 O for 60 minutes to allow for development of at least 20% of spontaneous tone from the original diameter. Once the vessel achieved proper tone, the endotheliumdependent vasodilator Ach (40 μl) was added to the bath to determine whether the endothelium was intact. Following the Ach test, the arterioles were washed one more time with PSS. After the 1-h equilibration period, intraluminal pressure was dropped down to 0 cmh 2 0 and then increased by increments of 10 cmh 2 O up to 140 cmh 2 O, by simultaneously raising both reservoirs in 10-cmH 2 O increments in order to alter myogenic tone in the absence of flow through the lumen. Intraluminal diameter was recorded at each pressure step. Changes in pressure were maintained for 3 min, which was sufficient to allow a steady vascular response to the change in pressure. After each step change in intraluminal pressure, diameter was recorded continuously, all pressure changes occurred in the absence of intraluminal flow. In order to determine maximal diameter at the end of the myogenic response, pressure was dropped back to 70 cmh 2 O, and then the vessel chamber was filled with calcium-free PSS, the bath was changed twice every 20 minutes to facilitate complete relaxation of the arteriolar smooth muscle. After the last rinse of calcium-free PSS, 40 μl 33

sodium nitroprusside (SNP) was also added to the bathing solution and maximal diameter was recorded. Data Presentation Data are presented in the same fashion as previously described in different studies done by Muller-Delp et al. (24, 37). Development of spontaneous tone was expressed as the percent constriction relative to maximal diameter and was calculated as follows: Spontaneous tone (%) = (ID max ID b )/ID max X 100 where ID max is the maximal inner diameter recorded at a pressure of 70 cmh 2 O and ID b is the steady-state baseline diameter. ID max was determined at the end of each experiment by incubating the vessel in Ca 2+ -free PSS with 40 μm sodium nitroprusside for 30 min. Active myogenic responses recorded in response to pressure changes were normalized according to the following formula: Normalized diameter = (ID s /ID max ) where ID s, is the steady-state diameter measured after each incremental pressure change. Diameter is normalized to account for differences in vessel size between young and old animals. Graphs were generated using SigmaPlot 11.0 Statistical Analysis Descriptive statistics were obtained in order to present the data as means ± standard errors. Body weight, maximal diameter and spontaneous tone were compared by two-way repeated measures ANOVA to identify significant differences between young and old, soleus vs. gastrocnemius or sedentary vs exercise trained. For the myogenic response a four-way repeated measures ANOVAs were performed to 34

compare variables such as young and old, exercise trained vs. sedentary, soleus vs. gastrocnemius, endothelium intact vs denuded vessels. Post-hoc analyses were performed using the Least Significant Difference (LSD). All statistical analyses were performed using SPSS and statistical significance was defined as P<0.05. 35

CHAPTER 3 RESULTS Animal Characteristics Body weight is reported in table 1-1. Body weight increased with age in sedentary as well as exercise trained animals, young sedentary rats had a weight of 364 ± 11 g. while old sedentary rats weighed 449 ± 10 g. In exercise trained animals there was also a significant increase in body weight, young ET weighed 389 ± 9 g and old ET weighed 430 ± 11.g. Table 1-1. Animal characteristics Young SED Young ET Old SED Old ET n 17 10 15 8 Age, mo 6.4 ± 0.2 6.9 ± 0.1 24.6 ± 0.1 * 24.75 ± 0.2 * Body Weight, g 364 ± 11 389 ± 9 449 ± 10 * 430 ± 11 * Values are means ± SE. n = no of animals. *significantly different from the young groups (P< 0.05) Vessel Characteristics Table 1-2 and table 1-3 report vessel characteristics. Spontaneous vascular tone developed at 70 cm H 2 O was significantly different with fiber type, age and condition (sedentary versus exercise trained). All the exercise trained animals developed more spontaneous tone when compared with their sedentary counterparts. Tone development was stronger in all the young animals, sedentary and exercise trained when compared to the old animals. Arterioles from the young soleus had greater spontaneous tone development than arterioles from the gastrocnemius in both exercise trained and sedentary groups. The same phenomenon was observed when the old soleus was compared with the old gastrocnemius from both groups. 36

Maximal inner arteriolar diameter was greater in old and young gastrocnemius when compared to the old and young soleus from both, ET and SED groups. There was no significant age-related difference found within the same muscle groups. Table 1-2. Vessel characteristics from exercise trained animals Soleus Muscle Gastrocnemius Muscle Young Old Young Old n 10 8 8 8 Maximal diameter, µm 133 ± 10 116 ± 7 162 ± 12 * 148 ± 14 * Spontaneous tone, % 48 ± 1 41 ± 2 32 ± 2 * 29 ± 1 * Values are means ± SE. n = no of animals. *P<0.05 significantly different from agematched soleus group. P<0.05 significantly different from respective young muscle group. Table 1-3. Vessel characteristics from sedentary animals Soleus Muscle Gastrocnemius Muscle Young Old Young Old n 11 12 12 12 Maximal diameter, µm 121 ± 7 115 ± 5 155 ± 6 * 169 ± 9 * Spontaneous tone, % 38 ± 2 30 ± 1 26 ± 1 * 21 ± 0 * Values are means ± SE. n = no of animals. *P<0.05 significantly different from agematched soleus group. P<0.05 significantly different from respective young muscle group. Myogenic Response All the figures depicting the myogenic response show a pressure - diameter relation as intraluminal pressure is raised from 0 to 140 cmh 2 O. 1A arterioles from soleus and gastrocnemius muscle displayed vascular constriction as pressure was increased within the examined range. Figure 2-1 shows the myogenic response from the soleus skeletal muscle arterioles with an intact endothelium. Results from this study 37

show that there is a stronger myogenic reaction in the arterioles from young animals than in those from old rats (P<0.05) (Fig 2-1-A), at the same time exercise training induces a stronger myogenic reaction in both old and young exercise trained animals (P<0.05) (Fig 2-1-C, Fig 2-1-D), being such response greater in young trained versus old trained throughout the examined pressure range (P<0.05)(Fig 2-1-B). Removal of the vascular endothelial cell layer eliminated the ageing and training-related changes found in the soleus muscle arterioles, which is shown in Figure 2-2-A and 2-2-B. Myogenic responses of the gastroncemius 1A arterioles with an intact endothelium are presented in Figure 2-3. It was observed that ageing reduces myogenic tone (P<0.05) (A). Exercise training did not have effect in old and young animals as seen in Figure 2-3 (A and B). Myogenic responses of the denuded arterioles from the superficial gastrocnemius are presented in Figure 2-4. There was no training effect in either the young or old animals (Fig 2-4-C and 2-4-D). Age difference is reversed in the sedentary group when compared to data with intact endothelium (P<0.05). It appears that the endothelium has a vasodilator effect in old and a vasoconstrictor effect in young rats. (Fig 2-3-A and 2-4- A) Legends used in the graphs Endothelium Intact: Young Control Old Control Old Trained Young Trained 38