Cardiovascular control during exercise and the role of the sympathetic nervous system in heart failure with reduced ejection fraction

The objective of this dissertation was to systematically investigate the hemodynamic response to exercise in heart failure with reduced ejection fraction (HFrEF) and healthy individuals of a similar age, with an emphasis on how the sympathetic nervous system (SNS) may contribute to the dysregulation of the cardiovascular system in this cohort. The first study aimed to determine how varying levels of metaboreceptor activation alters the mean arterial pressure (MAP) response as well as the degree in which cardiac output (CO) and systemic vascular conductance (SVC) contribute to the metaboreflex-induced increase in MAP. We observed similar increases in MAP induced by metaboreceptor activation in both groups; however, this response was driven primarily by increases in CO in the control group and reductions in SVC in the HFrEF group. These data suggest a preserved role of the metaboreflex-induced increase in MAP in HFrEF, but suggest that this response is governed by the peripheral circulation in this cohort, a maladaptation that may exacerbate systolic dysfunction through an increase in afterload. The second study of this dissertation was focused on investigating the peripheral vasodilatory and hyperemic response to exercise in isolation of central hemodynamic limitations in both the upper and lower limbs. This study documented an impaired hyperemic response to both static-intermittent handgrip exercise as well as dynamic single-leg knee-extensor exercise in HFrEF patients - impairments primarily attributed to vasodilatory dysfunction, as the increase in MAP induced by these exercise modalities was preserved compared to healthy individuals. Together, these findings have identified a significant attenuation of the exercise-induced hyperemic response during both upper and lower limb exercise, implicating maladaptions in the peripheral hemodynamic response to exercise as a potential contributor limiting exercise capacity in this patient group. The third study sought to address the contribution of the alpha-adrenergic receptor pathway in the regulation of blood flow to exercising skeletal muscle in HFrEF patients. At rest, alpha-1-adrenergic receptor vasoconstriction induced by local intra-arterial infusion of phenylephrine (PE) was reduced in HFrEF compared to control subjects. During exercise, the vasoconstrictor responsiveness to PE was significantly attenuated in the control group and preserved in HFrEF patients compared to rest. Additionally, nonspecific alpha-adrenergic receptor antagonism induced by local intra-arterial infusion of phentolamine increased blood flow to a greater degree in HFrEF compared to the control subjects, both at rest and during exercise. Together, these findings demonstrate a marked contribution of alpha-adrenergic receptor restraint of leg blood flow in HFrEF patients during exercise. Collectively, these three studies have provided new insight into the role the SNS and peripheral hemodynamics play in the maladaptive cardiovascular response to exercise displayed in patients with HFrEF, further implicating the peripheral expression of SNS activity as a primary contributor to impaired exercise capacity in this patient group.

CARDIOVASCULAR CONTROL DURING EXERCISE AND THE ROLE OF THE SYMPATHETIC NERVOUS SYSTEM IN HEART FAILURE WITH REDUCED EJECTION FRACTION by Zachary Barrett-O'Keefe A dissertation submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Exercise and Sport Science The University of Utah August 2015 Copyright © Zachary Barrett-O'Keefe 2015 All Rights Reserved The University of Utah Graduate School STATEMENT OF DISSERTATION APPROVAL The dissertation of Zachary Barrett-O'Keefe M.S. has been approved by the following supervisory committee members: D. Walter Wray , Chair 10/31/2014 Date Approved Russell S. Richardson , Member 10/31/2014 Date Approved J. David Symons , Member 10/31/2014 Date Approved Markus M. Amann , Member 10/31/2014 Date Approved John R. Halliwill , Member 10/31/2014 Date Approved and by Janet M. Shaw , Chair/Dean of the Department/College/School of Exercise and Sport Science and by David B. Kieda, Dean of The Graduate School. ABSTRACT The objective of this dissertation was to systematically investigate the hemodynamic response to exercise in heart failure with reduced ejection fraction (HFrEF) and healthy individuals of a similar age, with an emphasis on how the sympathetic nervous system (SNS) may contribute to the dysregulation of the cardiovascular system in this cohort. The first study aimed to determine how varying levels of metaboreceptor activation alters the mean arterial pressure (MAP) response as well as the degree in which cardiac output (CO) and systemic vascular conductance (SVC) contribute to the metaboreflex-induced increase in MAP. We observed similar increases in MAP induced by metaboreceptor activation in both groups; however, this response was driven primarily by increases in CO in the control group and reductions in SVC in the HFrEF group. These data suggest a preserved role of the metaboreflex-induced increase in MAP in HFrEF, but suggest that this response is governed by the peripheral circulation in this cohort, a maladaptation that may exacerbate systolic dysfunction through an increase in afterload. The second study of this dissertation was focused on investigating the peripheral vasodilatory and hyperemic response to exercise in isolation of central hemodynamic limitations in both the upper and lower limbs. This study documented an impaired hyperemic response to both static-intermittent handgrip exercise as well as dynamic single-leg knee-extensor exercise in HFrEF patients - impairments primarily attributed to vasodilatory dysfunction, as the increase in MAP iv induced by these exercise modalities was preserved compared to healthy individuals. Together, these findings have identified a significant attenuation of the exercise-induced hyperemic response during both upper and lower limb exercise, implicating maladaptions in the peripheral hemodynamic response to exercise as a potential contributor limiting exercise capacity in this patient group. The third study sought to address the contribution of the alpha-adrenergic receptor pathway in the regulation of blood flow to exercising skeletal muscle in HFrEF patients. At rest, alpha-1-adrenergic receptor vasoconstriction induced by local intra-arterial infusion of phenylephrine (PE) was reduced in HFrEF compared to control subjects. During exercise, the vasoconstrictor responsiveness to PE was significantly attenuated in the control group and preserved in HFrEF patients compared to rest. Additionally, nonspecific alpha-adrenergic receptor antagonism induced by local intra-arterial infusion of phentolamine increased blood flow to a greater degree in HFrEF compared to the control subjects, both at rest and during exercise. Together, these findings demonstrate a marked contribution of alpha-adrenergic receptor restraint of leg blood flow in HFrEF patients during exercise. Collectively, these three studies have provided new insight into the role the SNS and peripheral hemodynamics play in the maladaptive cardiovascular response to exercise displayed in patients with HFrEF, further implicating the peripheral expression of SNS activity as a primary contributor to impaired exercise capacity in this patient group. TABLE OF CONTENTS ABSTRACT ……………………………………………………………………………..iii LIST OF FIGURES……………………………………………………………………...vii LIST OF TABLES………………………………………………………………………..ix ACKNOWLEDGMENTS………………………………………………………………...x Chapter 1 INTRODUCTION……...…………………………………………………………1 Autonomic Dysfunction in Heart Failure with Reduced Ejection Fraction……………………………………………………………………2 The Metaboreflex Response to Exercise in Heart Failure with Reduced Ejection Fraction…………………………………………………………..4 Peripheral Hemodynamic Response to Exercise in Heart Failure with Reduced Ejection Fraction………………………………………………...8 Alpha-Adrenergic Receptor Control of Skeletal Muscle Blood Flow during Exercise in Heart Failure with Reduced Ejection Fraction………10 Hypotheses and Specific Aims…………………………………………..12 References………………………………………………………………..16 2 METABORECEPTOR ACTIVATION IN HEART FAILURE WITH REDUCED EJECTION FRACTION: LINKING CENTRAL AND PERIPHERAL HEMODYNAMICS……………………………………………………………..23 Abstract…………………………………………………………………..24 Introduction………………………………………………….……...……25 Methods…………………………………………………….……...….….27 Results……….…………………………………………………………...32 Discussion………………………………………………………………..34 References………………………………………………………………..42 3 HEMODYNAMIC RESPONSES TO SMALL MUSCLE MASS EXERCISE IN HEART FAILURE WITH REDUCED EJECTION FRACTION………………62 Abstract…………………………………………………………………..63 vi Introduction…….………………………….…...….…….….……………64 Methods…….…………………………………………………………….66 Results…….……………………….….…….…….….…….……….……71 Discussion………………………………………………………………..73 References…………………………….………………………………….84 4 ALPHA-ADRENERGIC RECEPTOR REGULATION OF SKELETAL MUSCLE BLOOD FLOW DURING EXERCISE IN HEART FAILURE WITH REDUCED EJECTION FRACTION………………………...………………...100 Abstract…………………………………………………………………101 Introduction…………………………….……………………………….102 Methods…………………………………………………………………104 Results………………………………….…………….…………………112 Discussion………………………………………………………………115 References………………………………………………………………125 5 CONCLUSION…………………………………………………………………149 References………………………………………………………………153 LIST OF FIGURES Figure 2.1 Metaboreflex-induced changes in mean arterial pressure (top), cardiac output (middle), and systemic vascular conductance (bottom) in control subjects and heart failure patients with reduced ejection fraction (HFrEF)…...……………………………………47 2.2 Metaboreflex-induced changes in pulse pressure (top) and total arterial compliance (bottom) in control subjects and heart failure patients with reduced ejection fraction (HFrEF)…………………………………………………………………………………..49 2.3 Metaboreflex-induced changes in effective arterial elastance in control subjects and heart failure patients with reduced ejection fraction (HFrEF)…………………………...51 2.4 Metaboreflex-induced changes in stroke work (top), end systolic pressure (middle), and stroke volume (bottom) in control subjects and heart failure patients with reduced ejection fraction (HFrEF)……………………………………………………..………….53 2.5 Relationship between metaboreflex-induced changes in stroke work and changes in effective arterial elastance in control subjects and heart failure patients with reduced ejection fraction (HFrEF)………………………………………………………………...55 2.6 Relationship between metaboreflex-induced changes in stroke work and changes in rate pressure product in control subjects and heart failure patients with reduced ejection fraction (HFrEF)…………………………………………………...…………………….57 3.1 Forearm blood flow (top) and mean arterial pressure (bottom) at rest and during static-intermittent handgrip exercise in control subjects and heart failure patients with reduced ejection fraction (HFrEF)……………………………………………………….91 3.2 Leg blood flow (top) and mean arterial pressure (bottom) at rest and during dynamic single-leg knee-extensor exercise in control subjects and heart failure patients with reduced ejection fraction (HFrEF)……………………………………………………….93 3.3 Relationship between leg blood flow at 15 W and maximal single-leg knee-extensor work rate in control subjects and heart failure patients with reduced ejection fraction (HFrEF)…………………………………………………………………………………..95 viii 4.1 Experimental timeline for phenylephrine (A) and phentolamine (B) infusion at rest and during exercise…………………………………………………………………......131 4.2 Changes (%) in leg blood flow (top) and leg vascular conductance (bottom) in response to local, intra-arterial infusion of phenylephrine (PE) in control subjects and heart failure patients with reduced ejection fraction (HFrEF)………………………….133 4.3Changes (%) in leg blood flow (top) and leg vascular conductance (bottom) in response to local, intra-arterial infusion of phentolamine (PHEN) in control subjects and heart failure patients with reduced ejection fraction (HFrEF)………………………….135 4.4 Absolute values (A) of: Leg blood flow (top), leg vascular conductance (middle), and leg perfusion pressure (bottom) in control subjects and heart failure patients with reduced ejection fraction (HFrEF) at rest and during knee-extensor exercise in control conditions (pre-infusion) and during phenylephrine (PE) infusion. PE-induced changes (%) (B) of: leg blood flow (top), leg vascular conductance (middle), and leg perfusion pressure (bottom) in control subjects and HFrEF patients at rest and during knee-extensor exercise……………...………………………………………………………………….137 4.5 Absolute values (A) of: Leg blood flow (top), leg vascular conductance (middle), and leg perfusion pressure (bottom) in control subjects and heart failure patients with reduced ejection fraction (HFrEF) at rest and during knee-extensor exercise in control conditions (pre-infusion) and during phentolamine (PHEN) infusion. PHEN-induced changes (%) (B) of: Leg blood flow (top), leg vascular conductance (middle), and leg perfusion pressure (bottom) in control subjects and HFrEF patients at rest and during knee-extensor exercise……………………..…………………………………………………………..139 4.6 Absolute values (A) of: Leg oxygen delivery (top), and leg oxygen consumption (bottom) in control subjects and heart failure patients with reduced ejection fraction (HFrEF) at rest and during knee-extensor exercise in control conditions (pre-infusion) and during phentolamine (PHEN) infusion. PHEN-induced changes () (B) of: Leg oxygen delivery (top) and leg oxygen consumption (bottom) in control subjects and HFrEF patients at rest and during knee-extensor exercise……………………………...141 4.7 Relationship between leg oxygen delivery at 10 W and relative intensity at 10 W during the control condition (pre-infusion) in control subjects and heart failure patients with reduced ejection fraction (HFrEF)………………………………………………...143 LIST OF TABLES Table 2.1 Subject characteristics……..…………………………………………………………58 2.2 Disease - specific characteristics and medications……..……………………………59 2.3 Central and peripheral hemodynamics at rest and during exercise…………………..60 2.4 Central and peripheral hemodynamics at rest and during metaboreceptor activation…………………………………………………………………………………61 3.1 Subject characteristics……..…………………………………………………………96 3.2 Disease - specific characteristics and medications……..……………………………97 3.3 Central and peripheral hemodynamics at rest and during handgrip exercise……..…98 3.4 Central and peripheral hemodynamics at rest and during single-leg knee-extensor exercise………..…………………………………………………………………………99 4.1 Subject characteristics..........………………………………………………………..144 4.2 Disease - specific characteristics and medications…………………………………145 4.3 Hemodynamic responses to propranolol and phenylephrine at rest..........................146 4.4 Hemodynamic responses to infusions of propranolol and phentolamine at rest…...147 4.5 Cardiovascular responses to intra-arterial infusions of phenylephrine and phentolamine at rest and during exercise……………………………………………….148 ACKNOWLEDGMENTS There are countless people to thank for their support and encouragement during the time it took me to complete this dissertation. I would like to take this opportunity to thank a few of these people who helped make the completion of this dissertation possible. I wish to express my sincere appreciation to my advisor, Dr. Wray, for bringing me to the University of Utah, sharing with me your knowledge, vision, and enthusiasm for physiology, as well as giving me the intellectual freedom to grow under your advising. This dissertation would not have been possible without your guidance and support. To my fortune, I have also benefitted from the advising of Dr. Richardson, Dr. Amann, Dr. Symons, and Dr. Halliwill. I am grateful for the time, effort, and unique perspective that you each brought to this endeavor. To the members of the Utah Vascular Research Laboratory (UVRL): thank you for sacrificing your time and energy to help me develop and complete the research studies comprising this dissertation. Additionally, work we do at the UVRL would not be possible without the subjects who volunteer their time, so I would like to thank all of the subjects who participated in my studies over the years. To Dr. Halliwill and the graduate students I had the privilege and honor of working with during my undergraduate years in the Department of Human Physiology at the University of Oregon: thank you for instilling in me the love for the pursuit of knowledge. xi Finally, I would like to dedicate this dissertation to my family. Throughout the years, your unconditional love and words of wisdom have helped keep me grounded and maintain a positive outlook on life. CHAPTER 1 INTRODUCTION 2 In the United States, chronic heart failure (CHF) constitutes one of the leading causes of mortality, accounting for roughly one third of all deaths from cardiovascular causes with a 50% 5-year mortality rate (52). Additionally, the American Heart Association estimates that more than 5 million people in the United States alone have CHF, with roughly 550,000 new cases diagnosed each year (52). CHF is a disease that has many different origins and is typically the most severe manifestation of almost every form of cardiac disease, including myocardial infarction, coronary atherosclerosis, hypertension, congenital heart disease, and cardiomyopathies (27). CHF is a pathology which is commonly characterized by left ventricular ejection fraction (LVEF), an expression of stroke volume (SV) as a percentage of left ventricular end-diastolic volume. While the crudity of LVEF as a measure of left ventricular function has been criticized, the association between LVEF and morbidity as well as mortality is well established (8, 67). However, CHF is a subjective clinical syndrome which is variably defined (34), and, in general, heart failure classification is poorly related to LVEF (34). For example, patients with a reduced LVEF may be asymptomatic, while severe CHF-related symptoms may be apparent in patients with a "preserved" LVEF. Thus, patients with CHF are now further delineated on the basis of LVEF, leading to the new nomenclature of heart failure with reduced ejection fraction (HFrEF) or heart failure with preserved ejection fraction (HFpEF) (29). Autonomic Dysfunction in Heart Failure with Reduced Ejection Fraction A hallmark characteristic of patients with HFrEF is heightened sympathetic nervous system (SNS) activity, quantified by resting muscle sympathetic nerve activity 3 (MSNA) (16, 32) and plasma norepinephrine (NE) concentration (17), both of which are associated with severity (16, 17) and predictive of mortality in HFrEF patients (3, 17). Additionally, observations from clinical trials have indicated that exaggerated SNS activation appears to precede the development of clinical signs and symptoms of HFrEF (17), suggesting that sympathoexcitation plays a role in the genesis and progression of the disease. While this increased SNS activity initially serves to improve cardiac function and support arterial blood pressure (ABP), it also provokes a sustained peripheral vasoconstriction that restrains skeletal muscle blood flow (63, 81). For this reason, the evolving model of pathophysiology in CHF has expanded to include the peripheral vasculature, where the disease manifests as vascular and skeletal muscle dysfunction related to underlying autonomic effects of the disease. One of the principal functional consequences of the exaggerated SNS activation and subsequent peripheral vasoconstriction in patients with HFrEF might be the worsening of symptoms upon exertion. Indeed, HFrEF patients have been documented to have severe exercise intolerance which is associated with the severity of the disease and triggers debilitating cases of dyspnea and fatigue (63). Interestingly, while central hemodynamic abnormalities are, by definition, the fundamental characterization of HFrEF, the degree of left ventricular dysfunction does not fully explain the degree of exercise intolerance or symptom status in this patient group (4, 22, 36, 65, 68). This supports the possibility that peripheral maladaptations may contribute to the exercise intolerance in this patient group. Early studies exploring the possible relation between exercise intolerance in HFrEF and SNS activity indicated that resting MSNA is negatively associated with aerobic capacity in this cohort (42), and patients with HFrEF 4 exhibit an exaggerated increase in MSNA during exercise (37, 43, 60, 62). These novel findings raise the question of the functional end-organ consequences of the persistent augmentation of MSNA during exercise in HFrEF. The Metaboreflex Response to Exercise in Heart Failure with Reduced Ejection Fraction As mentioned above, accumulating evidence indicates that exercise limitations in HFrEF patients are not predominantly due to inadequate left ventricular function, which has led to a shift in the investigations probing the mechanisms contributing to exercise intolerance in this patient population to the periphery. Recently, Drs. Coats and Piepoli have hypothesized that abnormalities arising from the skeletal muscle, specifically the sensory nerve fibers that mediate reflex changes triggered by exercise, may contribute to the exercise limitations in HFrEF (6, 48). This so-called "muscle hypothesis" of HF proposes another cycle of deterioration similar to that of neuroendocrine activation, whereby left ventricular dysfunction leads to abnormalities in skeletal muscle metabolism and function that contributes to an exaggerated reflex increase in SNS activity. Located within the skeletal muscle, the sensory afferent fibers mediating these reflex increases in SNS activity, often termed the "ergoreflex", consist of group III afferent fibers which are predominately mechanically sensitive (mechanoreceptors) and group IV afferent fibers (metaboreceptors) which are principally sensitive to metabolites produced during exercise (26). These afferent signals are carried to the cardiovascular center of the nucleus tractus solitarii (NTS), provoking specific efferent signals to the heart and peripheral circulation. Specifically, vagal withdrawal and an increase in SNS activity to the heart induces an increase in heart rate, which is accompanied by a 5 sympathetically-mediated increase in SV (1, 7, 44), collectively increasing the perfusion pressure generated by the heart. In the periphery, SNS activation helps divert blood flow to the exercising skeletal muscle through vasoconstriction of the vasculature perfusing less metabolically active tissue (2, 38, 39). Together, these cardiac and vascular responses work in concert to increase perfusion of the muscle from which the afferent signals emanate (1, 26, 45). The extent to which the mechano- and metaboreceptors contribute to the reflex increase in SNS activity (as measured by direct recordings of MSNA) in HFrEF patients has long been a topic of debate (40, 49). Implementing postexercise circulatory occlusion following handgrip exercise, previous studies have documented an increased sensitivity of both the mechanoreflex (37) and the metaboreflex (43, 60). However, conflicting evidence exists in regards to the metaboreflex, with studies indicating both a blunted (62) and a similar (37) increase in MSNA in HFrEF patients compared to healthy individuals. Under a closer lens, it appears that these conflicting results can be attributed to the modality of handgrip exercise and the intensity of exercise implemented. Indeed, Sterns et al. (62) had subjects perform static handgrip exercise to activate the metaboreflex, an exercise modality that is not rhythmic in nature, making it difficult to translate these finding to physical activity performed by HFrEF patients in everyday life. Furthermore, the intensity of rhythmic handgrip exercise implemented by Middlekauff et al. (37) failed to significantly increase MSNA during metaboreceptor activation in either HFrEF patients or healthy individuals, making it difficult to conclude that there is no difference in the metaboreflex control of MSNA between groups. Based on the conflicting findings on the role of the metaboreflex in increasing MSNA during physical stress in HFrEF, it is 6 not surprising that studies investigating the impact of this increase in SNS activity on ABP responses have been inconclusive as well. Indeed, studies have documented both exaggerated (48, 57) and similar (5, 30, 43, 62) increases in ABP during metaboreceptor activation in HFrEF patients compared to healthy individuals. The conflicting findings from these studies indicate that significant uncertainty still remains as to whether the metaboreflex-induced pressor response is altered in HFrEF. Additionally, very little is known about the roles cardiac output (CO) and systemic vascular conductance (SVC) play in increasing ABP during metaboreceptor activation in HFrEF. In a healthy animal model, O'Leary et al. (44) documented that the rise in ABP triggered by metaboreceptor activation was solely due to increases in CO during low to moderate exercise intensities. Additionally, during near maximal exercise, where increases in CO are minimal, Augustyniak et al. (2) documented that a further increase in ABP due to metaboreceptor activation was due to a reduction in SVC. This is in contrast to findings in an animal model of HF, where reductions in SVC predominately contribute to the metaboreflex-induced increase in ABP during all exercise intensities, a response likely due to impaired left ventricular function (19). In humans, even less is known about the degree in which CO and SVC contribute to the metaboreflex-induced increase in ABP in HFrEF. In one of the only studies exploring this topic, Crisafulli et al. (7) utilized one moderate intensity of rhythmic handgrip exercise to study this relationship in HFrEF and healthy controls, and observed a metaboreflex-induced increase in ABP which was predominantly driven by an increase in CO in healthy individuals and a reduction in SVC in patients with HFrEF. This has been complemented by work indicating an exaggerated increase in diastolic blood pressure during 7 metaboreceptor activation in HFrEF, indirectly indicating a reduction in SVC in this patient group (47). While these investigations have shed light on the differing avenues by which the metaboreflex increases ABP in HFrEF compared to their healthy counterparts, the inclusion of only one moderate intensity workload of handgrip exercise leaves unanswered whether the relative contribution of CO and SVC to the exercise pressor response varies as a consequence of exercise intensity. A more comprehensive understanding of the metaboreflex, and the relative contribution of CO and SVC to this reflex response, would add significantly to our understanding of the "muscle hypothesis" in HFrEF. Undeniably, an attenuation in the rise in CO coupled with an exaggerated reduction in SVC may collectively generate a scenario where blood flow to exercising skeletal muscle is potentially compromised due to central and peripheral hemodynamic alterations in order to preserve ABP. Indeed, reductions in SVC, a measure of systemic vascular tone which also represents the nonpulsatile component of arterial afterload (25, 80), may present a further stress to cardiac muscle, a stress which is likely vastly minimized in healthy individuals. It follows that knowing whether reductions in SVC play a primary role in the metaboreflex-induced increases in ABP across a wide array of exercise intensities will indicate at what level of work this reflex pathway might become detrimental in increasing perfusion of the exercising skeletal muscle. This concept was investigated in Specific Aim 1 of this dissertation. 8 Peripheral Hemodynamic Response to Exercise in Heart Failure with Reduced Ejection Fraction While an exaggerated metaboreflex response to exercise may contribute importantly to exercise intolerance in HFrEF, these patients may also exhibit a fundamental impairment in vasodilatory ability and hyperemia during physical activity. Initial evidence for a functional role of impaired peripheral hemodynamics in limiting exercise capacity in this cohort was documented during peak cycling exercise, where an apparent reduction in leg vascular conductance was observed in patients with HFrEF compared to healthy individuals (71-75). However, the use of a large muscle mass exercise paradigm presents limitations in distinguishing abnormalities in peripheral vasomotor function in HFrEF. When performing exercise, blood flow to exercising skeletal muscle is accomplished through an orchestrated series of events which includes contributions from both "central" (i.e. CO) and peripheral (i.e. SVC) portions of the cardiovascular system in order to divert blood away from less metabolically active tissue towards exercising skeletal muscle. During exercise which recruits a large fraction of total body muscle mass, central circulatory factors play an increasingly important role in the preservation of ABP (46, 51, 55, 56). Due to impaired left ventricular function, patients with HFrEF cannot increase CO to the same level as healthy individuals during exercise (63, 75). However, irrespective of cardiac function, there is no difference in the observed MAP during peak cycling exercise in HFrEF compared to healthy controls (63). This may indicate that a restraint of the exercise-induced increase in SVC plays a heightened role in ABP regulation in HFrEF, and may also indicate that the observed reduction in leg vascular conductance and associated reduction in perfusion of the 9 exercising skeletal muscle might solely be due to the maintenance of ABP versus limitations in vasodilatory capacity in this patient group (63, 75). In a small number of studies, the utilization of small muscle mass exercise such as dynamic single-leg knee-extensor (KE) and rhythmic handgrip exercise has allowed for a more thorough investigation of peripheral vasomotor responses to exercise in HFrEF in isolation of the significant cardiac stress and associated confounding effects of ABP regulation imposed by large muscle mass exercise. The results, however, have been equivocal. During handgrip exercise, patients with HFrEF have been documented to possess both similar (58) as well as attenuated (23, 81) vasodilatory and hyperemic responses in the exercising limb, differences which have been attributed to lack of standardization with respect to exercise cadence and intensity (35, 58, 69, 76, 81). In studies which utilized the single-leg KE exercise model, similar increases in leg vascular conductance and leg blood flow have been reported between HFrEF and healthy individuals across a range of exercise intensities (15, 33). However, since these studies were performed, the clinical treatment of HFrEF has evolved, with the inclusion of new pharmacological interventions (18, 21, 31, 66) which might indirectly alter vasomotor regulation in response to small muscle mass exercise. Understanding the peripheral vasomotor response to small muscle mass exercise in the presence of these recently implemented pharmacological interventions could enlighten clinicians on how the progression of clinical treatment of patients with HFrEF has affected vascular regulation during exercise. Thus, the objective of Specific Aim 2 of this dissertation was to utilize both arm and leg exercise models to provide a more definitive and comprehensive 10 assessment of the limb vasodilatory and hyperemic response during small muscle mass exercise in HFrEF patients on modern, optimized pharmacotherapy. Alpha-Adrenergic Receptor Control of Skeletal Muscle Blood Flow during Exercise in Heart Failure with Reduced Ejection Fraction In the periphery, MSNA is expressed though the stimulation of alpha-adrenergic receptors, located on the vascular smooth muscle, leading to vasoconstriction. A small number of prospective studies using acute alpha-adrenergic receptor agonist and antagonist drug administration have provided some insight concerning the expression of sympathoexcitation at the end organ in HFrEF. Zelis et al. (81) reported a qualitatively greater percent increase in forearm blood flow between HFrEF and control groups following intra-arterial infusion of phentolamine (PHEN), a nonselective alpha-adrenergic receptor antagonist. Similarly, brachial artery infusion of phenylephrine (PE) and BHT-933 (selective alpha-1 and alpha-2 agonists, respectively) in HFrEF patients and unmatched, healthy controls produced equivalent decreases in forearm blood flow, despite a substantially higher plasma NE concentration in the HFrEF patients (24). Findings from these previous studies in HFrEF are somewhat difficult to reconcile with more recent work in other populations with elevated SNS activity, where a downregulation and/or desensitization of alpha-adrenergic receptors is observed in response to chronic sympathoexcitation. For example, in the elderly, a population in which SNS activity is also elevated (10, 41, 64), Dinenno et al. (14) observed a substantially larger increase in leg blood flow to intra-arterial infusion of PHEN in healthy, older individuals compared to their younger counterparts. Our group (79) and 11 others (9, 61) have observed a blunted alpha-adrenergic receptor vasoconstriction in response to intra-arterial sympathomimetic infusions, which likely represents a protective response to the 200-300% increase in resting SNS activity and augmented endogenous contribution of the alpha-adrenergic receptors to vascular tone reported with advancing age. This lack of adaptation to high SNS activity in HFrEF likely predisposes these patients to an exaggerated cardiovascular response to acute sympathoexcitation, such as is seen during physical activity. Indeed, in healthy individuals, vascular conductance in the vessels perfusing exercising skeletal muscle is optimized in large part by exercise-induced reductions in alpha-adrenergic receptor sensitivity, a phenomenon termed "functional sympatholysis" (50). However, in HFrEF, the disease-related increase in SNS activity and sensitivity of alpha-adrenergic receptors may produce a level of vasoconstriction in the exercising muscle vasculature that cannot be entirely overcome, resulting in a sustained reduction in vascular conductance and blood flow in this cohort. Though a growing number of studies from our group (77-79) and others (11-13, 20, 28, 53, 54, 59, 70) have examined sympatholysis in healthy humans, very little is known concerning whether sympathetic vasoconstriction is altered in HF. In humans, early work performed by Zelis et al. (81) identified a substantial reduction in blood flow during rhythmic handgrip exercise in HFrEF patients, suggesting an "inadequate arteriolar vasodilation" that was partially restored when the exercise was repeated in the presence of intra-arterial infusion of PHEN. However, this response was not evident in a subsequent study using a similar pharmacologic approach during maximal upright cycling (72), and thus the evidence implicating sympathetic overactivity and subsequent 12 vasoconstriction of the exercising muscle vasculature of HFrEF patients remains equivocal. To our knowledge, no previous studies have directly tested whether sympatholysis is impaired in HFrEF patients, or determined if the observations concerning arm alpha-adrenergic receptor responses can be extended to the vasculature of large, locomotory muscle groups. Specific Aim 3 of this dissertation thus sought to close this important knowledge gap by examining the role the alpha-adrenergic receptor pathway plays in the regulation of peripheral vasomotor tone and blood flow at rest and during exercise in HFrEF patients. Hypotheses and Specific Aims The objective of this dissertation was to systematically examine the hemodynamic response to exercise in HFrEF and healthy control subjects of a similar age, and to determine the contribution of the metaboreflex and the alpha-adrenergic receptor pathway in the regulation of blood flow and vascular conductance in this cohort. Specifically, the first study determined how varying levels of metaboreceptor activation alters the ABP response in HFrEF patients, and established the degree to which CO and SVC contribute to the metaboreflex-induced pressor response. The second study utilized small muscle mass exercise in patients with HFrEF to further investigate the limb-specific peripheral vasodilatory and hyperemic response to exercise in isolation of central hemodynamic limitations. Building on these studies, the third study examined the contribution of the alpha-adrenergic receptor pathway as a mechanism responsible for the disease-related impairment in exercising muscle blood flow in HFrEF patients. 13 Specific Aim 1 Further elucidate the ABP response to varying levels of metaboreceptor activation and determine the relative contribution of CO and SVC to the metaboreflex response in HFrEF and healthy, age-matched controls. Hypothesis 1 HFrEF patients would exhibit similar increases in MAP across all levels of metaboreceptor activation compared to healthy controls. Hypothesis 2 HFrEF patients would exhibit a greater dependence on reductions in SVC than increases in CO to achieve the metaboreflex-induced pressor response compared to healthy controls. Hypothesis 3 HFrEF patients would exhibit greater increases in arterial afterload and attenuated increases in functional systolic work in response to metaboreceptor activation compared to healthy controls. Specific Aim 2 Investigate limb-specific peripheral hemodynamic responses to exercise in isolation of central hemodynamic limitations in patients with HFrEF and healthy controls. 14 Hypothesis 1 During both static-intermittent handgrip and single-leg KE exercise, patients with HFrEF would exhibit an attenuated hyperemic response driven by an impaired vasodilatory capacity compared to healthy controls. Specific Aim 3 Determine the degree to which chronically elevated SNA is expressed in the peripheral circulation, both at rest and during dynamic exercise, in patients with HFrEF and healthy, age-matched controls. Hypothesis 1 a) At rest, vasoconstriction in response to alpha-1-adrenergic receptor agonist drug infusion (PE) would reduce leg blood flow to a similar degree in HFrEF patients compared to healthy, age-matched controls. b) At rest, a greater vasodilation in response to alpha-adrenergic receptor antagonist drug infusion (PHEN) would contribute to an enhanced hyperemic response in HFrEF patients compared to healthy, age-matched controls. Hypothesis 2 a) During exercise, HFrEF patients would exhibit a sustained responsiveness to alpha-1-adrenergic receptor agonist drug infusions into the exercising limb, such that functional sympatholysis would be reduced in HFrEF patients compared to healthy, age-matched controls. b) During exercise, HFrEF patients would experience a greater vasodilation to alpha-adrenergic receptor antagonist drug infusion in the exercising limb, such that 15 sympathetic restraint of exercising limb blood flow would be greater in HFrEF patients compared to healthy, age-matched controls. 16 References 1. Amann M, Runnels S, Morgan DE, Trinity JD, Fjeldstad AS, Wray DW, Reese VR, and Richardson RS. On the contribution of group III and IV muscle afferents to the circulatory response to rhythmic exercise in humans. J Physiol 589: 3855- 3866, 2011. 2. Augustyniak RA, Collins HL, Ansorge EJ, Rossi NF, and O'Leary DS. Severe exercise alters the strength and mechanisms of the muscle metaboreflex. 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A comparison of regional blood flow and oxygen utilization during dynamic forearm exercise in normal subjects and patients with congestive heart failure. Circulation 50: 137-143, 1974. CHAPTER 2 METABORECEPTOR ACTIVATION IN HEART FAILURE WITH REDUCED EJECTION FRACTION: LINKING CENTRAL AND PERIPHERAL HEMODYNAMICS 24 Abstract The present study sought to evaluate the metaboreflex in heart failure patients with reduced ejection fraction (HFrEF), with an emphasis on the interaction between central and peripheral hemodynamics. In 23 HFrEF patients (63 ± 2 yrs) and 15 healthy, controls of a similar age (64 ± 3 yrs), we examined mean arterial pressure (MAP), cardiac output (CO), systemic vascular conductance (SVC), effective arterial elastance (Ea), and stroke work (SW) during metaboreceptor activation elicited by postexercise circulatory occlusion following static-intermittent handgrip exercise (15, 30, and 45% of maximal voluntary contraction (MVC)). Across workloads, the metaboreflex-induced increase in MAP was similar between groups. In controls, this was driven by increases in CO495 ± 155, 564 ± 156, 666 ± 217 ml/min); however, in HFrEF, this change was accomplished by reductions in SVC  ± 1.5,  ± 1.9, ± 1.8 ml/min/mmHg). This contributed to the exaggerated increases in Ea in HFrEF (0.16 ± 0.05, 0.32 ± 0.07, 0.36 ± 0.07 mmHg/ml) compared to controls (0.03 ± 0.05, 0.08 ± 0.07, 0.17 ± 0.06 mmHg/ml) which were associated with the attenuated increases in SW in this patient group (3,247 ± 1,446 mmHg*ml / mmHg/ml) compared to controls (13,853 ± 3,132 mmHg*ml /mmHg/ml). Together, these findings indicate a preserved role of the metaboreflex-induced pressor response in HFrEF, but suggest that this response is governed by the peripheral circulation in this cohort, a maladaptation that may exacerbate systolic dysfunction and contribute to exercise intolerance. 25 Introduction Heart failure with reduced ejection fraction (HFrEF) is a clinical syndrome which is commonly linked to exercise intolerance and capacity (36, 40). While there are many contributing factors to the exercise limitations in this patient population, the role of skeletal muscle maladaptations has been increasingly recognized, with specific interest focused on the muscle metaboreflex. Activation of this reflex pathway is mediated by metabolically sensitive group IV afferent fibers (metaboreceptors) originating in skeletal muscle, which increase efferent sympathetic nervous system (SNS) activity in an effort to augment the perfusion of the exercising skeletal muscle through increases in arterial blood pressure (ABP) (2, 9, 25). Whether the metaboreflex is altered in HFrEF patients remains a topic of ongoing debate (22, 28), with evidence for both exaggerated (6, 20, 23, 34) and similar (26-28, 32, 33) reflex increases in mean arterial blood pressure (MAP) during metaboreflex activation. The disparate findings from these studies suggest that significant uncertainty remains regarding disease-related changes in the muscle metaboreflex in HFrEF patients, as well as the contribution of this reflex in the cardiovascular response to exercise. Beyond the simple determination of the pressor response, further insight into the importance of metaboreceptor activation in patients with HFrEF may be gained by considering the relative contributions of changes in central (i.e. cardiac output (CO)) and peripheral (i.e. systemic vascular conductance (SVC)) hemodynamics to the increase in MAP in this patient group. Interestingly, in the animal model of heart failure (HF), the contributions of these factors to the overall metaboreflex-induced pressor response have been documented to be solely due to reductions in SVC across exercise intensities (13). 26 This is vastly different than the response observed in healthy animals, where the metaboreflex-induced increases in MAP were predominantly due to increases in CO at low to moderate intensities (25) and a shift towards a reliance on SVC to increase MAP only during high intensity exercise, when the ability to increase CO was compromised (4). In humans, only one study to date has examined central and peripheral contributions to the metaboreflex in HFrEF. Crisafulli et al. (9) reported a metaboreflex-induced increase in MAP which was predominantly driven by an increase in CO in healthy individuals and by a reduction in SVC in patients with HFrEF, suggesting a greater role of the peripheral vasculature in governing the pressor response. However, this study only included one level of metaboreceptor activation, leaving uncertainty regarding the graded nature of the response that has been demonstrated in an animal model of HF (13). Whether the metaboreflex-mediated increase in MAP is achieved by central or peripheral mechanisms may be of particular significance in HFrEF patients due to the potential of this reflex to further stress cardiac muscle through substantial increases in afterload. Indeed, SVC, a measure of systemic vascular tone, represents the nonpulsatile component of arterial afterload (16, 41), and considering that patients with HFrEF are known to be afterload-sensitive (3, 15, 31), this group can experience severe impairments in left ventricular systolic function when arterial afterload is increased (15). Thus, while the metaboreflex response is typically viewed as an effective way to increase perfusion of the exercising muscle via increases in perfusion pressure in healthy individuals, this reflex may exacerbate existing ventricular dysfunction in HFrEF patients if a marked reduction in SVC is elicited upon metaboreceptor activation. 27 Thus, the purpose of this study was to implement the use of postexercise circulatory occlusion (PECO) following static-intermittent handgrip exercise across a range of exercise intensities to comprehensively investigate the interaction between central and peripheral responses to metaboreceptor activation. We hypothesized that: 1) HFrEF patients would exhibit similar increases in MAP across all levels of metaboreceptor activation compared to healthy controls, 2) HFrEF patients would exhibit a greater dependence on reductions in SVC than increases in CO to achieve the metaboreflex-induced pressor response, and 3) HFrEF patients would exhibit a greater increase in arterial afterload and an attenuated increase in functional systolic work in response to metaboreceptor activation compared to healthy controls. Methods Subjects 23 New York Heart Association (NYHA) class II-III HFrEF patients (22 males and 1 female) and 15 healthy, control subjects (14 males and 1 female) of a similar age were recruited either by word of mouth or in the HF clinics at the University of Utah Health Sciences Center and the Salt Lake City VA Medical Center. All control subjects were nonsmokers, not taking any prescription medication, and were free of overt cardiovascular disease, as indicated by a health history questionnaire. Protocol approval and written informed consent were obtained according to University of Utah and Salt Lake City Veterans Affairs Medical Center Institutional Review Board requirements. All data collection took place at the Utah Vascular Research Laboratory located at the Veterans Affairs Salt Lake City Geriatric, Research, Education, and Clinical Center. All 28 studies were performed in a thermoneutral environment, with subjects reporting to the laboratory fasted, and not having performed any exercise within 24 hours of the study. Handgrip Exercise and Metaboreceptor Activation Subjects were instrumented with a Finometer (Finapres Medical Systems, Amsterdam, the Netherlands) on the nonexercising arm, a 3-lead ECG (Biopac, Goleta, CA, U.S.A.) to measure heart rate, and a pneumatic blood pressure cuff distal to the antecubital fossa on the exercising arm to isolate the metaboreflex following exercise. Subjects then rested supine for ≈20 minutes. First, baseline measurements were taken over the course of 1-minute. Second, maximal voluntary contraction (MVC) was established by taking the highest value recorded of three maximal contractions using a handgrip dynamometer (TSD121C, Biopac Systems, Goleta, CA). Static-intermittent handgrip exercise was performed at three intensities based on each subject's respective MVC (15, 30, and 45% of MVC). The subjects squeezed the dynamometer to the sound of a metronome (60 beats/min) and real-time force output was displayed on a computer monitor so that subjects could monitor their effort and make corrections when necessary. Each bout of handgrip exercise lasted 3 minutes, and was followed by 2 minutes of forearm ischemia to isolate the metaboreflex, with measurements taken during the final minute. Forearm ischemia was achieved through the inflation of the pneumatic blood pressure cuff on the exercising arm to suprasystolic pressures (>250 mmHg) 5 seconds before the end of the handgrip exercise. A 5-minute recovery period was given after each period of metaboreceptor activation to allow cardiovascular variables to return to resting values. If cardiovascular variables did not return to resting values after 5 minutes, additional rest was given. 29 Measurements Hemodynamic variables Stroke volume (SV), heart rate (HR), CO, and ABP were determined noninvasively (Finapres Medical Systems BV, Amsterdam, The Netherlands). SV was calculated using the Modelflow method which includes age, sex, height, and weight in its algorithm (Beatscope version 1.1; Finapres Medical Systems BV, Amsterdam, The Netherlands) (5), and has been shown to accurately track SV during a variety of experimental protocols including exercise (10, 11, 35, 38). Pulse pressure (PP), a measure of pulsatile load, the nonresistive oscillatory component of arterial afterload (7, 18), was calculated as: PP (mmHg) = systolic arterial pressure (SAP) - diastolic arterial pressure (DAP) Total arterial compliance (TAC), an index of pulsatile arterial afterload which takes into account the effect of SV (7, 29), was calculated as: TAC (ml/mmHg) = SV/PP MAP was calculated as: MAP (mmHg) = DAP + (PP*0.33) End systolic arterial pressure (Pes) was calculated as (18): Pes = 0.9*SAP. 30 CO was calculated as: CO (L/min) = SV*HR SVC, a measure of systemic vascular tone and the nonpulsatile (mean resistive) component of arterial afterload (16), was calculated as: SVC (ml/min/mmHg) = CO/MAP Systemic vascular resistance (SVR) was calculated as: SVR (mmHg/L/min) = MAP/CO Effective arterial elastance (Ea), an index of total arterial afterload (both pulsatile and nonpulsatile arterial afterload) (18, 29), was calculated as: Ea (mmHg/ml) = Pes/SV Stroke work (SW), a measure of functional systolic work performed by the left ventricle (16, 37), was calculated as: SW (mmHg*ml) = Pes*SV Rate pressure product (RPP), an index of myocardial oxygen consumption (19), was calculated as: RPP (AU) = SAP*HR 31 Near infrared spectroscopy To determine muscle microvascular deoxyhemoglobin (12) of one specific muscle group during exercise and metaboreceptor activation, in a subset of subjects (HFrEF = 13; control = 9), near infrared spectroscopy (NIRS) was employed on the belly of the brachioradialis and the flexor carpi radialis. A frequency-domain multidistance NIRS system was utilized (Oxiplex TS, ISS, Champaign, IL) that allows the absolute quantification of deoxyhemoglobin concentrations, expressed in μM (14). Prior to use, the probe was calibrated using a block with known absorption characteristics to calculate the absorption and scattering coefficients. Prior to placement, the skin covering the brachioradialis and the flexor carpi radialis was cleaned and double-sided adhesive tape was used to seat the diode, which was covered and further secured with coban (3M, St. Paul, MN). The data were acquired at 0.5 Hz, and 1-minute averages were calculated during the last minute of each exercise bout and during the final minute of PECO. Data Analysis Statistics were performed using commercially available software (SigmaStat 3.10; Systat Software, Point Richmond, CA). For both the exercise and metaboreceptor activation portion of the protocol, 2x4 repeated measures ANOVA (α < 0.05) (group: 2 levels; controls vs. HFrEF) (workload or metaboreflex activation: 4 levels; rest, 15, 30, and 45% of MVC) were utilized to determine the exercise and metaboreflex-induced alterations in hemodynamic measurements. The Holm-Sidak method was used for alpha adjustment and post hoc analysis. Linear regression analysis was performed on individual data, with the slope and y-intercept determined to evaluate the associations between the metaboreflex-induced changes in SW with Ea and RPP. Student t-tests were used to 32 compare the effect of metaboreflex-induced changes on slope and y-intercept values. All group data are expressed as means ± SEM. Results Subject Characteristics Baseline characteristics of the control subjects and HFrEF patients are displayed in Table 2.1. Disease-specific characteristics and medications of patients with HFrEF are presented in Table 2.2. Rest and Exercise Hemodynamics At rest, there were no significant differences in deoxyhemoglobin, MAP, CO, or SVC in HFrEF patients compared to controls (Table 2.3). Exercise elicited similar intensity-dependent increases in deoxyhemoglobin and MAP between groups (Table 2.3). The changes in MAP were solely driven by increases in CO across workloads in control subjects (Table 2.3) and reductions in SVC in HFrEF patients (Table 2.3). This was complemented by substantially attenuated increases in SAP and exaggerated increases in DAP in HFrEF compared to control subjects (Table 2.3). These differences resulted in a lower PP across exercise intensities in HFrEF, compared to controls (Table 2.3). However, when factoring in the differences in SV on PP as expressed by TAC, there were significant intensity-dependent reductions in TAC observed in control subjects and only a significant reduction at the high workload in HFrEF. Additionally, there were no significant differences in TAC between groups at any workload (Table 2.3). Ea was significantly increased across all workloads in both groups; however, the increases were significantly greater in HFrEF patients compared to controls at the two highest workloads 33 (Table 2.3). SW increased significantly across all workloads in the control subjects, with no significant difference from rest demonstrated by the HFrEF patients (Table 2.3). This was in light of similar RPP's across all workloads in both groups (Table 2.3). Metaboreflex-Induced Changes in Hemodynamics Metaboreceptor activation provoked similar increases in tissue deoxyhemoglobin (Table 2.4) and MAP (Figure 2.1, top and Table 2.4) across increasing levels of metaboreceptor activation between groups. However, the metaboreflex-induced increases in MAP were due exclusively to increases in CO in the control group (Figure 2.1, middle and Table 2.4), and reductions in SVC in HFrEF (Figure 2.1, bottom and Table 2.4). Similar to exercise, HFrEF patients exhibited a blunted increase in SAP across increasing levels of metaboreceptor activation and exaggerated increases in DAP compared to controls (Table 2.4). This led to a significantly attenuated increase PP in the HFrEF patients, who only established an increase in PP at the highest level of metaboreceptor activation (Figure 2.2, top and Table 2.4). However, when factoring in the significantly greater increases in SV induced by the metaboreflex exhibited by the control subjects compared to HFrEF patients (expressed as TAC), TAC was not significantly different between groups at any level of metaboreceptor activation (Figure 2.2, bottom and Table 2.4). Metaboreceptor activation provoked minimal increases in Ea in the control group, who only exhibited a significant increase at the highest level (Figure 2.3 and Table 2.4). In contrast, HFrEF patients displayed a significant increase in Ea across all levels of metaboreceptor activation and were significantly different from control subjects at the highest two levels (Figure 2.3 and Table 2.4). SW was significantly increased by metaboreceptor activation at every level of activation in the 34 control group, and only at the highest level in HFrEF patients (Figure 2.4, top and Table 2.4). This contributed to the significantly lower SW across all levels of metaborceptor activation in HFrEF compared to controls (Figure 2.4, top and Table 2.4). These differences in SW between groups were due to significantly blunted changes in Pes (Figure 2.4, middle) and SV (Figure 2.4, bottom and Table 2.4), induced by metaboreceptor activation in HFrEF patients compared to the control group. Across all levels of metaboreceptor activation, similar increases in RPP were observed between both groups (Table 2.4). The slope and the y-intercept of the relationships between metaboreflex-induced changes in SW and Ea were significantly greater in the control group compared to HFrEF (Figure 2.5). This demonstrates that for a given metaboreflex-induced change in Ea, there was less of a metaboreflex-induced increase in SW in HFrEF compared to the control group. The slope of the relationship between the metaboreflex-induced changes in SW and RPP were not significantly different between groups (Figure 2.6). However, the y-intercept was less in the HFrEF group compared to controls (Figure 2.6), representing a downward shift in the relationship. This indicates that for a given metboreflex-induced change in RPP, there was less of a metaboreflex-induced increase in SW in HFrEF compared to the control group. Discussion The present study sought to comprehensively evaluate the muscle metaboreflex in HFrEF patients and healthy control subjects of a similar age, with an emphasis on investigating the central and peripheral hemodynamic contributions to the metaboreflex-induced pressor response. Across multiple levels of metaboreceptor activation, the increase in MAP was similar between groups, providing new evidence against a disease- 35 related exaggeration of the muscle metaboreflex-induced pressor response in HFrEF. However, a discrete pattern of central and peripheral hemodynamic changes was observed between groups. In control subjects, the pressor response induced by metaboreceptor activation was driven by increases in CO, with no significant changes in SVC. In contrast, progressively greater reductions in SVC contributed to the pressor response in HFrEF patients, while CO remained unchanged. This metaboreflex-induced reduction in SVC in HFrEF patients contributed to the reduction in Ea, which was associated with a blunted increase in the metaboreflex-induced changes in SW. Additionally, a downward shift in the relationship between the metaboreflex-induced changes in SW and RPP was observed in HFrE patients compared to control subjects, indicating a likely reduction in myocardial efficiency during metaboreceptor activation. Together, these findings indicate a preserved role of the muscle metaboreflex-induced pressor response in HFrEF. However, the shift to increases in peripheral vasoconstriction driving this response in HFrEF patients represents a maladaptive process which places a substantial hemodynamic load on the heart, potentially exacerbating the underlying impairment in systolic function and myocardial efficiency, and thereby contributing to the exercise limitations present in this patient group. Metaboreflex Contribution to the Exercise-Induced Changes in Mean Arterial Pressure It is well-established that patients with HFrEF suffer from a nearly insurmountable intolerance to physical exertion (36, 40), which may be due, at least in part, to maladaptations in skeletal muscle. Indeed, Drs. Coats and Piepoli have hypothesized that abnormalities in sensory reflex activity in skeletal muscle may 36 contribute to the exercise limitations in HFrEF (8, 27), the so-called "muscle hypothesis" of HF. Located within the skeletal muscle are two distinct sensory afferent fiber types; group III afferent fibers which are predominately mechanically sensitive (mechanoreceptors) and group IV afferent fibers (metaboreceptors) which are principally sensitive to metabolites produced during exercise (17). Collectively, these reflex pathways serve to increase SNS activity, which ultimately increase perfusion pressure (2, 9, 25). In HF patients, some aspect of this reflex response appears to be dysfunctional. While difficult to completely isolate these respective reflex pathways, PECO has become a widely adopted approach whereby metabolic byproducts produced during exercise are trapped distal to the point of occlusion, thereby activating group IV afferent fibers with minimal input from group III fibers (1). Despite extensive use of this experimental technique over the past 80 years in both healthy humans and patient populations, the exact role of metaboreceptor activation in the cardiovascular response to exercise in HFrEF remains a topic of ongoing debate. Indeed, studies which have investigated the pressor response during PECO have documented both exaggerated (27, 32) and similar (6, 20, 23, 34) increases in MAP in HFrEF patients compared to healthy individuals. The conflicting findings from these studies indicate that significant uncertainty remains as to whether the metaboreflex-induced pressor response is altered in HFrEF. In the present study, we employed the PECO technique following three different handgrip exercise intensities in an effort to comprehensively evaluate the muscle metaboreflex in HFrEF patients compared to healthy control subjects of a similar age. As displayed in Figure 2.1 (top), we observed a metaboreflex-induced pressor response that 37 was almost identical between groups across all levels of metaboreceptor activation. These results are in disagreement with some of the earliest work on the topic (27, 32), and may be explained by differences in experimental protocols, including differing handgrip exercise paradigms and methods of activating the muscle metaboreflex (PECO vs. limb positive pressure). To our knowledge, this is the first study to perform PECO following multiple intensities of static-intermittent handgrip exercise, providing a systematic assessment of the pressor response across multiple levels of metaboreceptor activation. The present findings thus extend on the observations from previous work (6, 20, 23, 34), providing new evidence against a disease-related exaggeration of the pressor response induced by the muscle metaboreflex in HFrEF patients. Central and Peripheral Hemodynamic Contributions to Metaboreflex-Induced Changes in Mean Arterial Pressure While a large breadth of research has focused on elucidating the strength of the metaboreflex-induced pressor response in HFrEF, limited work has been undertaken to examine variables contributing to this rise in MAP. In a healthy animal model, O'Leary et al. (25) documented that the rise in MAP triggered by metaboreceptor activation was solely due to increases in CO during low to moderate exercise intensities. Subsequent work from the same group reported that the metaboreflex-induced increases in MAP in an animal model of HF were primarily due to reductions in SVC (13), indicating that the pressor response was achieved exclusively via peripheral vasoconstriction. Based on these findings, the authors concluded that the inability of the metaboreflex to increase CO 38 in HF is detrimental as a reduction in SVC is the sole variable this reflex pathway can alter in order to increase ABP and ultimately blood flow to exercising skeletal muscle. The present study extends these previous findings in animals to human HF, documenting metaboreflex-induced increases in MAP in patients with HFrEF (Figure 2.1, top) that were primarily accomplished through reductions in SVC (Figure 2.1, bottom), with virtually no changes in CO (Figure 2.1, middle). This was in marked contrast to the response observed in healthy control subjects, where increases in CO played a dominant role in increasing MAP during metaboreceptor activation (Figure 2.1, middle). To our knowledge, only one other study in humans has examined the roles of CO and SVC in increasing MAP during metaboreceptor activation in patients with HFrEF. Crisafulli et al. (9) reported a metaboreflex-induced increase in MAP which was predominantly driven by an increase in CO in healthy individuals and by a reduction in SVC in patients with HFrEF, suggesting a greater role of the peripheral vasculature in governing the metaboreflex-induced pressor response in the patient group. However, this previous study only investigated the hemodynamic alterations induced by one level of metaboreceptor activation, which somewhat limits interpretation. The importance of examining multiple levels of metaboreceptor activation should not be underestimated; in the animal model, both CO and SVC responses to metaboreceptor activation differed significantly with increasing exercise intensity (4, 25). The present study thus provides the first comprehensive investigation into the role of CO and SVC in the metaboreflex-induced pressor response in HFrEF in humans, identifying an intensity-dependent reduction in SVC during metaboreflex activation in HFrEF, and thus indicating a proportionally greater role of SVC in increasing MAP in this patient group. 39 Arterial Afterload and Systolic Function The manner by which metaboreceptor activation elicits an increase in MAP may be particularly significant when considering the relationship between the heart and the peripheral vasculature in HFrEF patients. Indeed, SVC represents the nonpulsatile component of arterial afterload (16, 41) and it is well-established that patients with HFrEF are afterload-sensitive (3, 15, 31) and face certain impairment in left ventricular systolic function if arterial afterload is increased (15). In the present study, at all levels of metaboreceptor activation, HFrEF patients exhibited an exaggerated increase in Ea, an index of total arterial afterload, compared to control subjects who only exhibited a significant augmentation in Ea at the highest level of metaboreceptor activation (Figure 2.3). Due to the lack of differences between groups in the reduction in TAC, a measure of the pulsatile component of arterial afterload (Figure 2.2, bottom), SVC likely is the primary contributor to the exaggerated increase in Ea and total arterial afterload induced by metaboreceptor activation exhibited in HFrEF. To our knowledge, this is the first study to definitively document an augmented arterial afterload induced by the metaboreflex-driven changes in SVC in HFrEF. This metaboreflex-induced increase in arterial afterload in HFrEF appears to have deleterious cardiac effects. In HFrEF patients, metaboreceptor activation provoked much smaller increases in SW (a measure of functional systolic work) compared to healthy control subjects (Figure 2.4, top), and it appears that arterial afterload may have contributed significantly to this response. Indeed, the attenuated slope and the downward shift in the relationship between metaboreflex-induced changes in SW, with the reflex changes in Ea (Figure 2.5), indicates a marked role of arterial afterload as a primary 40 restraining factor to the ability of the metaboreflex to increase CO and ultimately perfusion pressure in HFrEF. Compounded with these impairments in systolic function, HFrEF patients exhibited a reduction in myocardial efficiency, as estimated by the downward shift in the relationship between SW and RPP in HFrEF compared to control subjects (Figure 2.6). Taken together, these cardiac indices suggest that the metaboreflex-induced reductions in SVC and associated increases in afterload come at a steep cost to HFrEF patients, comprised of both reductions in systolic work and myocardial efficiency. Experimental Considerations The PECO technique is based on the principle that metabolites produced by the exercising skeletal muscle become trapped during circulatory occlusion, maintaining activation of the muscle metaboreflex. Though this well-established experimental approach provides a reproducible, intensity-dependent pressor response (Figure 2.1), we recognize the possibility that the metabolic milieu in the occluded tissue may differ between groups. However, this concern is somewhat mitigated by the inclusion of NIRS measurements of the occluded muscle, which indicated a similar level of muscle ischemia (as quantified by changes in deoxyhemoglobin) between HFrEF and control groups during all levels of metaboreceptor activation (Table 2.4). Additionally, we acknowledge that the arterial afterload calculations used in the current study are typically based on ABP measurements taken centrally, at the aorta (18). Due to the invasive nature and feasibility of collecting central aortic pressures in a large cohort of subjects, in the current study, ABP measurements were obtained noninvasively via finger photoplethysmography. Peripheral ABP measurements are often thought to not 41 completely describe central ABP due to the documented ABP wave amplification descending the arterial tree (30, 39). 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New York: Springer-Verlag, 1987, p. 406 p. 46 Figure 2.1 Metaboreflex-induced changes in mean arterial pressure (top), cardiac output (middle), and systemic vascular conductance (bottom) in control subjects and heart failure patients with reduced ejection fraction (HFrEF). * Significant difference from control, P<0.05; † Significant difference from rest, P<0.05. 47 Metaboreflex-Induced Change in Mean Arterial Pressure (mmHg) 0 5 10 15 20 25 † † † † † † Control HFrEF Metaboreceptor Activation (%MVC) 15% 30% 45% Metaboreflex-Induced Change in Systemic Vascular Conductance (ml/min/mmHg) -20 -15 -10 -5 0 5 10 *† *† *† Metaboreflex-Induced Change in Cardiac Output (ml/min) -600 -400 -200 0 200 400 600 800 1000 1200 † * † * † * 48 Figure 2.2 Metaboreflex-induced changes in pulse pressure (top) and total arterial compliance (bottom) in control subjects and heart failure patients with reduced ejection fraction (HFrEF). * Significant difference from control, P<0.05; † Significant difference from rest, P<0.05. 49 Metaboreflex-Induced Change in Pulse Pressure (mmHg) 0 5 10 15 20 25 30 † * † * † *† Control HFrEF Metaboreceptor Activation (%MVC) 15% 30% 45% Metaboreflex-Induced Change in Total Arterial Compliance (ml/mmHg) -0.9 -0.6 -0.3 0.0 † † † 50 Figure 2.3 Metaboreflex-induced changes in effective arterial elastance in control subjects and heart failure patients with reduced ejection fraction (HFrEF). * Significant difference from control, P<0.05; † Significant difference from rest, P<0.05. 51 Metaboreceptor Activation (%MVC) 15% 30% 45% Metaboreflex-Induced Change in Effective Arterial Elastance (mmHg/ml) 0.0 0.3 0.6 † *† † *† Control HFrEF 52 Figure 2.4 Metaboreflex-induced changes in stroke work (top), end systolic pressure (middle), and stroke volume (bottom) in control subjects and heart failure patients with reduced ejection fraction (HFrEF). * Significant difference from control, P<0.05; † Significant difference from rest, P<0.05. 53 Metaboreflex-Induced Change in Stroke Work (mmHg*ml) 0 1000 2000 3000 4000 5000 6000 † * † * † *† Control HFrEF Metaboreflex-Induced Change in End Systolic Arterial Pressure (mmHg) 0 10 20 30 40 † † † *† † *† Metaboreceptor Activation (%MVC) 15% 30% 45% Metaboreflex-Induced Change in Stroke Volume (ml/beat) -15 -10 -5 0 5 10 15 20 25 † * † * † * 54 Figure 2.5 Relationship between metaboreflex-induced changes in stroke work and changes in effective arterial elastance in control subjects and heart failure patients with reduced ejection fraction (HFrEF). * Significant difference from control, P<0.05. 55 Metaboreflex-Induced Change in Effective Arterial Elastance (mmHg/ml) -0.1 0.0 0.1 0.2 0.3 0.4 0.5 Metaboreflex-Induced Change in Stroke Work (mmHg*ml) 0 2000 4000 6000 Control: m = 13853 ± 3132 c = 1885 ± 463 HFrEF m = 3247 ± 1446* c = -254 ± 252* 56 Figure 2.6 Relationship between metaboreflex-induced changes in stroke work and changes in rate pressure product in control subjects and heart failure patients with reduced ejection fraction (HFrEF). * Significant difference from control, P<0.05. 57 Metaboreflex-Induced Change in Rate Pressure Product (AU) 0 500 1000 1500 2000 Metaboreflex-Induced Change in Stroke Work (mmHg*ml) -1000 0 1000 2000 3000 4000 5000 6000 Control: m = 1.97 ± 0.45 c = 505 ± 460 HFrEF m = 1.12 ± 0.43 c = -839 ± 542* 58 Table 2.1 Subject characteristics Control (n = 15) HFrEF (n = 23 ) Age, yrs 64 ± 3 63 ± 2 Height, cm 176 ± 2 175 ± 1 Weight, kg 80 ± 4 85 ± 4 Body mass index, kg/m2 26 ± 1 28 ± 1 Maximum voluntary contraction, kg 27 ± 2 25 ± 2 Glucose, mg/dl 85 ± 6 99 ± 4 Total cholesterol, mg/dl 192 ± 14 155 ± 11 Triglycerides, mg/dl 143 ± 28 131 ± 12 HDL, mg/dl 49 ± 4 39 ± 2* LDL, mg/dl 124 ± 11 96 ± 8 HFrEF, heart failure with reduced ejection fraction; HDL, high density lipoprotein; LDL, low density lipoprotein. Data are expressed as means ± SEM. * Significant difference from control, P <0.05. 59 Table 2.2 Disease - specific characteristics and medications HFrEF (n = 23) Disease-specific characteristics Left ventricular ejection fraction, % (means ± SEM) 22 ± 3 Diagnosis (ischemic) 14 / 23 Diagnosis (nonischemic) 9 / 23 NYHA class II 16 / 23 NYHA class III 7 / 23 Diabetic 4 / 23 Medications -Blocker 23 / 23 ACE inhibitor 17 / 23 Angiotensin receptor inhibitor 4 / 23 Statin 18 / 23 Diuretic 18 / 23 Aldosterone inhibitor 4 / 23 Calcium channel inhibitor 1 / 23 Digoxin 4 / 23 Anticoagulant 13 / 23 Antiarrhythmic 1 / 23 Erythropoiesis - stimulating agent 1 / 23 HFrEF, heart failure with reduced ejection fraction; NYHA, New York Heart Association; ACE, angiotensin-converting enzyme. 60 Table 2.3 Central and peripheral hemodynamics at rest and during exercise Workload (%MVC) Rest 15% 30% 45% Control Mean arterial pressure, mmHg 83 ± 2 92 ± 2† 96 ± 3† 103 ± 2† Systolic arterial pressure, mmHg 119 ± 3 140 ± 4† 143 ± 5† 154 ± 3 Diastolic arterial pressure, mmHg 66 ± 2 68 ± 2 73 ± 2† 77 ± 2† Pulse pressure, mmHg 53 ± 3 71 ± 4† 70 ± 4† 77 ± 3† Heart rate, beats/min 57 ± 2 64 ± 2† 65 ± 2† 68 ± 3† Stroke volume, ml/beat 109 ± 5 110 ± 5 111 ± 4 110 ± 4 Cardiac output, L/min 6.3 ± 0.3 7.0 ± 0.4† 7.2 ± 0.4† 7.4 ± 0.4† Systemic vascular conductance, ml/min/mmHg 75 ± 4 77 ± 5 75 ± 4 72 ± 4 Systemic vascular resistance, mmHg/L/min 14 ± 1 14 ± 1 14 ± 1 14 ± 1 Total arterial compliance, ml/mmHg 2.2 ± 0.2 1.6 ± 0.1† 1.7 ± 0.2† 1.5 ± 0.1† Effective arterial elastance, mmHg/ml 1.0 ± 0.0 1.2 ± 0.0† 1.2 ± 0.1† 1.3 ± 0.1† Stroke work, mmHg*ml 11,700 ± 592 13,920 ± 813† 14,357 ± 837† 15,308 ± 683† Rate pressure product, AU 6,821 ± 254 8,924 ± 339† 9,354 ± 513† 10,417 ± 440† Deoxyhemoglobin, μM (n=9) 27 ± 3 34 ± 4† 37 ± 6† 39 ± 5† HFrEF Mean arterial pressure, mmHg 84 ± 3 93 ± 3† 95 ± 4† 102 ± 3† Systolic arterial pressure, mmHg 117 ± 4 128 ± 5† 131 ± 5† 141 ± 5*† Diastolic arterial pressure, mmHg 67 ± 3 75 ± 3† 77 ± 3 † 83 ± 3† Pulse pressure, mmHg 50 ± 3 53 ± 3* 53 ± 4* 58 ± 4*† Heart rate, beats/min 67 ± 2* 69 ± 3† 71 ± 2† 73 ± 3† Stroke volume, ml/beat 83 ± 4* 81 ± 4* 79 ± 4* 76 ± 4* Cardiac output, L/min 5.4 ± 0.3 5.5 ± 0.2* 5.6 ± 0.3* 5.4 ± 0.3* Systemic vascular conductance, ml/min/mmHg 68 ± 4 62 ± 3*† 61 ± 4*† 55 ± 3*† Systemic vascular resistance, mmHg/L/min 17 ± 2 18 ± 2† 20 ± 3*† 21 ± 2*† Total arterial compliance, ml/mmHg 1.8 ± 0.1 1.6 ± 0.1 1.6 ± 0.1 1.4 ± 0.1† Effective arterial elastance, mmHg/ml 1.4 ± 0.1 1.6 ± 0.1† 1.6 ± 0.1*† 1.8 ± 0.2*† Stroke work, mmHg*ml 8,654 ± 464 9,253 ± 469* 9,192 ± 529* 9,562 ± 646* Rate pressure product, AU 7,677 ± 310 8,902 ± 450† 9,215 ± 455† 10,322 ± 578† Deoxyhemoglobin, μM (n=13) 28 ± 2 32 ± 2† 35 ± 2† 36 ± 3† MVC, maximum voluntary contraction; HFrEF, heart failure with reduced ejection fraction. Data are expressed as means ± SEM. * Significant difference from control, P<0.05; † Significant difference from rest, P<0.05. 61 Table 2.4 Central and peripheral hemodynamics at rest and during metaboreceptor activation Metaboreceptor activation Rest 15% 30% 45% Control Mean arterial pressure, mmHg 83 ± 2 90 ± 3† 93 ± 3† 100 ± 2† Systolic arterial pressure, mmHg 119 ± 3 132 ± 5† 139 ± 5† 150 ± 5† Systolic arterial pressure, mmHg - 13 ± 3† 19 ± 3† 34 ± 4† Diastolic arterial pressure, mmHg 66 ± 2 69 ± 2† 70 ± 2† 74 ± 1† Diastolic arterial pressure, mmHg - 4 ± 1† 4 ± 1† 8 ± 1† Pulse pressure, mmHg 53 ± 3 62 ± 3† 68 ± 4† 75 ± 5† Heart rate, beats/min 57 ± 2 58 ± 2 59 ± 2 58 ± 2 Stroke volume, ml/beat 109 ± 5 118 ± 5† 118 ± 5† 118 ± 5† Cardiac output, L/min 6.3 ± 0.3 6.8 ± 0.3† 6.9 ± 0.3† 6.8 ± 0.3† Systemic vascular conductance, ml/min/mmHg 75 ± 4 76 ± 3 75 ± 3 68 ± 3† Systemic vascular resistance, mmHg/L/min 14 ± 1 14 ± 1 14 ± 1 15 ± 1 Total arterial compliance, ml/mmHg 2.2 ± 0.2 2.0 ± 0.1 1.8 ± 0.2† 1.7 ± 0.2† Effective arterial elastance, mmHg/ml 1.0 ± 0.0 1.0 ± 0.1 1.1 ± 0.1 1.2 ± 0.1† Stroke work, mmHg*ml 11,700 ± 592 13,921 ± 695† 14,799 ± 896† 15,941 ± 1,008† Rate pressure product, AU 6,821 ± 254 7,659 ± 427† 8,217 ± 476† 8,680 ± 325† Deoxyhemoglobin, μM (n=9) 27 ± 3 48 ± 6† 50 ± 6† 51 ± 6† HFrEF Mean arterial pressure, mmHg 84 ± 3 90 ± 3† 94 ± 3† 100 ± 4† Systolic arterial pressure, mmHg 117 ± 4 125 ± 5† 129 ± 5† 140 ± 6† Systolic arterial pressure, mmHg - 8 ± 2† 12 ± 2*† 23 ± 3*† Diastolic arterial pressure, mmHg 67 ± 3 73 ± 3† 77 ± 3† 81 ± 3† Diastolic arterial pressure, mmHg - 7 ± 1† 9 ± 1*† 13 ± 1*† Pulse pressure, mmHg 50 ± 3 52 ± 4 53 ± 3* 59 ± 4*† Heart rate, beats/min 67 ± 2* 68 ± 2* 70 ± 2* 68 ± 2* Stroke volume, ml/beat 83 ± 4* 81 ± 4* 77 ± 4* 80 ± 4* Cardiac output, L/min 5.4 ± 0.3 5.5 ± 0.2* 5.3 ± 0.3* 5.4 ± 0.3* Systemic vascular conductance, ml/min/mmHg 68 ± 4 63 ± 4*† 58 ± 4*† 55 ± 4*† Systemic vascular resistance, mmHg/L/min 17 ± 2 18 ± 2† 20 ± 2*† 21 ± 3*† Total arterial compliance, ml/mmHg 1.8 ± 0.1 1.7 ± 0.1 1.6 ± 0.1 1.5 ± 0.1† Effective arterial elastance, mmHg/ml 1.4 ± 0.1 1.6 ± 0.2*† 1.7 ± 0.2*† 1.7 ± 0.2*† Stroke work, mmHg*ml 8,654 ± 464 9,021 ± 472* 8,896 ± 530* 9,982 ± 635*† Rate pressure product 7,677 ± 310 8,572 ± 469† 8,990 ± 426† 9,445 ± 477† Deoxyhemoglobin, μM (n=13) 28 ± 2 47 ± 4† 50 ± 4† 53 ± 4† MVC, maximum voluntary contraction; HFrEF, heart failure with reduced ejection fraction. Data are expressed as means ± SEM. * Significant difference from control, P<0.05; † Significant difference from rest, P<0.05. CHAPTER 3 HEMODYNAMIC RESPONSES TO SMALL MUSCLE MASS EXERCISE IN HEART FAILURE WITH REDUCED EJECTION FRACTION Reproduced with permission from Barrett-O'Keefe, Z., Lee, J.F., Berbert, A.,Witman, M.A.H, Nativi-Nicolau, J., Stelhlik, J., Richardson, R.S., and Wray, D.W. (2014). Hemodynamic responses to small muscle mass exercise in heart failure with reduced ejection fraction. Articles in Press. Am J Physiol Heart Circ Physiol (September 26, 2014). doi:10.1152/ajpheart.00527.2014. Copyright © 2014 by the American Physiological Society. 63 Abstract To better understand the mechanisms responsible for exercise intolerance in heart failure with reduced ejection fraction (HFrEF), the present study sought to evaluate the hemodynamic responses to small muscle mass exercise in this cohort. In 25 HFrEF patients (64 ± 2 yrs) and 17 healthy control subjects of a similar age (64 ± 2 yrs), mean arterial pressure (MAP), cardiac output (CO), and limb blood flow were examined during graded static-intermittent handgrip (HG) and dynamic single-leg knee-extensor (KE) exercise. During HG exercise, MAP increased similarly between groups. CO increased significantly (+1.3 ± 0.3 L/min) in the control group, but remained unchanged across workloads in HFrEF patients. At 15% maximum voluntary contraction (MVC), forearm blood flow was similar between groups, while HFrEF patients exhibited an attenuated increase at the two highest intensities compared to controls, with the greatest difference at the highest workload (352 ± 22 vs. 492 ± 48 ml/min, HFrEF vs. control, 45% MVC). During KE exercise, MAP and CO increased similarly across work rates between groups. However, HFrEF patients exhibited a diminished leg hyperemic response across all work rates, with the most substantial decrement at the highest intensity (1842 ± 64 vs. 2675 ± 81 ml/min, HFrEF vs. control, 15 W). Together, these findings indicate a marked attenuation in exercising limb perfusion attributable to impairments in peripheral vasodilatory capacity during both arm and leg exercise in patients with HFrEF, which likely plays a role in limiting exercise capacity in this patient population. 64 Introduction Heart failure with reduced ejection fraction (HFrEF) is associated with debilitating dyspnea and fatigue triggered by exercise, leading to a limited ability to perform everyday tasks and an impaired quality of life (52). Although central cardiac limitations are the paramount characteristic of HFrEF, impaired cardiac function does not fully explain the degree of exercise intolerance and symptom status in this patient population (8, 21, 34, 53, 60). This has steered studies focusing on exercise limitations in HFrEF towards potentially limiting factors in the periphery, with particular emphasis on disease-related changes in the regulation of skeletal muscle blood flow. Initial studies examining the hyperemic response to exercise in the skeletal muscle vasculature of HFrEF patients and healthy individuals utilized cycle ergometry, a large muscle mass exercise paradigm which results in the recruitment of the vast majority of lower limb muscle. These studies observed a marked reduction in blood flow, associated with an attenuation in leg vascular conductance, in HFrEF patients during exercise compared to their healthy counterparts (52, 62-66). However, due to the cardiac limitations associated with HFrEF, the engagement of such a large muscle mass during exercise may have outstripped cardiac pumping capacity (30), likely contributing to the observed attenuation in leg blood flow and vascular conductance in this patient population. These initial studies utilizing cycle ergometry led to the recognition that skeletal muscle hyperemic and vasodilatory capacity in HFrEF might be better studied by utilizing an exercise paradigm employing a smaller muscle mass, in order to control for cardiac limitations. In an animal model of heart failure, this was accomplished by utilizing an in situ spinotrapezius muscle preparation and investigating capillary red blood cell flux, which is indicative of capillary blood flow, during electrically elicited 65 muscle contractions (46). Results from this study convincingly displayed an attenuation in capillary red blood cell flux in HF animals compared to control animals. In human HF, the two most widely incorporated exercise modalities to study the peripheral hemodynamic responses to exercise while controlling for confounding cardiac limitations are static-intermittent handgrip (HG) and dynamic single-leg knee-extensor (KE) exercise. Despite the implementation of these exercise modalities in a small number of studies, results have not definitively determined the extent of peripheral hemodynamic limitations in this cohort. Indeed, studies using static-intermittent HG exercise have documented both similar (50) and blunted (22, 67) hyperemic responses, linked to impairments in vasodilation, in HFrEF patients compared to healthy individuals, differences likely attributable to the variations in rhythmicity, duration, and intensity of the exercise performed. Using the dynamic single-leg KE exercise model, Esposito et al. (14) identified an attenuated hyperemia during maximal effort in HFrEF patients, a response that was likely due to the significantly lower maximal KE work rate in the patient group compared to healthy individuals. In one of the only studies to utilize KE exercise at submaximal work rates in HFrEF patients, Magnusson et al. reported similar increases in leg blood flow and vascular conductance between HFrEF and healthy individuals (30), suggesting that impaired peripheral hemodynamics may not contribute to the exercise intolerance in this cohort. Thus, significant controversy remains regarding the HFrEF-associated alteration of peripheral hemodynamics during exercise modalities that minimally challenge central hemodynamics. It is noteworthy that significant advances in pharmacotherapy have been made since these previous small muscle mass exercise studies were performed (19, 20, 27, 55). 66 Indeed, the average HFrEF patient now receives a pharmacologic regimen that includes an average of 10 medications, with beta blockers and a host of peripheral vasodilators now included as standard of care (45). Thus, while exercise intolerance remains a key clinical presentation in HFrEF, uncertainty exists regarding the possible role of impaired peripheral hemodynamics in patients who are optimally medicated in this "modern era" of differentiated drug treatment. Consequently, we sought to systematically examine peripheral responses to small muscle mass exercise in optimally medicated HFrEF patients. To comprehensively assess the hemodynamic response to small muscle mass exercise in HFrEF patients and healthy control subjects of a similar age, we utilized both upper and lower limb exercise paradigms across a wide range of intensities. We hypothesized that compared to healthy controls, HFrEF patients would exhibit an attenuated hyperemic response driven by an impaired vasodilatory capacity during both static-intermittent HG and single-leg KE exercise. Methods Subjects A total of 25 New York Heart Association (NYHA) class II-III HFrEF patients (24 males and 1 female) and 17 healthy controls (16 males and 1 female) of a similar age were recruited to partake in this study either by word of mouth or in the HF clinics at the University of Utah and the Salt Lake City VA Medical Center. All control subjects were nonsmokers, not taking any prescription medication, and were free of overt cardiovascular disease, as indicated by a health history questionnaire. Protocol approval and written informed consent were obtained according to University of Utah and Salt 67 Lake City Veterans Affairs Medical Center Institutional Review Board requirements. All data collection took place at the Utah Vascular Research Laboratory located at the Veterans Affairs Salt Lake City Geriatric, Research, Education, and Clinical Center. All studies were performed in a thermoneutral environment, with subjects reporting to the laboratory fasted, and not having performed any exercise within 24 hours of the study. Subjects reported to the laboratory on a preliminary day to complete health histories, physical examinations, and perform a graded single-leg knee-extensor test to determine maximal work rate. Handgrip Exercise Hemodynamic responses to static-intermittent HG exercise were assessed in 15 control subjects and 23 patients. Subjects rested in the supine position for ≈20 minutes prior to the start of data collection with the right arm abducted at 90°. The elbow joint was extended at heart level to allow subjects to perform HG exercise. First, maximal voluntary contraction (MVC) was established by taking the highest value recorded of 3 maximal contractions using a handgrip dynamometer (Biopac Systems, Goleta, CA). Static-intermittent HG exercise was performed at 3 workloads based on each subject's respective MVC (15, 30, and 45% of MVC). Each exercise level was performed for 3 minutes to ensure the attainment of steady-state hemodynamics. The subjects squeezed the dynamometer to the sound of a metronome at a rate of 1 Hz, and real-time force output was displayed to provide visual feedback to the subjects. A 5 minute recovery period was given between each exercise bout. 68 Single-Leg Knee-Extensor Exercise Hemodynamic responses to dynamic single-leg KE exercise were assessed in 16 controls and 16 patients. The KE paradigm implemented in this study has been described in detail previously (3, 4, 7, 26). Briefly, subjects were seated in a semi-recumbent position on an adjustable chair with a cycle ergometer (model 828E; Monark Exercise AB, Vansbro, Sweden) positioned behind them. Resistance was created by applying friction to the flywheel, which was turned by the subject via a metal bar connecting the crank arm of the ergometer to a metal boot in which the subject's foot was placed. Subjects exercised for 3 minutes at 4 work rates (0, 5, 10, and 15 W) while maintaining 60 contractions per minute. A 5 minute recovery period was given between each exercise bout. Measurements Ultrasound Doppler assessments Measurements of brachial and common femoral artery blood velocity and vessel diameter were performed using a Logiq 7 ultrasound Doppler system (GE Medical Systems, Milwaukee, WI) operating in duplex mode. The Logic 7 was equipped with a linear array transducer operating at a Doppler frequency of 5 MHz in high-pulsed repetition frequency mode (2-25 kHz). All blood velocity measurements were obtained with the probe appropriately positioned to maintain an insonation angle of 60º or less. The sample volume was maximized according to vessel size and centered within the vessel based on real-time ultrasound visualization. Mean velocity values (angle-corrected, and intensity weighted area under the curve) were automatically calculated using commercially available software (Logic 7). Vessel diameter was obtained during 69 end diastole (corresponding to an R wave documented by the simultaneous ECG signal; Logic 7) using the same transducer at an imaging frequency ranging from 9 to 14 MHz. The brachial artery of the right arm was insonated approximately midway between the antecubital and axillary regions, medial to the biceps brachii muscle, while the common femoral artery was insonated 2-3 cm proximal to the bifurcation of the common femoral artery into the superficial and deep branches. Vessel diameter was determined at a perpendicular angle along the central axis of the scanned area. Analysis of brachial artery diameter was performed using off-line automatic edge-detection brachial analyzer software (Medical Imaging Applications, LLC, Coralville, IA), which is described in detail elsewhere (39). Ultrasound Doppler measurements were performed continuously, with the last 60 seconds of each exercise intensity used for the determination of limb blood flow. Using arterial diameter and Vmean, forearm and leg blood flow were calculated as: Limb blood flow (ml/min) = (Vmean * π (arterial diameter/2)2 * 60) Hemodynamic variables Stroke volume (SV), arterial blood pressure (ABP), and heart rate (HR) were determined noninvasively. SV was calculated using the Modelflow method which includes age, gender, height, and weight in its algorithm (Beatscope version 1.1; Finapres Medical Systems BV, Amsterdam, The Netherlands) (9), and has been documented to accurately track SV during a variety of experimental protocols including exercise (12, 13, 51). ABP was measured continuously via photoplethysmography (Finometer, Finapres 70 Medical Systems BV, Amsterdam, The Netherlands), and mean arterial pressure (MAP) was calculated as: MAP (mmHg) = diastolic arterial pressure + (pulse pressure * 0.33) Heart rate was monitored from a standard 3 lead electrocardiogram recorded in duplicate on the data acquisition system (Biopac, Goleta, CA) and the Logic 7. Cardiac output (CO) was calculated as: CO (L/min) = SV * HR Systemic vascular conductance (SVC) was calculated as: SVC (ml/min/mmHg) = CO / MAP Systemic vascular resistance (SVR) was calculated as: SVR (mmHg/L/min) = MAP / CO Forearm and leg vascular conductance were calculated as: Limb vascular conductance (ml/min/mmHg) = blood flow / MAP Data Analysis Statistics were performed using commercially available software (SigmaStat 3.10; Systat Software, Point Richmond, CA). For HG exercise, 2x4 repeated measures ANOVA (α < 0.05) (group: 2 levels, controls vs. HFrEF) (workload, 4 levels: rest, 15, 71 30, and 45% of MVC) were performed to determine the hemodynamic responses in controls and HFrEF during exercise of increasing intensity. For KE, 2x5 repeated measures ANOVA (α < 0.05) (group: 2 levels, controls vs. HFrEF) (work rate, 5 levels: rest, 0, 5, 10, 15 W) were utilized to determine the hemodynamic responses in age-matched controls and HFrEF during exercise of increasing intensity. The Holm-Sidak method was used for alpha adjustment and post hoc analysis. A Person Product Moment Correlation (α < 0.05) was performed to evaluate the association between leg blood flow at 15 W and maximum KE work rate. All group data are expressed as means ± SEM. Results Subject Characteristics Baseline characteristics of the control subjects and HFrEF patients are displayed in Table 3.1. Disease-specific characteristics and medications of patients with HFrEF are presented in Table 3.2. Handgrip Exercise During baseline, prior to HG exercise, there were no significant differences in resting MAP, forearm blood flow, forearm vascular conductance, brachial artery diameter, or CO between groups (Figure 3.1 and Table 3.3). However, resting HR was significantly higher and SV significantly lower in HFrEF patients compared to controls (Table 3.3). In both groups, MAP and HR increased from resting values in an exercise intensity-dependent manner, with no difference between groups (Figure 3.1 and Table 3.3). CO increased from rest at all exercise intensities in the control group, but remained 72 unchanged in the HFrEF patients (Table 3.3). This significant difference in the CO response in HFrEF patients appears to be the result of a tendency for decreased SV across workloads, though this reduction did not reach statistical significance (Table 3.3). During HG exercise, SVC decreased and SVR increased in HFrEF patients, but remained unchanged in the control group (Table 3.3). At the lowest intensity (15% MVC), forearm blood flow and vascular conductance increased to a similar degree between groups; however, at the higher workloads (30 and 45% MVC), HFrEF patients exhibited significantly lower forearm blood flow (30 and 45% MVC) and vascular conductance (45% MVC) compared to controls (Figure 3.1 and Table 3.3). Single-Leg Knee-Extensor Exercise During the baseline period prior to KE exercise, there were no significant differences in any indices of central or peripheral hemodynamics between groups (Figure 3.2 and Table 3.4). In both groups, MAP and CO increased from resting values in an exercise intensity-dependent manner, with no differences between groups (Figure 3. 2 and Table3. 4). HR also increased in an intensity-dependent manner, with a significantly elevated HR in HFrEF patients compared to controls (5, 10, and 15W) (Table 3.4). SV, SVC, SVR remained unchanged in both groups across all KE exercise intensities (Table 3.4). In contrast to HG exercise, both leg blood flow and leg vascular conductance were markedly reduced at all exercise intensities in HFrEF patients compared to controls (Figure 3.2 and Table 3.4), with the greatest reduction (25-35%) present at the highest exercise intensity (15 W). Additionally, there was a strong positive association, between 73 leg blood flow at 15 W and the maximum KE work rate in HFrEF which was not apparent in the control group (Figure 3.3). Discussion This study sought to comprehensively evaluate the hemodynamic responses induced by limb-specific, small muscle mass exercise across a wide range of exercise intensities in HFrEF patients and healthy, age-matched controls. During HG exercise, a divergent hemodynamic response was observed across HG intensities in the HFrEF group. Specifically, both groups exhibited a similar forearm hyperemic and vasodilatory response during lower intensity (15% MVC) HG exercise, but HFrEF patients exhibited a 15-25% attenuation in forearm blood flow at higher intensities (30 and 45% MVC), due to an impaired vasodilatory capacity. During KE exercise, HFrEF patients exhibited a 20- 35% lower leg blood flow and vascular conductance during KE exercise compared to control subjects, with the most substantial decrements at the highest exercise intensity (15 W). Together, these findings indicate that HFrEF patients on modern, optimized pharmacotherapy exhibit a severely compromised ability to vasodilate vasculature of both the upper and lower limbs, thus restricting perfusion of the exercising skeletal muscle and likely limiting exercise capacity in this patient group. Regulation of Skeletal Muscle Blood Flow during Exercise in HFrEF A hallmark symptom of patients with HFrEF is an impaired exercise tolerance and an associated reduction in maximal exercise capacity (16, 40, 60, 65). Interestingly, the degree of left ventricular dysfunction does not fully explain the degree of exercise intolerance or symptom status in this patient group (8, 21, 34, 53, 60), supporting the 74 possibility that peripheral hemodynamic dysfunction may contribute to exercise intolerance. Evidence from previous studies (62-66) indicate a functional role for impaired peripheral hemodynamics in limiting exercise capacity in this cohort during peak cycling exercise, where an apparent reduction in leg vascular conductance was observed in patients with HFrEF compared to healthy individuals, contributing to the documented impairment in perfusion. However, the use of a large muscle mass exercise paradigm presents limitations in distinguishing abnormalities in peripheral hemodynamics in HFrEF. During exercise that recruits a large fraction of total body muscle mass, central circulatory factors play an increasingly important role in the preservation of MAP (41, 47, 49). Due to impaired left ventricular function during whole body exercise, patients with HFrEF largely depend on systemic vasoconstriction (i.e. reductions in SVC) in order to maintain MAP (52). This may indicate that the markedly lower leg vascular conductance and associated decrement in perfusion of the exercising skeletal muscle during cycling exercise might solely be due to the maintenance of MAP versus limitations in vasodilatory capacity in this patient group (52, 66). In order to investigate how altering the total amount of muscle recruited during exercise might affect the peripheral hemodynamic responses to exercise in this patient group, LeJemtel et al. (28) examined the differences in leg blood flow during maximal single- and double-legged upright cycling in HFrEF patients and healthy control subjects. Interestingly, they observed similar leg blood flow values in the HFrEF patients during both single-and double-legged exercise modalities, which they attributed to an impaired vasodilator response in the patient group (28). These results were indirectly supported by Jondeau et al. (23), who utilized maximal arm cycling exercise superimposed on maximal 75 leg cycling to examine the impact of HF on cardiopulmonary reserve. In this study, addition of arm exercise while cycling provoked an increase in peak oxygen (O2) consumption in severe HF patients, but not in healthy controls, which was interpreted as evidence for an inadequate vasodilator response to exercise resulting in impaired O2 delivery, as the arterial-venous O2 difference is near maximal in HFrEF during maximal leg cycling (25). While these novel studies were some of the first to investigate the hemodynamic response to exercise which recruited differing amounts of muscle mass during all combinations of arm and leg cycling, it is likely that some degree of cardiac limitation still confound these results. This is even the case during maximum single-legged cycling, where Martin et al. (31) has documented that the CO achieved in HFrEF patients is similar compared CO values during maximum double-legged cycling in this patient group. Thus, in the present study, we employed two limb-specific small muscle mass exercise modalities (static-intermittent HG and dynamic single-leg KE exercise) across a range of submaximal exercise intensities in order to more thoroughly investigate the peripheral hemodynamic response to exercise in HFrEF in isolation of the significant confounding effects of CO limitations and MAP regulation imposed by large muscle mass exercise. Hemodynamic Responses to Handgrip Exercise Due to the accessibility and limited cardiorespiratory stress associated with this modality, handgrip exercise has been utilized in a number of studies over the past several decades to investigate the peripheral hemodynamic response in HFrEF patients. Indeed, Zelis et al. (67) investigated the peripheral blood flow response to graded static- 76 intermittent handgrip exercise in HFrEF patients compared to control subjects in the early 1970's, and documented an impaired exercise hyperemia in this patient group. However, it is noteworthy that this study utili