Regulation of Cardiac Contractility

Regulation of Cardiac Contractility

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Integrated Systems Physiology: From Molecule to Function to Disease Editors D. Neil Granger, Louisiana State University Health Sciences Center-Shreveport Joey P. Granger, University of Mississippi Medical Center Physiology is a scientific discipline devoted to understanding the functions of the body. It addresses function at multiple levels, including molecular, cellular, organ, and system. An appreciation of the processes that occur at each level is necessary to understand function in health and the dysfunction associated with disease. Homeostasis and integration are fundamental principles of physiology that account for the relative constancy of organ processes and bodily function even in the face of substantial environmental changes. This constancy results from integrative, cooperative interactions of chemical and electrical signaling processes within and between cells, organs, and systems. This eBook series on the broad field of physiology covers the major organ systems from an integrative perspective that addresses the molecular and cellular processes that contribute to homeostasis. Material on pathophysiology is also included throughout the eBooks. The state-of the-art treatises were produced by leading experts in the field of physiology. Each eBook includes stand-alone information and is intended to be of value to students, scientists, and clinicians in the biomedical sciences. Since physiological concepts are an ever-changing work-in-progress, each contributor will have the opportunity to make periodic updates of the covered material. Published titles (for future titles please see the Web site, www.morganclaypool.com/page/lifesci)

Copyright © 2011 by Morgan & Claypool Life Sciences All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means—electronic, mechanical, photocopy, recording, or any other except for brief quotations in printed reviews, without the prior permission of the publisher. Regulation of Cardiac Contractility R. John Solaro www.morganclaypool.com ISBN: 9781615041749 paperback ISBN: 9781615041756 ebook DOI: 10.4199/C00030ED1V01Y201104ISP018 A Publication in the Morgan & Claypool Publishers Life Sciences series INTEGRATED SYSTEMS PHYSIOLOGY: FROM MOLECULE TO FUNCTION TO DISEASE Book #18 Series Editor: D. Neil Granger, LSU Health Sciences Center, and Joey P. Granger, University of Mississippi Medical Center Series ISSN ISSN 2154-560X

print

ISSN 2154-5626

electronic

Regulation of Cardiac Contractility R.  John Solaro Department of Physiology and Biophysics University of Illinois at Chicago College of Medicine Chicago, IL

INTEGRATED SYSTEMS PHYSIOLOGY: FROM MOLECULE TO FUNCTION TO DISEASE #18

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Abstract Contractility describes the relative ability of the heart to eject a stroke volume (SV) at a given prevailing afterload (arterial pressure) and preload (end-diastolic volume; EDV). Various measures of contractility are related to the fraction as the SV/EDV or the ejection fraction, and the dynamics of ejection as determined from maximum pressure rise in the ventricles or arteries or from aortic flow velocities determined by echocardiography. At the cellular level, the ultimate determinant of contractility is the relative tension generation and shortening capability of the molecular motors (myosin cross-bridges) of the sarcomeres as determined by the rates and extent of Ca activation, the turnover kinetics of the cross-bridges, and the relative Ca responsiveness of the sarcomeres. Engagement of the regulatory signaling cascades controlling contractility occurs with occupancy and signal transduction by receptors for neurohumors of the autonomic nervous system as well as growth and stress signaling pathways. Contractility is also determined by the prevailing conditions of pH, temperature, and redox state. Short-term control of contractility is fully expressed during exercise. In long-term responses to stresses on the heart, contractility is modified by cellular remodeling and altered signaling that may compensate for a time but which ultimately may fail, leading to disorders.

Keywords heart, afterload, preload, length-dependent activation, Ca regulation, sarcomere, protein phosphorylation

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Contents Introduction: Contractility and the Integrative Biology of the Myocardium.....................1 Contractility in the Modern Context............................................................................. 1 Control of Cardiac Contractility Is Critical to the Matching of Cardiac Output to Venous Return During Exercise with Little Change in End Diastolic Volume and with Tuning of the Dynamics of Contraction and Relaxation to Heart Rate............................................................................... 2 Control of Contractility Is at the Cellular Level of Organization.....................................8 Left Ventricular Diastolic and Systolic Pressure, Ejection, and Relaxation Reflect Sarcomeric Mechanical Properties............................................................................... 12 Integration of Sarcomere Mechanics with Cardiac Function Clarifies the Meaning of Preload, Afterload, and Contractility........................................................................... 16 Pressure Volume Loops Provide a Quantification of Contractility.................................. 19 Phosphorylations of Regulatory Proteins in Excitation Contraction Coupling Modify Contractility by Controlling Cellular Ca2+ Fluxes, the Response of the Myofilaments to Ca2+, and the Kinetics of the Cross-Bridge Cycle................................. 23 Contractility May Be Altered by a Variety of Mechanisms Not Involving a Prominent Role for the Autonomic Nervous System..................................................... 27 Cardiac Function Curves Provide a Compact Graphical Representation of Regulation of  CO and SV............................................................................................ 28 Heart Failure as a Failure of  Contractility.................................................................... 31 References.................................................................................................................. 33 Author Biography....................................................................................................... 35



Introduction: Contractility and the Integrative Biology of the Myocardium Contractility in the modern context The use of the term contractility goes back well over a 125 years, and was used to simply describe a property of assorted tissues to shorten. The term has something to do with the ability of heart tissue to shorten, but has taken on new connotations in current thinking. Moreover, with the state of detailed knowledge of molecular and cellular control of the level of activity and dynamics of the heart, assigning a strict definition does not seem appropriate inasmuch as the relative performance of the heart may take on different dimensions including the relative peak pressure in the cardiac chambers at relatively constant volume (peak tension in an isometric contraction of muscle fibers), changes in the rate of pressure (tension) development, and the slope of the relation between chamber volume and chamber end systolic pressure. There has also been the designation of changes in contractility as promoted by extrinsic control mechanisms such as neuro-humoral signaling in contrast to those promoted by intrinsic control mechanisms such as the end diastolic fiber length (Frank-Starling relation). As will be evident here, consideration of the mechanism by which contractility is controlled indicates that this is an artificial separation. Whatever the case, it is apparent that the term contractility remains useful to permit succinct written and oral communication between and among scientists and clinicians. However, as described here, detailed understanding of the control mechanisms altering contractility in health and disease demands flexibility in the interpretation of the meaning of a statement regarding the relative contractility of the heart. In approaching this detailed understanding, we first consider the pressure and volume dynamics of the heart beat and how these change with changes in contractility. These altered dynamics constrain theories as to the mechanisms accounting for altered contractility at the molecular and cellular levels. We then discuss current understanding of these molecular and cellular mechanisms. In considering these mechanisms, we focus on the left ventricle (LV). Chapters in monographs (Page et al., 2001; Bers, 2001; Opie, 2004, and Katz, 2010) provide more details and extended discussions of contractility.

  REGULATION OF CARDIAC CONTRACTILITY

Control of cardiac contractility is critical to the matching of cardiac output to venous return during exercise with little change in end diastolic volume and with tuning of the dynamics of contraction and relaxation to heart rate Revelations on the state of contractility of the left ventricle come from measures of the changes in the LV volume and pressure in the transition from rest to exercise, a short term modification in cardiac performance, Straight forward and useful expressions related to cardiac performance are the following equations:

CO = HR × SV

(1)



CO = HR × (EDV − ESV)

(2)

where CO is cardiac output; HR is heart rate, SV is stroke volume, EDV is end diastolic volume, and ESV is end-systolic volume. Figure 1 provides a simple illustration of the heart beat demonstrating these volumes and includes a definition of three major variables that determine cardiac function. The dashed line around the circumference of the ventricular chamber indicates that during filling and ejection there are length changes in the fibers of the chamber. The EDV is viewed as the pre-load, a term that arose from studies of isolated strips of heart muscle in which a load had to be added to stretch the fibers in a way mimicking the stretch occurring with filling of the ventricle to the EDV. Similarly afterload denotes the pressure against which the ventricle must develop pressure for ejection to occur. In the illustration the ESV is equated roughly to contractility and end-systolic sarcomere length, i.e., the extent of cellular shortening. Although the ESV is also affected by the afterload, we will clarify the interplay of the three variables and how contractility may be assessed apart from the effects of afterload. Figure 2 demonstrates the application of equations (1) and (2) in the changes in HR, CO, SV, EDV, and ESV in the transition from a resting steady state to a steady state during a bout of moderate exercise. Applying the mathematics in the above equation could provide a quick assessment of how one might alter CO, i.e., simply by a change in HR, EDV and/or ESV. However, the variables are not independent. For example, increases in HR beyond a certain level induce a depression in SV by limiting EDV, i.e., filling time. In addition classical ideas suggest that increases or decreases in EDV (pre-load), which engage the Frank-Starling relation alter CO with no effect on contractility, which was presumed to be independent of diastolic mechanisms. It is now recognized though that mechanisms engaged during diastole do affect contractility and thus systolic events.

Introduction  

Aorta

Afterload

Body Lungs

RA

LA

RV

LV

EDV

ESV

Preload

Contractility

120 ml

60 ml

Figure 1: Schematic illustrating the cardiac cycle. Illustrations at the top and middle represent the heart at end diastolic volume (EDV) and end systolic volume (ESV) emphasizing circumferential shortening of the ventricular chambers responsible for ejection. The scheme also emphasizes that a correlate of ventricular volume is length of the sarcomere, the functional unit, ultimately responsible for ejection. Stroke volume (SV) is EDV − ESV and cardiac output is computed from the relation: CO = HR × SV.

Figure 2 provides an illustration of the interplay of HR, SV and CO during an episode of exercise. The data were determined for a healthy young adult before and after administration of the beta-adrenergic blocking agent, propranolol. Comparison of the changes in the ventricular volumes and HR before and after blockage of adrenergic input to the heart provides a measure of how modifications in contractility and HR affect the functional readouts. Comparisons shown in Figure 2 also provide insights into the effects of physiological aging on the heart, which resemble the effects of beta-blockade. The data in Figure 2 reveal important aspects of the control of cardiac output during the most common physiological stress on the cardiovascular system. There are several important revelations provided by the data in Figure 2. Note that CO is essentially a linear function of the work load, and does not change with blockade of beta receptors. This result reflects the necessity and capability of the cardiovascular system to meet the demands of the tissues. A critical driving force is tissue oxygen needs, which in the case demonstrated in Figure 2 changed many fold in a relatively short time. Oxygen extraction as blood flows through the tissues is about 20% of the arterial concentration, making oxygen among the most flow limited components of the blood

200

HR 150

HR + Pro SV + Pro

100

SV 50

0

50

100

Workload (watts)

150

Ventricular Volumes (ml)

HR (bpm) or SV (ml)

  REGULATION OF CARDIAC CONTRACTILITY 200 150

EDV + Pro

100

EDV ESV + Pro

50 0

ESV 0

50

100

150

Workload (watts)

CO (L/min)

15

CO CO + Pro

10

5

0

0

50

100

Workload (watts)

150

Figure 2: Changes in heart rate, cardiac output, and left ventricular volumes during increasing work load during exercise. Data (courtesy of Dr. Edward Lakatta) were collected on a normal young healthy male exercising on a stationary bicycle. During a repeat of the exercise, the subject administered either vehicle or propranol, a beta-adrenergic blocking agent. See text for further description.

together with carbon dioxide and heat. Other components of blood have extraction ratios much less than oxygen and are much less flow limited. Thus, meeting tissue oxygen demands automatically provides all of the needs of the cell for exchange of other nutrients and wastes. The coupling of tissue oxygen needs to CO involves neural, mechanical, and chemical control mechanisms, which will be elucidated as we take up the theme of control of cardiac contractility. Although not extensively discussed here an important variable providing the tissue oxygen needs is venous return (VR). Inasmuch as the cardiovascular circuit is closed, it is difficult to separate CO from VR since they must be the same in the steady state. Certainly the increase in CO noted in Figure 2 came about not only because the heart rate is increased but because return flow to the heart, the VR, has increased as the contraction and relaxation of the muscles squeeze blood vessels holding large volumes of blood (capacitance vessels), and because of neural inputs to the resistors in the circuit and the walls of the capacitance vessels. This return of blood is critical to the

Introduction  

increase in flow out of the heart to the lungs and eventually to the body. Yet, imagine what would happen to the ventricular volumes in Figure 2 if the return of the volume of blood added to the chambers of the heart from the veins during diastole was not completely transferred to the arteries. This might happen over a few transitional beats, but cannot be sustained for very long. Thus, an important function of the heart in which altered contractility comes into play is the matching of CO to VR, while maintaining an overall economy of oxygen use by the pump. As we consider the molecular and cellular mechanisms of control of contractility, we will emphasize how alterations at the level of heart are integrated into the overall homeostasis of blood flow. Failure of these mechanisms to provide a robust response to exercise induces alterations in the ventricular volumes that will limit exercise capacity and promote compensatory alterations in contractility that are adaptive for a time, but in the long term become maladaptive and fatal. These long term adaptive and maladaptive responses to stresses affect contractility and induce significant disorders of the heart. A second revelation of the data in Figure 1 is that in the control conditions (no beta-blocker) the nearly 3 fold increase in CO occurred not by increasing the loading volume i.e. elevated EDV, but with a drop in both EDV and ESV. SV increased with the work load of exercise indicating that the ESV fell a bit more than the EDV. As we will see, the fall in ESV reflects molecular mechanisms promoting the ventricle to reduce the volume at end systole. The ratio of SV/EDV provides a measure to the relative ability of the ventricle to eject. This ratio is called the ejection fraction and is commonly employed to assess relative cardiac function i.e. contractility. Activation of the sympathetic nervous system (SNS) is an inevitable consequence of exercise either by conscious activity of the cerebral cortex affecting the brainstem or by involuntary neural mechanisms typical of the autonomic nervous system. An important aspect is that the ability of the heart to match an increase in VR to an increase in CO with a reduction in EDV and ESV is energetically favorable. The favorable energetic profile of the heart in the control condition in the data illustrated in Figure 2 is couched in the law of LaPlace. This law, which is illustrated in Figure 3, states that wall tension is related to the pressure in the ventricular chamber times the radius of curvature of the chamber (equation 3),

Tension = (Pressure × Radius)/2h

(3)



Pressure = (Tension × 2h)/Radius

(4)

where h is the thickness of the walls of the chamber. Equation (4) is a simple rearrangement of equation (3). One important aspect of the LaPlace relation is that an increase in wall tension will increase pressure in the chamber at constant radius and wall thickness. This is close to what happens with isovolumetric pressure development in the left ventricle, when the muscle cells that form the chamber are activated to contract, and develop tension and pressure before the aortic valve opens.

  REGULATION OF CARDIAC CONTRACTILITY Wall tension in the heart and the Law of La Place T = Pi x r 2h Pi

Pi

h

radius

h

radius

If Pi is the same in both hearts, then the wall tension that muscles must develop to eject blood is greater for the heart on the right.

Figure 3: Schematic illustrating the law of Laplace. The Laplace relation emphases the favorable economy of cardiac ejection at relatively small EDV compared to relative big EDV. See text for further discussion.

As we will discuss the tension is developed by molecular motors in the sarcomeres in a chemomechanical process linking a mechanical change to a splitting of ATP. The more motors working the more ATP hydrolyzed. The more ATP that is split the more oxygen is required to induce the mechanical change. The second important aspect is that the wall tension required to develop a given pressure is related to the radius of the chamber. Elevations in EDV increase this radius; and reductions in EDV decrease the radius. To develop the same ventricular pressure in a ventricle with a relatively big EDV requires more wall tension than a ventricle with a relatively small EDV (Fig. 3). Thus the ability of the heart to increase CO with a fall in EDV is economic with regard to the oxygen cost of tension generation. The corollary is that increasing CO with increases in EDV is a threat to the economy of cardiac function and a threat to homeostasis. The increase in CO occurred in the control condition largely because of an increase in HR and a fall in ESV. Both of these major control mechanisms are subject to regulation by the autonomic nervous system (ANS). This role of the ANS is emphasized by data in Figure 2 following blockade of adrenergic beta receptors with propranolol in which there is inhibition of the adrenergic signaling to the heart. Note that the increase in CO associated with the increased work load is not affected by propranolol. Matching of CO to the increase in VR was not compromised, but there was a trade off in making sure the system supplies the tissue oxygen needs. The fractional increase in HR was less in the presence of beta blockade, and thus according to equation (1) with the constant CO,

Introduction  

SV must be increased. With out beta-receptor stimulation the increase in SV occurred largely due to an increase in EDV, and this result indicates the significance of the ANS in matching CO to VR while maintaining hemeostatic control over the EDV. The values presented in Figure 2, which are steady state values of HR, CO and ventricular volumes, do not emphasize the alterations in cardiac dynamics that occur during increased work load and activity of the ANS. Changes and levels of contractility are often assessed and understood in terms of these dynamics. The data in Figure 4 illustrate these dynamics of cardiac function as the time dependence of left ventricular volume before and after an episode of exercise. This view of cardiac function not only emphasizes the decrease in ESV with little change in EDV that occurred with exercise, but indicates the abbreviation of the contraction/relaxation cycle. The reduction in overall cycle time is critical to tuning the heart beat to the increased HR and thus maintaining an adequate duration for ventricular filling during diastole. Figure 1 also illustrates that the volume changes are associated with shortening of the cells making up the left ventricular chamber and that the changes in cell length reflect changes in sarcomere length. We next consider the cellular and molecular mechanisms responsible for the changes in cardiac function demonstrated in Figures 2 and 3.



Control of Contractility Is at the Cellular Level of Organization Figure 5 shows a schematic of the major components of the working cardiac myocytes involved in excitation, contraction, and relaxation of heart muscle cells. Consideration of the mechanisms of control of contractility in heart muscle involves regulatory elements at the level of the working myocytes. In contrast to the case with skeletal muscle there is no spatial summation (motor unit recruitment) in generating tension in heart muscle. Heart cells operate as a syncytium in which tight junctions of low electrical resistance ensure that when one cell is activated (depolarized), all cells become activated. Thus we focus on regulation at the level of the cells themselves. Figure 5 depicts the T-tubule containing channels and transporters as an invagination of the surface membrane or sarcolemma. The sarcolemma membrane houses alpha- and beta-receptors for nor-epinephrine, epinephrine, and muscarinic receptors for acetylcholine together with G-proteins, which form a molecular apparatus for transducing the signal established when these ligands bind to their receptor. A critical membrane system, the sarcoplasmic reticulum (SR), forms an internally enclosed network of tubules in which high concentrations of Ca2+ are stored in diastole. Electrical excitation of the cell from action potentials arising from the sino-atrial node induce membrane depolarization that promotes gating of Ca2+ channels, which open and cause a small release of Ca2+ into the cytoplasm. The small Ca2+ current induces a release of Ca2+ from the SR by a process called Ca-induced Ca-release. The release occurs through Ca2+ release channels commonly referred to as ryanodine receptors (RyR2). Depolarization-induced influx of Ca2+ current (ICa) through the L-type channels contributes approximately 20–25% of the free Ca2+ in a cardiac twitch. The release of Ca2+ through the RyRs contributes the remaining 75–80% of Ca2+ necessary for cardiac contraction. Experiments employing fluorescent indicators that sense Ca2+ have revealed the activity of local clusters of RyR receptors. These experiments demonstrate elementary events known as Ca2+ sparks that reflect the activity of small groups of RyRs. Enhanced ICa influx increases localized Ca2+ accumulation, which increases Ca2+ spark frequency, and produces a graded stimulation of RyR Ca2+ release from the SR. Release into the cytosol increases the local concentration of Ca2+ surrounding the myofilaments and promotes Ca2+ binding to TnC on the thin filaments. With Ca-bound to TnC thin filaments are no

Control of Contractility is at the Cellular Level of Organization   End Diastolic Length

LV Volume EDV

Rest Exercise EDV

ESV ESV sec

End Systolic Length

Figure 4: Changes in dynamics of the cardiac cycle as illustrated by the time dependence of left ventricular volume before and during exercise. The panel on the left demonstrates the relation between myocyte and sarcomere length at end diastole and end systole. The data illustrate the reduction in cycle time associated with the increased HR. See text for further description.

longer inhibited with regard to its reactivity with myosin. The myosin cross-bridges react with actin and impel the thin filaments towards the center of the sarcomere as illustrated in Figures 1 and 5. Ca2+ is removed from the cytoplasm by a Ca2+ activated MgATPase (SERCA2a), which refills the SR with Ca returning the diastolic state. To fully restore the diastolic state and maintain a steadystate, the net movement of Ca2+ from the extracellular space that entered with voltage gating of the Ca2+ channels must be and is extruded in exchange for Na+ by the action of the Na+,Ca2+ exchange protein (NCX), which is driven by the Na+ gradient across the sarcolemma. A key membrane protein not illustrated in Figure 5 is the Na, K ATPase responsible for pumping these ions and establishing the gradient, which is critical for electrical responses and for the establishing the gradient of Na. Relatively small and slow processes for Ca2+ efflux from the cytosol include transport via a sarcolemmal Ca2+ pump and transport into the mitochondrial space. The proportion of Ca2+ flux through the NCX and these slow processes is species dependent, with a lower proportion of Ca2+ handled by these mechanisms in rodents compared to humans. This species difference requires consideration when applying results obtained in rodent studies to human cardiac function. Phospholamban (PLB) is a small but critically important proteo-lipid that interacts with SERCA and inhibits transport of Ca2+ (MacLennan and Kranias, 2003). The inhibition is released

10  REGULATION OF CARDIAC CONTRACTILITY Beta R Alpha R Muscarinic R Ca2+ T-tubule (ECF)

SERCA2a Ca2+

RyR

PLB SR

Ca-Channel K-Channel Na+

Ca2+

NCX

Z-Disc

Mitochondrion

Thin Filament

Thick Filament

Titin

Figure 5: Essential elements responsible for excitation contraction coupling (ECC) in a cardiac myocyte. Surface membranes are illustrated surrounding the myofilaments and mitochondria and include invaginations called T-tubules. The surface membrane and T-tubules contain channels and transporters are shown as well as alpha- and beta-receptors for nor-epinephrine, epinephrine, and muscarinic receptors for acetylcholine. Surrounding the myofilaments is sarcoplasmic reticulum (SR), which is an internally enclosed network of tubules in which high concentrations of Ca2+ are sequestered during diastole and released in systole. Action potentials trigger ECC by voltage gating of L-type Ca2+ channels which induce a net current of Ca2+ into the sarcoplasm. These Ca2+ ions promote gating of Ca2+-release channels in the SR (RyR2) by a process termed Ca2+ induced Ca2+ release (CICR). Ca2+ concentration in the sarcoplasm transiently increases and there is diffusion of Ca2+ to the myofilaments. Ca2+-binding to thin filament troponin C (TnC) triggers force and shortening of the sarcomeres. Relaxation is associated with transport of Ca2+ from the sarcoplasm by Ca2+ activated MgATPase (SERCA2a). The Ca2+ entering the cell through Ca2+ channels is returned to the extracellular space in exchange for Na+ by the action of the Na+,Ca2+ exchange protein (NCX). SERCA2a activity is controlled by proteolipid, phospholamban (PLB), which inhibits transport. Phosphorlation of PLB releases this inhibition, thereby enhancing Ca2+ uptake rate. See text for details.

Control of Contractility is at the Cellular Level of Organization  11

when PLB becomes phosphorylated most commonly by cAMP dependent protein kinase (PKA). An important concept with regard to control of cardiac function is that during a basal resting state, the amount of Ca released into the cytoplasm does not saturate the thin filament TnC sites. Rather only about 20–25% of these sites bind Ca2+. This leaves a reserve of thin filament sites, which may be recruited. Clearly therefore one way to increase or decrease force producing cross-bridges is to vary the amounts of Ca2+ delivered to the myofilaments. As we will see, this is one of the significant mechanisms occurring during the episode of exercise illustrated in Figure 2.

12

Left Ventricular Diastolic and Systolic Pressure, Ejection, and Relaxation Reflect Sarcomeric Mechanical Properties The fundamental structural unit of the cardiac muscle cell responsible for force and shortening capability is the sarcomere (Reviewed in Kobayashi and Solaro, 2005). Figure 6 illustrates the molecular makeup of the region of over lap between thin and thick filaments. The reaction of the myosin cross-bridges, which are molecular motors housed in the sarcomeric thick filaments, with the actins of the thin filaments generates active cellular force, shortening, and power. In Figure 6 the cross-bridge is demonstrated during diastole, when reaction with actin is blocked by tropomyosin (Tm) and during systole, when cross-bridges are at the end of a power stroke after reacting with actin and splitting ATP. The reaction cycle of the cross-bridge with actin begins with attachment followed by a movement of the lever arm of the myosin head, which impels the thin filament in each half-sarcomere to slide toward the center. The cycle ends with detachment (reviewed in Hinken and Solaro, 2007). MgATP hydrolysis powers the force generation and shortening and provides the energy for these movements and it is generally agreed that one ATP is split per cycle. In entering the diastolic state ATP binds very rapidly cross-bridge and is rapidly split into ADP and Pi, which remains bound. In this state the cross-bridge is highly reactive with actin, but the actins are not available owing to a steric block imposed by the position of Tm on the thin filament. As shown in Figure 6 binding of Ca to TnC promotes the movement of Tm, making actin sites available. The cross-bridge can now attach and engage a catalytical cycle in which there are step-wise releases of Pi and ADP, and isomerization of the cross-bridge that is geared into a progressive change in mechanical state of the cross-bridge leading to thin filament sliding. With the release of ADP and Pi, the cross-bridges complete the power stroke and transfer into a nucleotide-free rigor state, which is strongly bound. Detachment requires binding of MgATP and the cycle may begin again depending on actins availability.

sarcomeric mechanical properties  13

Figure 6 also illustrates the complex protein-protein interactions triggered by Ca2+ binding to the thin filament. TnC is one component of troponin (Tn) a hetero-trimeric protein complex consisting also of TnI, named for its prominent inhibitory activity, and TnT, named for its Tm binding activity. As shown in the left panel of Figure 6, in diastole, both Tn and Tm are situated on the thin filament in positions that block actin sites or hinder the actin-cross-bridge reaction. Tm is

Diastole

Systole

Actin

TnI TnC

Ca

Tm

TnT Crossbridge MLC1 MLC2

ADP + Pi ATP

MyBP-C Titin

Figure 6: Molecular mechanisms of sarcomere activation. The left panel shows the diastolic state of a region of overlap of thin and thick filaments illustrating detailed structure and location of major protein strands consisting of actin and tropomyosin (Tm). Attached to the thin filament is a the heterotrimeric troponin (Tn) complex. TnC is a dumbbell shaped protein with the N-lobe containing a single regulatory Ca-binding site. cTnI is shown tethered to actin on an actin strand by an inhibitory peptide and a second actin binding region flanking a switch peptide, which interacts with cTnC upon Ca-activation. cTnI has a unique stretch of N-terminal amino acids, which contain phosphorylation sites at S23, S24. The N-peptide interacts with the N-lobe of cTnC, but is released upon phosphorylation. Tm is wedged and immobile in a position that blocks the actin –cross-bridge reaction. Tm is held in this blocking position by the Ip of cTnI on one side, and by the N-terminal tail of troponin T (cTnT) from the Tn in register on the adjacent actin strand. cTnT from the Tn complex on the adjacent actin strand is shown with stripes. The right panel shows Ca2+-binding to TnC that triggers alterations in thin filament structure and promotion of the actin-cross-bridge reaction. Myosin binding protein C (MyB-C) and myosin light chains (MLC1 and MLC2) modulate cross-bridge activity. Titin is a major structural protein responsible for passive tension. (See text for further discussion).

14  REGULATION OF CARDIAC CONTRACTILITY

immobilized in the blocking position by cTnI, which is tethered to actin, and by the tail of TnT, which comes from the Tn complex in register on the opposite actin strand. The binding of cTnI to actin occurs via two regions, a highly basic inhibitory peptide and a second actin binding region. These regions flank a switch peptide, which binds to cTnC when Ca2+ binds to the N-lobe of cTnC, which houses a single regulatory Ca2+ -binding site, thereby participating in the mechanism by which Tn releases the thin filament from inhibition. An important domain of  TnI is a unique N-terminal peptide, which has phosphorylation sites for PKA. In the absence of phosphorylation, the peptide reacts with TnC and enhances the Ca-affinity of  TnC. With phosphorylation, the peptide no longer interacts with TnC and there is a suppression of the affinity of  TnC for Ca2+. The reaction of the switch peptide with the N-lobe of TnC is promoted by Ca-binding and exposure of a hydrophobic patch on TnC. The result is an induction of release of TnI from its inhibitory position. In association with this release of TnI, there are movements of TnT as the Tn complex pivots on the thin filament releasing Tm, which is now mobile. Actin sites for reaction with cross-bridges are exposed and the force and shortening producing cross-bridge cycle proceeds. There is also evidence that the reaction of cross-bridges with the thin filament can promote more actincross-bridge reactions by cooperative, feedback mechanisms. The strongly reacting cross-bridge has been demonstrated to increase the affinity of TnC for Ca2+ as well as to move Tm further away from the region of actin that reacts with the cross-bridges. Interactions of regulatory proteins with thick filament myosin also affect the ability of crossbridges to react with the thin filament, especially the kinetics of the cross-bridge cycle. The major mechanisms involve influences of the regulatory myosin light chain (MLC2) and myosin binding protein C (MyBP-C), both of which are illustrated in Figure 5 as interacting with the lever arm region of the cross-bridge. These proteins affect the radial disposition of the cross-bridge i.e. its movement relative to the main thick filament backbone. For example removal of MyBP-C by genetic approaches or phosphorylation by PKA causes the cross-bridge to move in a radial direction away from the backbone of the thick filament. Phosphorylation of MLC2 by a Ca-calmodulin dependent kinase (MLCK) also induces this radial movement. Other important determinants of the number of cross-bridges reacting with the thin filaments are the sarcomere length, and the load (velocity of shortening). Elements in the sarcomeres are important determinants of passive elastic properties of the cell and thus diastolic properties of the ventricular chambers (Krueger and Linke, 2011). They also appear to affect signaling cascades, systole and contractility. A major molecular spring is the giant protein, titin. Interactions of titin with proteins of the sarcomere are illustrated in Figures 5 and 6. Titin extends from the mid-line of the sarcomere to the Z-disk. The region near the Z-line houses a stretch of amino acids that are coiled and act as a spring. As the sarcomere is passively stretched in diastole by the VR, titin elongates giving rise to passive tension. The spring like action of titin

sarcomeric mechanical properties  15

may be important also as the sarcomeres shorten and generate a restoring force producing a sucking action that is likely to be important in early diastole. Moreover, regions of titin in the overlap region of the sarcomere interact with myosin binding protein C. This interaction may affect the radial movement of the cross-bridges described above. These findings have led to new concepts regarding how diastolic state i.e. the stretch on titin, may affect systolic state and thus contractility. Thus, the conformational changes in titin with the stretch during diastole may also affect cross-bridge disposition. Although not discussed here, there is emerging evidence that the stress-strain relation of titin is not a constant but a variable controlled by phosphorylation. Although not depicted in detail in the figures, the Z-disc of the sarcomere not only anchors the thin filaments, but also links sarcomeres in series by titin and thin filaments interactions. There are also lateral connections linking the sarcomere to the surface membrane. In addition to its role in force transmission, the Z-disc is emerging as a significant locus of communication within the cells.

16

Integration of Sarcomere Mechanics with Cardiac Function Clarifies the Meaning of Preload, Afterload, and Contractility Ultimately the properties of the sarcomeres described above are responsible for establishing and altering cardiac contractility. The data and illustrations in Figure 6 provide a means to integrate the passive and active properties of the sarcomeres into the heart beat, and to understand pre-load, afterload, and contractility. The left panel of Figure 7 schematically depicts mechanical changes of one sarcomere during a heart beat. The panels on the right show the read out of this sarcomeric activity in the form of tension and length changes at the level of a muscle strip, and in the form of pressure and volume changes at the level of the heart. The correlate of sarcomere and cellular tension is the pressure (by the Law of LaPlace), and the correlate of sarcomere and cellular length is the ventricular volume. The sarcomere schematic in Figure 7 illustrates elements, the contractile element and a spring. The spring is shown in series with the contractile element and is a lumped element representing titin, collagen, and cytoskeletal proteins. Yet in the working range of the cardiac myocytes titin is the major source of diastolic tension. Diastolic filling, represented by a load attached to the end of the sarcomere, establishes the sarcomere length before activation and this rationalizes the term “preload” as the correlate for EDV. The stretching of the passive elements as the ventricles fill in diastole gives rise to an end diastolic tension shown in the tension trace and an end diastolic pressure (EDP) shown in the ventricular pressure trace. The load the sarcomere encounters with activation is illustrated in the sarcomere in Figure 7 and termed the “afterload,” which suggests the sarcomere does not sense this load until after tension begins to develop with activation. This is roughly the course of events in the intact ventricle with afterload and rationalizes the “afterload” as the correlate of aortic pressure, which is, in essence, not sensed by the sarcomere until the aortic valve opens. With coupling of excitation to a release of Ca2+ into the myofilament space, cross-bridges are recruited into force generating cycles and the cell develops tension, shortens, and

Integration of Sarcomere Mechanics  17

EDV

Isometric

ESV

Isotonic

IsoVol Pressure

120

mm Hg

EDV

Tension

ESV

60

LV Pressure

0

Pre Load

EDL

EDV

ESL

ESV AP

mV

After Load

LV Vol

Length

sec

ECG mV sec

Figure 7: Correlates of sarcomere mechanics in ventricular function. In the muscle preparation, depicted from the perspective of a single sarcomere, a weight added to the muscle in diastole in the analogue of the EDV is a weight, the preload, added prior to activation. The addition of the preload, establishes sarcomere length. A second weight is the analogue of the load the sarcomere discovers it must lift after activation, i.e., the afterload. Activation is triggered by the action potential and viewed at the body surface by the ECG. Tension develops lifting the afterload as cycling cross-bridges react with actin, and develop force isometrically until the tension developed matches the afterload. This sarcomeric activity is reflected in the ventricle as an increase in wall tension, isovolumic pressure development followed by opening of the aortic valve and ejection of blood against the rising pressure in the aorta. Dashed lines in the figure represent measurements in which muscle length was held constant or in which the aorta was clamped to produce an isovolumic beat. The peak amplitude of pressure or tension provides a measure of contractility. See text for further description.

stretches the elastic element. As shown in Figure 7, when the tension matches the afterload, the sarcomere and muscle strip shortens lifting the afterload. With a constant afterload, tension develops in an isotonic twitch. In the case of the ventricle, pressure rises with no change in volume (isovolumetric) until the valve opens and ejection commences. The ejection of the SV occurs against an increasing pressure head (afterload) as the aortic walls are stretched and recoil. Thus in the heart afterload is not constant and ejection is referred to as an auxotonic. With repolarization of the myocytes and re-uptake of Ca2+ by the SR, activation of the thin filament wanes and force generating cross-bridge cycling also wanes as cross-bridges detach and do not re-enter the force generating state. The cell and sarcomere return to the diastolic state ready for the next cycle. Tension is shown to fall in the isotonic twitch, and as pressure in the LV falls below

18  REGULATION OF CARDIAC CONTRACTILITY

the pressure in the aorta, the one-way aortic valve closes and LV pressure falls with no change in ventricular volume. Holding the sarcomere or muscle length constant and the ventricular volume isovolumic (dashed lines in Fig. 7), provides an approach for revealing the state of contractility in the heart function shown in Figure 7. This maneuver permits isolating the contractility from changes in sarcomere length and afterload, both of which affect the number of force generating cross-bridges reacting in a force generating cycle. In this case the sarcomere and muscle strip cannot lift the load and develop the maximum isometric tension at the particular length and the ventricle develops maximum pressure at the particular constant volume (dashed lines in the right panel of Fig. 6). The approach (in animal experiments) to establishing the isovolumetric beat shown in Figure 6 is to cross-clamp the aorta making resistance and afterload infinite. This can be done reversibly to provide a snapshot of the peak pressure in an isovolumetric beat and a measure of the contractility in essentially the same way that peak tension is a measure of contractility in the sarcomere. The num‑ ber of force generating cross-bridges under these conditions is thus determined by this maneuver and provides a read out by contractility. A common definition of contractility is the peak tension when the sarcomeres are neither lengthening nor shortening. As we will discuss, a useful correlate of this point in the normal beat of the heart is at the end systolic pressure at the end of ejection and as the heart enters into isovolumetric relaxation. In an isometric twitch (isovolumic beat), peak tension (peak pressure) reflects relative number of  Tn sites binding Ca2+, the relative length of the sarcomeres, and the response of the myofilaments to Ca2+. As we will see these amounts of Ca2+ delivered to the myofilaments and thus the number of  Tn units and cross-bridges recruited into the beat are a regulated variable in heart muscle cells. However, as we will also discuss, the response of the myofilaments to Ca2+ is also a variable affecting peak tension that must be considered in the generation of peak tension (pressure) or contractility in the isometric twitch (isovolumetric beat).

19

Pressure Volume Loops Provide a Quantification of Contractility Assessing changes in contractility in the human and comparing the state of contractility (absolute contractility) between and among groups of humans represents a significant problem. Clearly this must be done in the human as non-invasively as possible. In discussing the illustrations and data in Figures 1–3, we alluded to the concept of ESV (end systolic length) as a measure of the ability of the ventricle to contract. Plotting of the relation between ventricular volume and pressure during a beat, referred to as P-V loops provides a powerful approach for determination of contractility in the in situ beating heart. Although not discussed here this relation forms a theoretical basis for interpretation of non-invasive echo-cardiographic assessment of changes in ventricular volumes. Figure 8 compares the P-V measurement to the time course of volume and pressure changes in a beat of the heart and indicates the relation between the ventricular volumes during points in the cycle (EDV and ESV) and sarcomere length. Afterload is reflected in the ventricular pressure during ejection. Note that at end systole, the myofilaments contain bound Ca, are at a length that is neither lengthening nor shortening and are at roughly a constant afterload. This point thus has the properties for providing measure related to contractility. The illustrations in Figure 9A and 9B aids in understanding the utility of the P-V loop when analyzing the integral effects of pre-load, afterload, and contractility in controlling cardiac out put. The figure shows a series of beats in response to an increase in VR to a new steady-state. Beat #1 is the resting steady state; and, as exemplified by the data in the exercise example of Figure 2, VR has increased as indicated during beat #2. In beat #2 the SV matches the loading volume. However in beat #3 the SV does not match the loading volume because the after load has increased in response to the increased SV ejected into the stiff aorta. Thus the shortening of the sarcomeres is less in beat #3 versus beat #2. In beat #4 loading volume and SV are matched, but this occurs with an increase in EDV and ESV. Note that the line drawn through the ESP values (ESV-ESP relation) in the various beats provides a curve of contractility, which has not changed in example shown in Figure 9A. In experiments with the objective of deriving contractility from the ESV-ESP curve maneuvers similar to that described in Figure 9 are carried out altering VR either by rapid infusion of an intra-venous

CaCaCa

CaCaCa

CaCaCa

mm Hg

CaCaCa

(mm Hg)

LV Pressure

20  REGULATION OF CARDIAC CONTRACTILITY

120

Aortic Pressure

60

LV Pressure

LA Press

0

Vol

(ml)

EDV

ESV

Volume

120

75 ECG

0.0

0.4

0.8

Time (s)

Figure 8: Plot of the relation between ventricular pressure as a function of ventricular volume. The right panel illustrates the time course of changes in volumes and pressures during the cardiac cycle. The left panel illustrates the pressure-volume relation emphasizing the end diastolic sarcomere length established by the EDV and the end systolic sarcomere length with Ca2+ bound to TnC. The pressure at end systole provides an analogue of contractility.

solution to increase VR or in animal experiments by compression of the vena cava to decrease VR. The assumption is that the alterations in contractility do not occur rapidly enough to seriously affect the ESV-ESP relation, thus permitting a snapshot of contractility. We see in Figure 9B (extension of the beats shown in Figure (A) that shifts in the curve induced by induction of mechanisms (e.g., beta-adrenergic signaling) for increasing contractility shift and rotate the ESV-ESP relation to the left, thus permitting a reduction in ESV at the elevated VR. Figure 9 shows the beginning point as the same steady state as in beat #4 of Figure 9. However, with an increase in contractility, in beat #5, SV is now greater than loading volume, and with VR constant at the elevated level, ESV may be reduced by the increase in contractility thereby permitting the increased SV and CO to occur with little change in EDV from beat #1 (Fig. 9A). As indicated in Figures 9A and 9B and stressed in the previous sections, a surrogate of muscle and sarcomere length is ventricular volume and the surrogate of muscle tension is pressure. Thus, the pressure-volume relations essentially reflect length tension properties of muscle. This is emphasized

Pressure Volume Loops Provide a Quantification of Contractility  21

EDV SV 1 Pressure in mm Hg (Tension)

A

4

3

2

EDV ESV

VR

ESV

SV after increase in VR and Afterload

200

Control SV

3 4

100

2

1

30

60

90

120

150

Ventricular Volume (Length)

B

4

5

6

Pressure in mm Hg (Tension)

Contractility ESV-ESP Relation

200

(Contractility)

100

30

60

90

120

150

Ventricular Volume (Length)

Figure 9: Pressure volume (P-V) relations of a series of cardiac cycles following an increase in VR to a new steady state. A. As discussed more fully in the text, with an increase in VR to a new steady level at constant HR, there is a transient mismatch between the loading volume and the stroke volume and in the new steady state indicated in beat #4 CO matches VR, but the system is operating at an elevated EDV. The end systolic pressure (ESP) points, designated with filled circles, represent points reflecting contractile state (contractility). B. Illustration of the effect of increased contractility to permit the increase in VR to be matched by a CO operating with an EDV similar to that in beat #1.

22  REGULATION OF CARDIAC CONTRACTILITY

by the illustrations in Figure 9, which indicates the with the dashed P-V loops changes occurring after an increase in VR achieved by rapidly increasing the loading volume through an increase in VR. These ESP points reflect isovolumetric pressure or isometric cellular tension and are points rooted in the sarcomere length tension relation. It is important to realize that a line connecting these points represents a constant state of contractility. It is also important to realize that the relation between ventricular pressure and tension is related to the Frank-Starling relation (Starling’s law of the heart; also classically referred to as hetero-metric auto-regulation). The pressure or tension is different at each of the points because the muscle length, with each of the beats depicted in Figure 9 most likely occurred with the same amounts of Ca2+ released to the myofilaments. However it is now well accepted that the slope of the P-V loop is also related to a variation in the response of the myofilaments to Ca2+ also known as length dependent activation. With increases in sarcomere length tension increases not only because the region of overlap between thin and thick filaments is increasing, but also because the response to Ca2+ increases. Mechanisms for length dependent acti‑ vation are not fully agreed upon and may be related to an effect of a force generating cross-bridge in one unit of the thin filament (Fig. 6) to promote activation of a near neighbor unit. Increases in length, which stretch titin, and modify the titin-MyBP-C-cross-bridge interaction, may also affect radial movements of the cross-bridges increasing their local concentration in the functional unit. Note also that the ESP reflects the extent of shortening as previously discussed. As contractility increases, the ESP-volume relation will rotate and shift and thus at a given afterload the sarcomere will be able to shorten to a greater extent, circumferential shortening will occur to a greater extent, and the SV will be increased at a given afterload. We take this concept up again, after we consider the cellular mechanisms by which contractility can be altered.

23

Phosphorylations of Regulatory Proteins in Excitation Contraction Coupling Modify Contractility by Controlling Cellular Ca2+ Fluxes, the Response of the Myofilaments to Ca2+, and the Kinetics of the Cross-Bridge Cycle Adrenergic and cholinergic signals in the autonomic nervous system are major controllers determining the state of contractility in the myocardium. The effects of adrenergic stimulation at the level of the cardiac myocytes are indicated in isometric twitches shown in Figure 10A and in the pressure traces in Figure 10B. The data show there is an increase in the peak amplitude of force and pressure with adrenergic stimulation and in the peak amplitude of the Ca-transient. With adrenergic stimulation There is also abbreviation of the action potential duration and an abbreviation of the over cycle time of the both tension, pressure, and the Ca transient. Linkage of signals arising from the transmitters of the autonomic nervous system as well as blood levels of neurohumors such as epinephrine and acetylcholine affect EC coupling via an elaborate signaling cascade. In Figure 2 the activity of this system was revealed by propranolol treatment of the subject performing the exercise. The binding of neurotransmitters, neurohumors, or pharmacological agonists to adrenergic or cholinergic receptors triggers the cascade or in the case of propranolol blocks the cascade. Gproteins, which are GTP binding proteins, transduce the receptor binding signal to an alteration of the enzyme activity of adenylyl cyclase, which is responsible for the generation of cyclic AMP from ATP. Stimulatory G proteins (Gs) linked to adrenergic beta-receptors promote the formation of cAMP, whereas inhibitory G proteins (Gi) inhibit adenylyl cyclase. cAMP activates protein kinase A, which phosphorylates key proteins that regulate the entry and exit of Ca2+ from the myofilament

24  REGULATION OF CARDIAC CONTRACTILITY Basal State

mV

Ca2+

mN

Adrenergic Stimulation

B.

Action Potential

Catransient

Force

Time

Basal State Adrenergic Stimulation Adrenergic inhibition

Pressure (mm Hg)

A.

Isovolumic LV Pressure

120

60

0

Time

Figure 10: Effects of adrenergic stimulation on cardiac dynamics. A. Data from measurements of action potential, intracellular Ca2+ transients and isometric tension in a single cardiac myocyte in a basal state and during stimulation with an adrenergic agonist. B. Readout of the changes at the cellular level in the isovolumetric beats of the left ventricle. The effects of adrenergic stimulation are to abbreviate the action potential, increase the amplitude and dynamics of both the Ca2+ transient, twitch tension, and isovolumetric pressure development.

space. As mentioned above in the case of the SR, PLB is the major PKA substrate; which, in its dephosphorylated state, inhibits the rate of transport of SR Ca-transport through its interaction with SERCA2a. PLB is also a substrate for Ca2+ activated calmodulin dependent kinase (CAMK). This Ca2+ dependent phosphorylation appears important in a “staircase” effect in which force generated by the myocardium increases with HR. With the increased frequency associated with an increase in HR, there is a greater influx of Ca2+ due to increased amplitude and delayed inactivation of ICa. With phosphorylation of PLB by either PKA or CAMK, there is suppression of the PLBSERCA2a interaction and a release of the Ca2+ pump activity from inhibition; Ca2+ affinity of the pump increases, without changes in the maximum velocity. This increase in Ca2+ uptake increases Ca2+ loaded into the SR and induces an accelerated relaxation of the myocytes. This increase in rate of Ca2+ removal from the cytoplasm and myofilaments accounts substantially for the enhanced relaxation and abbreviated contraction/relaxation cycle during adrenergic stimulation. PKA also phosphorylates a subunit in the oligomeric assembly of proteins that make up the L-type Ca2+

Phosphorylations of Regulatory Proteins  25

channel of the heart. The phosphorylation enhances the probability that the channel will open upon depolarization, but apparently does not affect the unitary conductance. Thus, “sleeping” Ca2+ channels awaken when phosphorylated. This increase in the trigger for Ca2+ release together with the increased Ca2+ loading associated with PLB phosphorylation essentially accounts for the increase in the systolic Ca2+ transient. Regulation of the release of Ca2+ through SR RyR2s by PKA also provides a mechanism to control delivery of Ca2+ to the myofilaments and thus to control contractility. In this case the mechanism involves PKA-mediated phosphorylation of the binding protein, FKBP12.6. In the absence of phosphorylation, FKBP12.6 stabilizes clusters of RyR and enhances coupled gating within the clusters. This coupling determines the “gain” of the Ca2+ release mechanism, and in the absence of phosphorylation of FKBP12.6, the gain is high for SR Ca2+ release. In this context, the term “gain” means the amount of Ca2+ released for a given change in Ca2+ current. Phosphorylation of FKBP12.6 removes this stabilizing effect on the clusters of RyRs, and results in reduced SR Ca2+ release due to decreased CICR gain, and increased Ca2+ leak from the SR during diastole. PKA dependent phosphorylation of an isotype of K+ channels is also important as a determinant of the duration of the ventricular action potential, which is reduced with adrenergic stimulation (Fig. 10A). It is now recognized that autonomic nervous system signaling to the sarcomere is also an important factor in controlling contractility (Solaro, 2008). Two major substrates for PKA are TnI and MyBP-C. The major impact of phosphorylation of  TnI, which is a unique property of the adult cardiac isoform, is to increase the off rate for Ca-exchange with TnC. Thus, not only does phosphorylation of PLB increases removal of Ca2+ from the cytoplasm, but phosphorylation of TnI also promotes release of Ca2+ from its TnC binding sites. There is also evidence that phosphorylation of MyBP-C by PKA acts to increase cross-bridge kinetics. This increase in kinetics promotes the entrance and the exit of the cross-bridges into and out of the force generating cycle. Increases in the rate of tension development are related to this increase in cross-bridge kinetics and to increases in the rate of release of Ca into the cytoplasm. In view of the kinetic changes, measurement of the maximum rate of rise of pressure (+dp/dt max) during systole provides a way of assessing contractility. Moreover, –dp/dt max provides a measure a measure of relaxation kinetics or lusitropy. The maximum rate of pressure development occurs before the opening of the aortic valve and has proved a useful index of the contractile state of the myocardium. Enhanced cross-bridge cycling rates are also well documented to promote the rate of relaxation. Figure 11 summarizes the integrated effects of modifying properties of membrane and myofilament proteins by phosphorylation, which tightly control contractility. As emphasized above, the phosphorylations are critical to the ability of the heart to tune its activity cycle to the fast heart rates during adrenergic stimulation and to accommodate the increasing VR without a significant change in EDV.

26  REGULATION OF CARDIAC CONTRACTILITY

Autonomic Nervous System NE Ach

G pr o te in s

AC ATP PKA off

cAMP

PDE AMP PLB

Ca-Channel

RyR

MyBP-C

RyR- P

MyBP-C -P

PKA on PLB- P Ca-Channel- P SR CaUptake Rate

SR CaRelease

X-Bridge Kinetics

TnI

TnI -P TnC - CaRelease

Figure 11: Autonomic signal transduction and signaling in cardiac muscle cells. Binding of neurotransmitters acetylcholine (ACH) and nor-epinephrine (NE) to the receptors induces an activation (NE) or inhibition (ACH) of adenylyl cyclase (AC), elaboration of cAMP, and activation of protein kinase A (PKA). Levels of cAMP are also regulated by the activity of phosphodiesterases (PDE) that convert cAMP to AMP. PKA phosphorylates phospholamban (PLB), L-type Ca2+ channel subunits, and ryanodine receptors (RyR) as well as K channels (not shown). There is also PKA dependent phosphorylation of myosin binding protein C (MyBP-C, and troponin I (TnI)). The phosphorylations form an integrated signaling cascade with effects on Ca2+ uptake and release and uptake as well as altered cross-bridge kinetics with a net result of increased contractility and dynamics of contraction and relaxation. See text for further discussion.

27

Contractility May Be Altered by a Variety of Mechanisms Not Involving a Prominent Role for the Autonomic Nervous System Phosphatases are important through their action to de-phosphorylate the regulatory proteins affecting contractility. Although once pictured to be constitutively active and not significantly regulated, it is now clear that phosphatases are highly regulated by signaling pathways. This is an active and evolving area of investigation. Control of the breakdown of cAMP by phospho-diesterases (PDE) is also an important mechanism for control of contractility. There is also involvement of cGMP dependent kinases. Another major mechanism for control of contractility is the chemical environment in the cytoplasm. Intracellular pH, ionic milieu, reduction/oxidation state, and levels of nitric oxide and its metabolites also affect the variety of protein-protein interactions underlying EC coupling. This occurs via direct modification (oxidation, nitrosylation, acetylation) of key membrane or myofilament proteins or via indirect action on the enzymes (kinases/phosphatases, nitric oxide synthase, acetylases, de-acetylases etc.) controlling post-translational modifications of the proteins affecting contractility. An emerging area of significance is the role of lipid signaling in the control of contractility. This has become increasingly important to understand clinically with the increase in metabolic syndrome and cardiac lipotoxicity. Pharmacological agents that affect contractility, which are known as inotropic agents, represent important therapeutic approaches. Some agents such as dobutamine, mimic adrenergic neuro-transmitters, and some agents such as digitalis, indirectly increase Ca2+ loading into the SR by inhibiting the Na, K ATPase, reducing the Na gradient and thus inhibiting Ca2+ extrusion through the Na,Ca2+ exchanger. There are also inotropic agents, which work directly by inhibiting PDE or altering the myofilament response to Ca2+.

28

Cardiac Function Curves Provide a Compact Graphical Representation of Regulation of CO and SV Another logical and useful view of regulation of cardiac output that has stood the test of time are so-called “Starling Curves” or “The Starling Relation” or simply “Cardiac Function Curves.” Although these curves may take on different forms, a useful and straight forward relation is the plot of a measure of the filling of the heart versus a measure of ejection. For example, plots relate a variety of measures of ventricular filling such as right atrial pressure (RAP) or EDP to a variety of measures of ejection referred to in some cases as “ventricular performance” usually pressure/volume work. The relation between the volume/pressure curves shown in previous figures and the generation of a relation between EDV and SV is illustrated in Figure 12. The three beats shown at different pre-loads in Figure 12 occurred with no change in contractility (same vol-ESP curve) or afterload. The curves emphasize that cardiac function curves relate measures of filling and ejection at constant afterload and contractility. Shifts in the EDV-SV relation induced by changes in contractility and afterload are illustrated in the left panel of Figure 13. With an increase in contractility the curve is rotated up and to the left and demonstrates new steady states operating at reduced EDV. The opposite occurs with a decrease in contractility or an increase in afterload as illustrated in the left panel of  Figure 13. Figure 14 shows another form of cardiac function in which CO is the dependent variable, which is easily generated from the knowledge of the HR. If one substitutes RAP, the ultimate determinant of EDV (EDP), the cardiac function becomes one emphasized by Guyton and colleagues. Inasmuch as RAP is common to VR and CO, this permitted plots of venous return curves and cardiac function curves on the same set of coordinates. See other texts for more discussion of this useful concept in the interpretation of cardiac function (Guyton and Hall, 2006). The emphasis here is on the molecular control mechanisms underpinning the cardiac function curves. In thinking about the application of cardiac function curves, it useful to realize that at a particular steady state that one is operating on a particular point in the relation at steady state contractil-

Cardiac Function Curves  29

Pressure in mm Hg

1

2

EDV at constant afterload and contractility

3

200

3

2 1

SV

100

30

60

90

120

150

50

Ventricular Volume (ml)

100

EDV

150

Figure 12: Generation of cardiac function curves from pressure-volume relations. Three beats are shown with increases in preload at constant contractility and afterload. A plot of stroke volume (SV) at each of the end diastolic volumes (EDV) associated with each beat generates a common form of the cardiac function curve or Starling relation.

contractility at constant afterload or afterload at constant contractility

SV

contractility at constant afterload or afterload at constant contractility

SV

50

100 EDV

150

50

100 EDV

150

Figure 13: Shifts in cardiac function curves with alteration in contractility and afterload. The figure emphasizes that each curve represents a state of constant contractility and afterload. Shifts in the cardiac function curve from a physiological basal state (solid line) as indicated occur with changes in after load and contractility.

30  REGULATION OF CARDIAC CONTRACTILITY

SV (ml)

oad ility erl t Aft trac n o C

CO (L/min)

200

12

100

6

100

ad Afterlo tility c a tr n o C

200

EDV (ml)

300

Figure 14: Generation of cardiac function curves relating CO to EDV. As in Figure 14, this figure emphasizes that each curve represents a state of constant contractility and afterload. Shifts in the cardiac function curve from a physiological basal state (solid line) as indicated occur with changes in after load and contractility.

ity and afterload. One must view a cardiac function curves as one of a family of curves with the heart moving from curve to curve with altered contractility and afterload and up and down the same curve with a change in preload. For example, the hemodynamic data in Figure 2 can be perceived in terms of cardiac function curves. Before the exercise episode one imagines the subject at a point of a curve at a resting EDV and SV. With the anticipation and beginning of exercise adrenergic nerves to the heart and blood vessels were activated. In the case of  VR, flow of blood to the heart increased owing to the influence of neural inputs on the wall tension of the blood vessels, and the mechanical action of the muscles to impel blood venous blood in the large vessels toward the heart. The heart thus faced the task of reacting to the increased VR. Major control mechanism came into play to match the CO to the VR economically. These mechanisms included an increase in HR, and an increase in the capability of the myocytes and sarcomere to shorten i.e. an increase in contractility. At the same time the adrenergic activation may have transiently increased resistance, and therefore afterload, to redistribute flow from the gut and kidneys to skeletal muscles. Thus steady states points describing the EDV-SV relation switch to a different curve dictated by the elevated contractility and afterload. A useful exercise is to pick a point on the control curve in Figure 13 and predict where this point moves during the time course of the changes shown in Figure 2. This exercise will emphasize and clarify the integration of pre-load, after load and contractility in homeostasis of blood flow.

31

Heart Failure as a Failure of Contractility The term heart failure describes a complex set of signs and symptoms reflecting a failure in many cases of a number physiological systems, including the heart, kidney, and blood vessels. Primary disorders of the heart related to a reduction in coronary flow and elevated afterload (hypertension) are common stresses leading a change in cardiac function. When viewed in the context of cardiac function curves, heart failure is quantified by a downward shift of the EDV-SV relation. In this situation, maintenance of a SV sufficient to meet tissue oxygen needs may occur in the resting state, but at a severe cost in terms of elevated EDV and EDP. With elevated LV EDP, atrial pressure must increase to fill the LV during diastole and pulmonary venous pressure must increase to move blood from the lungs to the left atrium. The increase in pulmonary venous pressure is a major factor inducing a transudation of fluid into the interstitial space of the lungs, thereby hindering gas exchange at the alveoli. This exacerbates the inefficient use of ATP at relative high EDV described in Figure 3 in the context of Laplace’s law. That is, with a loss of a reserve of contractility reflected in the downward shift of the EDV-SV relation maintaining tissue oxygen needs becomes severely stressed. To maintain life the heart must match CO to VR, and that task will be met at the expense of all else. In the case of heart failure the expense is a severe limit on exercise. Thus the patient with symptomatic heart failure has a severe limitation in exercise capability and quality of life. Discussion of the mechanism of transition of hearts into failure is beyond the scope of this consideration of control of contractility. However, understanding the basic mechanisms controlling cardiac contractility provides a strong base for understanding the theories and experiments relating to the mechanisms of heart failure. For example, there is evidence that with long standing increased afterload (hypertension) heart cell begin to remodel. They cannot divide, but they grow bigger (hypertrophy). With hypertrophy there are shifts in the isoform population and abundance of some of the key proteins in EC coupling. For example one common result is a reduced concentration of SERCA2a in the myocytes compared to the myofilaments. The reduction in Ca2+ delivery to TnC reduces contractility and limits the recruitment of cross-bridges during exercise. Another example is the idea that an activation of kinases that promote cardiac hypertrophy have an effect to inhibit

32  REGULATION OF CARDIAC CONTRACTILITY

the ability of Ca2+ to activate the myofilaments through a maladaptive phosphorylation of key sarcomeric regulatory proteins. In the case of coronary artery disease or in the response to a myocardial infarction, there is also evidence that apart from the reduced nutrient and gas exchange, the heart attack signals cardiac remodeling with changes similar to those with hypertension. More recently genetic linkage analysis has identified mutations in key proteins of cardiac EC coupling that induce cardiac remodeling and sudden death. In this case there is no doubt the disorder arises in the heart from a change in the heart muscle and not the blood vessels. Disorders of the heart what ever the cause remain epidemic in world heath, and detailed understanding of control of cardiac contractility is essential in the quest for measures to prevent, diagnose, and treat these disorders (Katz, 2010; Opie, 2004).

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References Bers, DM. Excitation-Contraction Coupling and Cardiac Contractile Force. 2nd edition. Kluwer Academic Publishers, Dordrecht/Boston/London, 2001. Guyton AC, Hall JE. Textbook of Medical Physiology. 11th edition. Elsevier Saunders, Philadelphia, 2006. Hinken AC, Solaro RJ. A dominant role of cardiac molecular motors in the intrinsic regulation of ventricular ejection and relaxation. Physiology (Bethesda). Apr 2007;22:73–80. doi:10.1152/ physiol.00043.2006 Katz, AM. Physiology of the Heart. Lippincott, Williams, and Wilkins, Philadelphia, 2010. Kobayashi T, Solaro RJ. Calcium, thin filaments, and the integrative biology of cardiac contractility. Annu Rev Physiol. 2005;67:39–67. doi:10.1146/annurev.physiol.67.040403.114025 Kruger M, Linke WA. The giant protein titin: a regulatory node that integrates myocytes signaling pathways. J Biol Chem. 2011 Jan 21. MacLennan DH, Kranias EG. Phospholamban: a crucial regulator of cardiac contractility. Nat Rev Mol Cell Biol. Jul 2003;4(7):566–577. doi:10.1038/nrm1151 Opie, LH. Heart Physiology. From Cell to Circulation. 4th Edition, 2004. Page E, Fozzard, H, Solaro RJ, Eds. Handbook of Physiology: Section 2: The Cardiovascular System. Volume 1. The Heart. Oxford University Press, New York, 2001. Solaro RJ. Multiplex kinase signaling modifies cardiac function at the level of sarcomeric proteins. J Biol Chem. Oct 3 2008;283(40):26829–26833. doi:10.1074/jbc.R800037200

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Author Biography R. John Solaro is a distinguished university professor and head of the Department of Physiology and Biophysics at the College of Medicine, University of Illinois at Chicago. In 1975–1976, as a British-American Heart Fellow, he studied with Professor S. V. Perry in Birmingham, England. In 1977, he joined the faculty at the University of Cincinnati, where he was supported by an NIH Research Career Development Award and was offered an AHA Established Investigator award. In 1987, he was a Fogarty International Fellow at University College, London. He is the past director and founder of the UIC Center for Cardiovascular Research. At UIC, Dr. Solaro has received the University Scholar Award, the Faculty of the Year Award, and the Mentor of the Year Award. Dr. Solaro’s major research interest is in the general area of cellular and molecular mechanisms controlling the contraction of the heart and how these mechanisms are altered in pathological conditions and by pharmacological interventions. He has published more than 300 peer-reviewed papers.