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Biochemistry and physiology of cardiac muscle
THE NORMAL HEART
Biochemistry and physiology of cardiac muscle
Key points
Adam Nabeebaccus
C
Intracellular calcium regulation and its interaction with the myofilaments is central to the normal contractile function of the heart
C
Physiological contractile regulation involves the Franke Starling response, heart rate-dependent regulation, autonomic control and the effects of autocrine/paracrine factors such as nitric oxide
C
Calcium regulates other important processes within myocytes, including excitationetranscription coupling and mitochondrial function; this is achieved through spatial and temporal compartmentation of the calcium signal in cellular microdomains
C
Cross-talk between different cell types within the heart (cardiomyocytes, endothelial cells, fibroblasts, immune cells) is important both for normal physiological function and in the diseased heart
C
Overall, cardiac function is also influenced by other cellular processes including the turnover of cell constituents, metabolic function, redox state, epigenetic control and regulation of cardiomyocyte viability
Ajay M Shah
Abstract The heart is composed of muscle cells (cardiomyocytes) that account for most of the heart mass and generate its pumping force. Other cell types (fibroblasts, vascular endothelial cells, vascular smooth muscle cells, immune cells) and the extracellular matrix also play key roles in cardiac function, in both health and disease. Excitatione contraction coupling links the electrical activation of cardiomyocytes to cellular contraction. Calcium is a key second messenger in this process; its entry into the cell triggers further calcium release from the sarcoplasmic reticulum, which then activates the contractile machinery. Subsequent reduction in calcium concentration brings about cardiac relaxation, which is necessary for the heart to refill. Calcium also regulates other critical processes in the heart, including transcription of genes and the matching of energy supply from the mitochondria with cellular demand. In health, the contractile function of the heart is regulated by several factors, including its loading conditions, autonomic influences and many locally produced autocrine/paracrine agents. These factors alter contractile strength through two main mechanisms: modulation of the calcium transient within cardiomyocytes, and/or changes in myofilament sensitivity to calcium.
contractility, dysfunction of which is implicated in disease states such as heart failure.
Keywords Calcium; cardiomyocyte; contractile function; excitatione contraction coupling; fibroblasts; myofilament
Structure of the myocardium The heart is composed of cardiomyocytes, fibroblasts, endocardial and endothelial cells, immune cells, coronary vessels and ECM.1
Introduction
Cardiomyocytes account for most of the cardiac mass and volume but only approximately 30% of cardiac cell numbers. They are connected to each other via specialized gap junctions, which provide electrical coupling and allow an action potential to spread between adjacent cardiomyocytes by the intercellular movement of ions. This is vital for synchronized contraction of myocytes. Gap junction channels are formed from a family of proteins known as connexins. The sarcolemmal membrane of cardiomyocytes has invaginations that form an extensive T-tubule network, regions of which lie in close apposition to the sarcoplasmic reticulum (SR). SR is the major intracellular store of calcium and a central regulator of cardiac contractility. The fundamental contractile unit, the sarcomere, is formed from contractile myofibrils, which comprise interdigitating thin filaments (actin and associated regulatory proteins, tropomyosin, troponins C, I and T) and thick filaments (myosin). The sarcomere also contains numerous noncontractile proteins (e.g. titin, myomesin, telethonin) that have important structural and signalling functions. Interspersed between the myofibrils are numerous mitochondria, which
The synchronous contraction of cardiomyocytes during ventricular systole generates the power required to pump blood out of the heart. Conversely, active myocyte relaxation and passive mechanical properties of the ventricles (the latter largely dependent on the extracellular matrix (ECM)) determine filling of the heart during diastole. Several interacting regulatory processes operate to ensure that cardiac performance is finely tuned to match changing circulatory requirements. In this article, we provide an overview of the mechanisms that regulate cardiac
Adam Nabeebaccus PhD MRCP is a Clinical Lecturer at King’s College London, UK. His research interest is the mechanisms of cardiac remodelling in response to chronic cardiac stress. Competing interests: none declared. Ajay M Shah FRCP FMedSci is British Heart Foundation (BHF) Professor of Cardiology and Director of the King’s College London BHF Centre of Excellence, UK. His research interest is the pathophysiology of cardiac remodelling. Competing interests: none declared.
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THE NORMAL HEART
generate the energy (in the form of adenosine triphosphate (ATP)) to fuel contraction.
capillary level, there is a close apposition between endothelial cells and cardiomyocytes. These endothelial cells not only provide the lining of blood vessels, but also modulate cardiac function through the release of diffusible factors (described below).
Fibroblasts are the most numerous cells in the heart. They are responsible for the continual production and turnover of the ECM of the heart. In response to injury, such as myocardial infarction, fibroblast increase in number and undergo a phenotypic change to so-called myofibroblasts, which play a crucial role in organ repair and healing by fibrosis. In experimental models of cardiac injury, some of these myofibroblasts may be recruited from circulating bone marrow-derived cells or from local endothelial cells that have undergone a phenotypic change known as endothelialemesenchymal transition.
Immune cells also reside in the healthy myocardium and interact with cardiomyocytes, fibroblasts and ECM to help maintain normal myocardial structure and function. In the injured myocardium (e.g. after myocardial infarction, in chronic heart failure), a change in immune cell number and subtype makes an important contribution to the overall myocardial remodelling process. Damage-associated molecular patterns, comprising components of injured cells and tissues, are involved in stimulating immune responses. The effect of immune activation ranges from damaging inflammatory responses to tissue reparative processes whose overall balance dictates the acute and chronic response to cardiac injury.
ECM is a complex array of molecules that provides structural support to the cellular components of the heart. The ECM also allows appropriate transmission of the mechanical forces generated by cardiomyocytes. The major components of the ECM are types I and III collagen. The ECM also contains various protease enzymes, which allow degradation of matrix components. Important among these are the matrix metalloproteinases, of which there are over 20 known subtypes.
Excitationecontraction coupling and contractile function Electrical excitation of the cardiomyocyte initiates a dramatic transient rise in intracellular calcium concentration (the so-called calcium transient). The events that couple sarcolemmal depolarization to elevation of calcium concentration and initiation of contraction are known as excitationecontraction coupling (Figure 1).2
The main coronary arteries, which provide the heart with its blood supply, sit on the epicardial surface of the heart. They divide into smaller blood vessels that penetrate the myocardium. At
Excitation–contraction coupling in cardiac myocytes Na+–Ca2+ exchanger
L-type Ca2+ Ca2+ channel
3Na+ Sarcolemma The wave of depolarization spreading along the sarcolemma and T-tubule system initiates calcium entry via L-type calcium channels (the calcium current), which stimulates further calcium release from the sarcoplasmic reticulum. The resulting rise in intracellular calcium concentration activates the contractile machinery (actin Following contraction, the cytoplasmic calcium concentration is reduced again by transport back into the sarcoplasmic reticulum (Ca2+-ATPase) and across the sarcolemma (Na+–Ca2+ exchange and sarcolemmal Ca2+-ATPase), thus allowing relaxation.
Ca2+
Ca2+
T-tubule
Sarcoplasmic reticulum
Mitochondrion Ca2+
Ca2+
Sarcolemmal Ca2+-ATPase
Sarcoplasmic reticulum Ca2+-ATPase
Ca2+ Sarcoplasmic reticulum Ca2+-release channel
(Ca2+) Z line
Actin
Myosin
One sarcomere
Figure 1
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THE NORMAL HEART
Calcium is involved in matching mitochondrial energy production (by oxidative phosphorylation) to cardiac work. Various sites within mitochondria, including key dehydrogenases (e.g. pyruvate dehydrogenase and a-ketoglutarate dehydrogenase) and also the F1F0 ATPase, are susceptible to changes in activity determined by local calcium concentration. An abnormality in calcium handling therefore affects not only excitatione contraction coupling, but also mitochondrial metabolism, leading to a cellular energetic disturbance and oxidative stress. The consequent ATP deficit and redox imbalance can further impair excitationecontraction coupling and contractile function. The many described actions of calcium within the cardiomyocyte are feasible because of distinct local calcium concentrations within the cell, in so-called microdomains. For example, excitationecontraction coupling (involving sarcolemmal ICa and SR ryanodine receptors in close proximity) is likely to involve a local free calcium transient signal that is spatially distinct from that seen in perinuclear pathways identified in excitationetranscription coupling.
During each heartbeat, the depolarization wave spreads across the sarcolemma and T-tubule system, and initiates calcium influx through voltage-gated L-type calcium channels. This calcium influx or calcium current (ICa) initiates further calcium release from the SR (calcium-induced calcium release) via the ryanodine receptor. The elementary unit of SR calcium release, the calcium spark, represents calcium released locally from the opening of a few calcium-release channels. According to the local control theory of excitationecontraction coupling, the cell calcium transient induced by an action potential represents the spatial and temporal summation of individual calcium sparks. The contractile machinery is switched on by binding of calcium to troponin C on the thin filament, which enables projections (S1 heads) on the myosin molecules to interact with actin filaments, forming cross-bridges. This energy-requiring process involves ATP hydrolysis by myosin ATPase. Repetitive cross-bridge cycles of attachment and detachment continue as long as the cytosolic calcium concentration is high. The power stroke generated by the cross-bridge cycle is responsible for force generation or muscle shortening. Cross-bridge interactions show cooperativity; in other words, force-generating cross-bridges promote further binding of more cross-bridges, which effectively amplifies the contractile force. Muscle relaxation is governed by lowering of the cytoplasmic calcium concentration, consequent dissociation of calcium from troponin C, and switching off of the actinemyosin interaction. This involves active transport of calcium back into the SR (via SR Ca2þ-ATPase) and extrusion across the sarcolemma, by both the NaþeCa2þ exchanger and (less importantly) sarcolemmal Ca2þATPase. Mitochondria can also accumulate calcium, particularly when cytosolic concentrations become excessively high (e.g. during severe ischaemia). In addition to the reduction in cytosolic Ca2þ, recoil of elastic elements within the myocyte (notably within titin molecules in the sarcomere) may also be involved in the process of relaxation. The events that comprise excitationecontraction coupling influence the size and kinetics of the calcium transient. An abnormally low calcium transient can lead to depressed contractility. Reduction in SR Ca2þ-ATPase activity and abnormalities of SR calcium release (e.g. calcium leak) occur in heart failure and are generally accompanied by diastolic calcium overload; this can contribute to delayed relaxation and diastolic dysfunction, triggering of ventricular arrhythmias and chronic changes in cell structure (e.g. altered gene expression) as a result of activation of downstream calcium-dependent signalling pathways. Up-regulation of the NaþeCa2þ exchanger activity may, to some extent, compensate for reduced SR Ca2þ-ATPase activity. Independent of excitationecontraction coupling, changes in myofilament properties (e.g. responsiveness to calcium) are also implicated in heart failure, ischaemiaereperfusion injury and hypertrophic cardiomyopathy. Calcium concentrations within the cardiomyocyte also influence other cellular processes. They have been found to be involved in the control of gene transcription (so-called excitationetranscription coupling) and may in part mediate processes such as cardiac hypertrophy. The binding of calcium to calmodulin leads to activation of certain protein kinases (e.g. calmodulin kinase) or phosphatases (e.g. calcineurin), which then modulate signal transduction pathways and/or transcription factors to alter the expression of specific genes.
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Contractile reserve and regulation Considerable contractile reserve (Figure 2) is normally available to meet variations in circulatory demand, for example during exercise. At a cellular level, the recruitment of this contractile reserve involves changes in the cytosolic calcium transient and/or myofilament responsiveness to calcium, and is regulated by several important pathways as outlined below. There is usually significant impairment of these pathways in heart failure, which is a major reason for the exercise intolerance characteristic of this condition. The FrankeStarling response describes an increase in contractile force that occurs with increasing myocyte length or stretch (the latter brought about by increased ventricular diastolic volume). The main underlying mechanism is an increase in myofilament responsiveness to calcium, but a length-dependent release of autocrine/paracrine factors may also be involved. The FrankeStarling response is thought to be preserved at a cellular level in human heart failure, but in a heart that is dilated and stiff, it may be functionally impaired by limitation in the ability to stretch myocytes. An increase in heart rate enhances contractile force primarily by increasing sarcolemmal calcium influx per unit time, with consequent increased calcium loading of the SR. This normal positive forceefrequency relationship is greatly blunted or even becomes negative in heart failure. Autonomic control is centrally involved in contractile regulation. Sympathetic activation, involving catecholamine release, has both positive inotropic and chronotropic effects via b-adrenoceptors. These actions are antagonized by parasympathetic release of acetylcholine. The inotropic effect of b-stimulation results from an increase in the intracellular calcium transient caused by increases in ICa and SR calcium release. b-Stimulation also accelerates relaxation (lusitropic action) by stimulating SR calcium uptake, promoting faster dissociation of calcium from the myofilaments, and accelerating cross-bridge cycling. The predominant mechanism for enhanced SR calcium reuptake here is phosphorylation of phospholamban, thereby relieving tonic inhibition on the SR ATPase by this protein. Reduced responsiveness to b-adrenergic stimulation is a fundamental feature of human heart failure.
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THE NORMAL HEART
Contractile reserve –
β-stimulation Heart rate
Activation
Calcium current
Nitric oxide Acetylcholine +
Ca2+
+
Sarcoplasmic Ca2+ reticulum Ca2+ release
Ca2+ transient Ca2+
Negative inotropic effect
– +
Angiotensin II Endothelin 1 Length
+ Positive inotropic effect
Relaxation Sarcoplasmic reticulum Ca2+ uptake
+ β-stimulation +
β-stimulation Nitric oxide Angiotensin II Endothelin 1 Length
Sarcolemmal Ca2+ extrusion
Decreased cytoplasmic Ca2+ concentration
+
+ –
Ca2+ dissociation
Positive lusitropic effect
– Negative lusitropic effect
These are the major pathways by which muscle length, heart rate, autonomic control ( β-adrenergic stimulation) and paracrine factors (nitric oxide, endothelin 1 and angiotensin II) produce changes in: • level of activation and contractile strength (inotropic effects, top) • rate of relaxation (lusitropic effects, bottom). Blue arrows indicate an increase and red arrows a decrease in the components of excitation–contraction coupling. β-adrenergic stimulation. Figure 2
Autocrine/paracrine regulation e the endothelial cells within the coronary microvasculature, fibroblasts and cardiomyocytes themselves all release bioactive factors that can regulate cardiac contraction as well as having chronic effects on cardiac structure. The physiological release of such factors is likely to involve local signals such as mechanical forces, oxygen tension and local autacoids. In the physiological setting, nitric oxide can have direct actions on cardiomyocytes, independent of its vasodilator effects. These include: an acceleration of myocyte relaxation and reduction in diastolic tone, resulting from a reduction in myofilament calcium responsiveness; modulation of excitationecontraction coupling; and a ‘damping down’ of responses to b-adrenergic stimulation. Both endothelial and neuronal isoforms of nitric oxide synthase
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(eNOS, nNOS) are expressed in the heart and exert isoformspecific actions. Abnormal nitric oxide bioactivity (excessively low or high) contributes to contractile dysfunction in cardiac hypertrophy, heart failure and myocarditis. Other local factors such as endothelin 1, angiotensin II and reactive oxygen species (ROS) also modulate contractile properties and remodelling, particularly in the diseased heart. ROS production has been shown to modulate the physiological effects of increased stretch to enhance contraction. Endothelin 1 has potent hypertrophic effects and can also stimulate release of angiotensin II, which has similar actions. Increased angiotensin II production through increased local angiotensin-converting enzyme activity is a cardinal feature of hypertrophy and heart failure, contributing to inappropriate hypertrophy, fibrosis and slowed ventricular relaxation.
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THE NORMAL HEART
multiple mRNAs encoding proteins in the same signalling pathway, and can therefore have profound effects on cellular function. Epigenetic and miRNA regulatory mechanisms that alter contractile function may act through diverse effects, including on calcium- and sodium-handling proteins and contractile proteins. Targeting individual miRNAs could be a powerful therapeutic approach because of the potential to influence entire signalling pathways. Finally, the regulation of cardiomyocyte viability and cell number also impacts on heart function. When individual cardiomyocyte survival is compromised (e.g. after ischaemia), cells can undergo programmed cell death (apoptosis or necrosis), which leads to a decline in overall function. Cardiomyocytes are considered terminally differentiated cells and have very low proliferative potential, apart from early in life. However, it is experimentally possible to induce the division of adult cardiomyocytes, and identifying methods that could achieve significant regeneration of cardiomyocytes in the heart in vivo (and thereby improve cardiac function) is currently an area of major research efforts.5 A
The increased generation of ROS, such as superoxide and hydrogen peroxide, is involved in many of the effects of angiotensin II within the heart. ROS can induce abnormal SR calcium release or activate detrimental signalling pathways that contribute to the development of heart failure. Interestingly, some ROS sources can also mediate adaptive responses in the chronically stressed heart. These pathways could provide novel therapeutic targets in heart failure.3
Other cellular processes important in cardiac muscle function Beyond contractile regulation, several other processes influence optimal cardiomyocyte and heart function. Cellular proteins can be damaged over time and need to be efficiently removed and replaced. Intracellular processes such as autophagy and proteasomal degradation of defective proteins ensure that the cell’s environment for contraction is preserved. Cell organelles such as mitochondria also undergo turnover. In particular, mitophagy is a process that degrades dysfunctional mitochondria and limits damage caused by such mitochondria. Cardiac metabolism is crucial for generating the energy required for both heart contraction and turnover of cellular constituents. The heart can use multiple substrates to generate ATP for contraction, including carbohydrates, fatty acids, ketone bodies and proteins. The ability to adjust substrate use in response to demand, hormonal cues and nutrient status is important in maintaining ATP levels and contractile function. The metabolic status is also crucial in the maintenance of an appropriate redox environment, which affects multiple cardiac functions. Epigenetic regulation also has a role in contractile regulation, cardiac hypertrophy and fibrosis.4 The term refers to modulation of gene expression through mechanisms such as DNA methylation, ATP-dependent chromatin remodelling and other histone modifications (acetylation, methylation), rather than transcription factors. Non-coding RNAs including microRNAs (miRNAs) play important regulatory roles at a post-transcriptional level by binding to messenger RNAs (mRNAs) to silence their translation or promote degradation. Individual miRNAs generally target
KEY REFERENCES 1 Katz AM. Physiology of the heart. 5th edn. Philadelphia: Lippincott Williams & Wilkins, 2010. 2 Bers DM. Calcium cycling and signaling in cardiac myocytes. Annu Rev Physiol 2008; 70: 23e49. 3 Shah AM, Mann DL. In search of new therapeutic targets and strategies for heart failure: recent advances in basic sciences. Lancet 2011; 378: 704e12. 4 Nabeebaccus A, Sag CM, Webb I, Shah AM. Cellular basis for heart failure. In: Mann D, Felker M, eds. Heart failure: a companion to Braunwald’s heart disease. 3rd edn. Philadelphia: Elsevier, 2015; 28e41. Chapter 2. 5 Laflamme MA, Murry CE. Heart regeneration. Nature 2011; 473: 326e35.
TEST YOURSELF To test your knowledge based on the article you have just read, please complete the questions below. The answers can be found at the end of the issue or online here. Question 1 A 64-year-old white woman was found on two occasions to have a raised blood pressure of 162/115 mmHg. She was advised to take amlodipine.
Question 2 A 70-year old woman is admitted in cardiogenic shock. She is commenced on an intravenous infusion of dobutamine. What effect at cellular level can be expected from this treatment? A Increased force of contraction of the myofilaments B Decrease in SR calcium release C Decreased inward calcium current (ICa) D Decreased phosphorylation of phospholamban E Decreased calcium uptake into the sarcoplasmic reticulum during diastole
What is the site of action of this drug in the cardiac myocyte? A Voltage-gated L-type calcium channels B Ryanodine receptor C Sarcoplasmic reticulum-Ca2þ ATPase (SERCA) D NaþeCa2þ exchanger E T-type calcium channels
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THE NORMAL HEART
What peptide, present in excess, is likely to be responsible for the hypertrophy seen in this patient? A Angiotensin ll B Insulin C Acetylcholine D Atrial natriuretic peptide E Troponin I
Question 3 A 58-year-old man presented with a 2-month history of breathlessness on exertion and lying flat. On clinical examination, his heart rate was 96 beats/minute, blood pressure 115/78 mmHg, and jugular venous pressure raised 5 cm. Peripheral oedema was noted. The heart was clinically enlarged, but there were no abnormal heart sounds. Investigations Chest X-ray showed cardiomegaly Echocardiogram showed dilation and hypertrophy of the wall of the left ventricle
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Biochemistry and physiology of cardiac muscle
Key points
Adam Nabeebaccus
C
Intracellular calcium regulation and its interaction with the myofilaments is central to the normal contractile function of the heart
C
Physiological contractile regulation involves the Franke Starling response, heart rate-dependent regulation, autonomic control and the effects of autocrine/paracrine factors such as nitric oxide
C
Calcium regulates other important processes within myocytes, including excitationetranscription coupling and mitochondrial function; this is achieved through spatial and temporal compartmentation of the calcium signal in cellular microdomains
C
Cross-talk between different cell types within the heart (cardiomyocytes, endothelial cells, fibroblasts, immune cells) is important both for normal physiological function and in the diseased heart
C
Overall, cardiac function is also influenced by other cellular processes including the turnover of cell constituents, metabolic function, redox state, epigenetic control and regulation of cardiomyocyte viability
Ajay M Shah
Abstract The heart is composed of muscle cells (cardiomyocytes) that account for most of the heart mass and generate its pumping force. Other cell types (fibroblasts, vascular endothelial cells, vascular smooth muscle cells, immune cells) and the extracellular matrix also play key roles in cardiac function, in both health and disease. Excitatione contraction coupling links the electrical activation of cardiomyocytes to cellular contraction. Calcium is a key second messenger in this process; its entry into the cell triggers further calcium release from the sarcoplasmic reticulum, which then activates the contractile machinery. Subsequent reduction in calcium concentration brings about cardiac relaxation, which is necessary for the heart to refill. Calcium also regulates other critical processes in the heart, including transcription of genes and the matching of energy supply from the mitochondria with cellular demand. In health, the contractile function of the heart is regulated by several factors, including its loading conditions, autonomic influences and many locally produced autocrine/paracrine agents. These factors alter contractile strength through two main mechanisms: modulation of the calcium transient within cardiomyocytes, and/or changes in myofilament sensitivity to calcium.
contractility, dysfunction of which is implicated in disease states such as heart failure.
Keywords Calcium; cardiomyocyte; contractile function; excitatione contraction coupling; fibroblasts; myofilament
Structure of the myocardium The heart is composed of cardiomyocytes, fibroblasts, endocardial and endothelial cells, immune cells, coronary vessels and ECM.1
Introduction
Cardiomyocytes account for most of the cardiac mass and volume but only approximately 30% of cardiac cell numbers. They are connected to each other via specialized gap junctions, which provide electrical coupling and allow an action potential to spread between adjacent cardiomyocytes by the intercellular movement of ions. This is vital for synchronized contraction of myocytes. Gap junction channels are formed from a family of proteins known as connexins. The sarcolemmal membrane of cardiomyocytes has invaginations that form an extensive T-tubule network, regions of which lie in close apposition to the sarcoplasmic reticulum (SR). SR is the major intracellular store of calcium and a central regulator of cardiac contractility. The fundamental contractile unit, the sarcomere, is formed from contractile myofibrils, which comprise interdigitating thin filaments (actin and associated regulatory proteins, tropomyosin, troponins C, I and T) and thick filaments (myosin). The sarcomere also contains numerous noncontractile proteins (e.g. titin, myomesin, telethonin) that have important structural and signalling functions. Interspersed between the myofibrils are numerous mitochondria, which
The synchronous contraction of cardiomyocytes during ventricular systole generates the power required to pump blood out of the heart. Conversely, active myocyte relaxation and passive mechanical properties of the ventricles (the latter largely dependent on the extracellular matrix (ECM)) determine filling of the heart during diastole. Several interacting regulatory processes operate to ensure that cardiac performance is finely tuned to match changing circulatory requirements. In this article, we provide an overview of the mechanisms that regulate cardiac
Adam Nabeebaccus PhD MRCP is a Clinical Lecturer at King’s College London, UK. His research interest is the mechanisms of cardiac remodelling in response to chronic cardiac stress. Competing interests: none declared. Ajay M Shah FRCP FMedSci is British Heart Foundation (BHF) Professor of Cardiology and Director of the King’s College London BHF Centre of Excellence, UK. His research interest is the pathophysiology of cardiac remodelling. Competing interests: none declared.
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THE NORMAL HEART
generate the energy (in the form of adenosine triphosphate (ATP)) to fuel contraction.
capillary level, there is a close apposition between endothelial cells and cardiomyocytes. These endothelial cells not only provide the lining of blood vessels, but also modulate cardiac function through the release of diffusible factors (described below).
Fibroblasts are the most numerous cells in the heart. They are responsible for the continual production and turnover of the ECM of the heart. In response to injury, such as myocardial infarction, fibroblast increase in number and undergo a phenotypic change to so-called myofibroblasts, which play a crucial role in organ repair and healing by fibrosis. In experimental models of cardiac injury, some of these myofibroblasts may be recruited from circulating bone marrow-derived cells or from local endothelial cells that have undergone a phenotypic change known as endothelialemesenchymal transition.
Immune cells also reside in the healthy myocardium and interact with cardiomyocytes, fibroblasts and ECM to help maintain normal myocardial structure and function. In the injured myocardium (e.g. after myocardial infarction, in chronic heart failure), a change in immune cell number and subtype makes an important contribution to the overall myocardial remodelling process. Damage-associated molecular patterns, comprising components of injured cells and tissues, are involved in stimulating immune responses. The effect of immune activation ranges from damaging inflammatory responses to tissue reparative processes whose overall balance dictates the acute and chronic response to cardiac injury.
ECM is a complex array of molecules that provides structural support to the cellular components of the heart. The ECM also allows appropriate transmission of the mechanical forces generated by cardiomyocytes. The major components of the ECM are types I and III collagen. The ECM also contains various protease enzymes, which allow degradation of matrix components. Important among these are the matrix metalloproteinases, of which there are over 20 known subtypes.
Excitationecontraction coupling and contractile function Electrical excitation of the cardiomyocyte initiates a dramatic transient rise in intracellular calcium concentration (the so-called calcium transient). The events that couple sarcolemmal depolarization to elevation of calcium concentration and initiation of contraction are known as excitationecontraction coupling (Figure 1).2
The main coronary arteries, which provide the heart with its blood supply, sit on the epicardial surface of the heart. They divide into smaller blood vessels that penetrate the myocardium. At
Excitation–contraction coupling in cardiac myocytes Na+–Ca2+ exchanger
L-type Ca2+ Ca2+ channel
3Na+ Sarcolemma The wave of depolarization spreading along the sarcolemma and T-tubule system initiates calcium entry via L-type calcium channels (the calcium current), which stimulates further calcium release from the sarcoplasmic reticulum. The resulting rise in intracellular calcium concentration activates the contractile machinery (actin Following contraction, the cytoplasmic calcium concentration is reduced again by transport back into the sarcoplasmic reticulum (Ca2+-ATPase) and across the sarcolemma (Na+–Ca2+ exchange and sarcolemmal Ca2+-ATPase), thus allowing relaxation.
Ca2+
Ca2+
T-tubule
Sarcoplasmic reticulum
Mitochondrion Ca2+
Ca2+
Sarcolemmal Ca2+-ATPase
Sarcoplasmic reticulum Ca2+-ATPase
Ca2+ Sarcoplasmic reticulum Ca2+-release channel
(Ca2+) Z line
Actin
Myosin
One sarcomere
Figure 1
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THE NORMAL HEART
Calcium is involved in matching mitochondrial energy production (by oxidative phosphorylation) to cardiac work. Various sites within mitochondria, including key dehydrogenases (e.g. pyruvate dehydrogenase and a-ketoglutarate dehydrogenase) and also the F1F0 ATPase, are susceptible to changes in activity determined by local calcium concentration. An abnormality in calcium handling therefore affects not only excitatione contraction coupling, but also mitochondrial metabolism, leading to a cellular energetic disturbance and oxidative stress. The consequent ATP deficit and redox imbalance can further impair excitationecontraction coupling and contractile function. The many described actions of calcium within the cardiomyocyte are feasible because of distinct local calcium concentrations within the cell, in so-called microdomains. For example, excitationecontraction coupling (involving sarcolemmal ICa and SR ryanodine receptors in close proximity) is likely to involve a local free calcium transient signal that is spatially distinct from that seen in perinuclear pathways identified in excitationetranscription coupling.
During each heartbeat, the depolarization wave spreads across the sarcolemma and T-tubule system, and initiates calcium influx through voltage-gated L-type calcium channels. This calcium influx or calcium current (ICa) initiates further calcium release from the SR (calcium-induced calcium release) via the ryanodine receptor. The elementary unit of SR calcium release, the calcium spark, represents calcium released locally from the opening of a few calcium-release channels. According to the local control theory of excitationecontraction coupling, the cell calcium transient induced by an action potential represents the spatial and temporal summation of individual calcium sparks. The contractile machinery is switched on by binding of calcium to troponin C on the thin filament, which enables projections (S1 heads) on the myosin molecules to interact with actin filaments, forming cross-bridges. This energy-requiring process involves ATP hydrolysis by myosin ATPase. Repetitive cross-bridge cycles of attachment and detachment continue as long as the cytosolic calcium concentration is high. The power stroke generated by the cross-bridge cycle is responsible for force generation or muscle shortening. Cross-bridge interactions show cooperativity; in other words, force-generating cross-bridges promote further binding of more cross-bridges, which effectively amplifies the contractile force. Muscle relaxation is governed by lowering of the cytoplasmic calcium concentration, consequent dissociation of calcium from troponin C, and switching off of the actinemyosin interaction. This involves active transport of calcium back into the SR (via SR Ca2þ-ATPase) and extrusion across the sarcolemma, by both the NaþeCa2þ exchanger and (less importantly) sarcolemmal Ca2þATPase. Mitochondria can also accumulate calcium, particularly when cytosolic concentrations become excessively high (e.g. during severe ischaemia). In addition to the reduction in cytosolic Ca2þ, recoil of elastic elements within the myocyte (notably within titin molecules in the sarcomere) may also be involved in the process of relaxation. The events that comprise excitationecontraction coupling influence the size and kinetics of the calcium transient. An abnormally low calcium transient can lead to depressed contractility. Reduction in SR Ca2þ-ATPase activity and abnormalities of SR calcium release (e.g. calcium leak) occur in heart failure and are generally accompanied by diastolic calcium overload; this can contribute to delayed relaxation and diastolic dysfunction, triggering of ventricular arrhythmias and chronic changes in cell structure (e.g. altered gene expression) as a result of activation of downstream calcium-dependent signalling pathways. Up-regulation of the NaþeCa2þ exchanger activity may, to some extent, compensate for reduced SR Ca2þ-ATPase activity. Independent of excitationecontraction coupling, changes in myofilament properties (e.g. responsiveness to calcium) are also implicated in heart failure, ischaemiaereperfusion injury and hypertrophic cardiomyopathy. Calcium concentrations within the cardiomyocyte also influence other cellular processes. They have been found to be involved in the control of gene transcription (so-called excitationetranscription coupling) and may in part mediate processes such as cardiac hypertrophy. The binding of calcium to calmodulin leads to activation of certain protein kinases (e.g. calmodulin kinase) or phosphatases (e.g. calcineurin), which then modulate signal transduction pathways and/or transcription factors to alter the expression of specific genes.
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Contractile reserve and regulation Considerable contractile reserve (Figure 2) is normally available to meet variations in circulatory demand, for example during exercise. At a cellular level, the recruitment of this contractile reserve involves changes in the cytosolic calcium transient and/or myofilament responsiveness to calcium, and is regulated by several important pathways as outlined below. There is usually significant impairment of these pathways in heart failure, which is a major reason for the exercise intolerance characteristic of this condition. The FrankeStarling response describes an increase in contractile force that occurs with increasing myocyte length or stretch (the latter brought about by increased ventricular diastolic volume). The main underlying mechanism is an increase in myofilament responsiveness to calcium, but a length-dependent release of autocrine/paracrine factors may also be involved. The FrankeStarling response is thought to be preserved at a cellular level in human heart failure, but in a heart that is dilated and stiff, it may be functionally impaired by limitation in the ability to stretch myocytes. An increase in heart rate enhances contractile force primarily by increasing sarcolemmal calcium influx per unit time, with consequent increased calcium loading of the SR. This normal positive forceefrequency relationship is greatly blunted or even becomes negative in heart failure. Autonomic control is centrally involved in contractile regulation. Sympathetic activation, involving catecholamine release, has both positive inotropic and chronotropic effects via b-adrenoceptors. These actions are antagonized by parasympathetic release of acetylcholine. The inotropic effect of b-stimulation results from an increase in the intracellular calcium transient caused by increases in ICa and SR calcium release. b-Stimulation also accelerates relaxation (lusitropic action) by stimulating SR calcium uptake, promoting faster dissociation of calcium from the myofilaments, and accelerating cross-bridge cycling. The predominant mechanism for enhanced SR calcium reuptake here is phosphorylation of phospholamban, thereby relieving tonic inhibition on the SR ATPase by this protein. Reduced responsiveness to b-adrenergic stimulation is a fundamental feature of human heart failure.
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THE NORMAL HEART
Contractile reserve –
β-stimulation Heart rate
Activation
Calcium current
Nitric oxide Acetylcholine +
Ca2+
+
Sarcoplasmic Ca2+ reticulum Ca2+ release
Ca2+ transient Ca2+
Negative inotropic effect
– +
Angiotensin II Endothelin 1 Length
+ Positive inotropic effect
Relaxation Sarcoplasmic reticulum Ca2+ uptake
+ β-stimulation +
β-stimulation Nitric oxide Angiotensin II Endothelin 1 Length
Sarcolemmal Ca2+ extrusion
Decreased cytoplasmic Ca2+ concentration
+
+ –
Ca2+ dissociation
Positive lusitropic effect
– Negative lusitropic effect
These are the major pathways by which muscle length, heart rate, autonomic control ( β-adrenergic stimulation) and paracrine factors (nitric oxide, endothelin 1 and angiotensin II) produce changes in: • level of activation and contractile strength (inotropic effects, top) • rate of relaxation (lusitropic effects, bottom). Blue arrows indicate an increase and red arrows a decrease in the components of excitation–contraction coupling. β-adrenergic stimulation. Figure 2
Autocrine/paracrine regulation e the endothelial cells within the coronary microvasculature, fibroblasts and cardiomyocytes themselves all release bioactive factors that can regulate cardiac contraction as well as having chronic effects on cardiac structure. The physiological release of such factors is likely to involve local signals such as mechanical forces, oxygen tension and local autacoids. In the physiological setting, nitric oxide can have direct actions on cardiomyocytes, independent of its vasodilator effects. These include: an acceleration of myocyte relaxation and reduction in diastolic tone, resulting from a reduction in myofilament calcium responsiveness; modulation of excitationecontraction coupling; and a ‘damping down’ of responses to b-adrenergic stimulation. Both endothelial and neuronal isoforms of nitric oxide synthase
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(eNOS, nNOS) are expressed in the heart and exert isoformspecific actions. Abnormal nitric oxide bioactivity (excessively low or high) contributes to contractile dysfunction in cardiac hypertrophy, heart failure and myocarditis. Other local factors such as endothelin 1, angiotensin II and reactive oxygen species (ROS) also modulate contractile properties and remodelling, particularly in the diseased heart. ROS production has been shown to modulate the physiological effects of increased stretch to enhance contraction. Endothelin 1 has potent hypertrophic effects and can also stimulate release of angiotensin II, which has similar actions. Increased angiotensin II production through increased local angiotensin-converting enzyme activity is a cardinal feature of hypertrophy and heart failure, contributing to inappropriate hypertrophy, fibrosis and slowed ventricular relaxation.
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THE NORMAL HEART
multiple mRNAs encoding proteins in the same signalling pathway, and can therefore have profound effects on cellular function. Epigenetic and miRNA regulatory mechanisms that alter contractile function may act through diverse effects, including on calcium- and sodium-handling proteins and contractile proteins. Targeting individual miRNAs could be a powerful therapeutic approach because of the potential to influence entire signalling pathways. Finally, the regulation of cardiomyocyte viability and cell number also impacts on heart function. When individual cardiomyocyte survival is compromised (e.g. after ischaemia), cells can undergo programmed cell death (apoptosis or necrosis), which leads to a decline in overall function. Cardiomyocytes are considered terminally differentiated cells and have very low proliferative potential, apart from early in life. However, it is experimentally possible to induce the division of adult cardiomyocytes, and identifying methods that could achieve significant regeneration of cardiomyocytes in the heart in vivo (and thereby improve cardiac function) is currently an area of major research efforts.5 A
The increased generation of ROS, such as superoxide and hydrogen peroxide, is involved in many of the effects of angiotensin II within the heart. ROS can induce abnormal SR calcium release or activate detrimental signalling pathways that contribute to the development of heart failure. Interestingly, some ROS sources can also mediate adaptive responses in the chronically stressed heart. These pathways could provide novel therapeutic targets in heart failure.3
Other cellular processes important in cardiac muscle function Beyond contractile regulation, several other processes influence optimal cardiomyocyte and heart function. Cellular proteins can be damaged over time and need to be efficiently removed and replaced. Intracellular processes such as autophagy and proteasomal degradation of defective proteins ensure that the cell’s environment for contraction is preserved. Cell organelles such as mitochondria also undergo turnover. In particular, mitophagy is a process that degrades dysfunctional mitochondria and limits damage caused by such mitochondria. Cardiac metabolism is crucial for generating the energy required for both heart contraction and turnover of cellular constituents. The heart can use multiple substrates to generate ATP for contraction, including carbohydrates, fatty acids, ketone bodies and proteins. The ability to adjust substrate use in response to demand, hormonal cues and nutrient status is important in maintaining ATP levels and contractile function. The metabolic status is also crucial in the maintenance of an appropriate redox environment, which affects multiple cardiac functions. Epigenetic regulation also has a role in contractile regulation, cardiac hypertrophy and fibrosis.4 The term refers to modulation of gene expression through mechanisms such as DNA methylation, ATP-dependent chromatin remodelling and other histone modifications (acetylation, methylation), rather than transcription factors. Non-coding RNAs including microRNAs (miRNAs) play important regulatory roles at a post-transcriptional level by binding to messenger RNAs (mRNAs) to silence their translation or promote degradation. Individual miRNAs generally target
KEY REFERENCES 1 Katz AM. Physiology of the heart. 5th edn. Philadelphia: Lippincott Williams & Wilkins, 2010. 2 Bers DM. Calcium cycling and signaling in cardiac myocytes. Annu Rev Physiol 2008; 70: 23e49. 3 Shah AM, Mann DL. In search of new therapeutic targets and strategies for heart failure: recent advances in basic sciences. Lancet 2011; 378: 704e12. 4 Nabeebaccus A, Sag CM, Webb I, Shah AM. Cellular basis for heart failure. In: Mann D, Felker M, eds. Heart failure: a companion to Braunwald’s heart disease. 3rd edn. Philadelphia: Elsevier, 2015; 28e41. Chapter 2. 5 Laflamme MA, Murry CE. Heart regeneration. Nature 2011; 473: 326e35.
TEST YOURSELF To test your knowledge based on the article you have just read, please complete the questions below. The answers can be found at the end of the issue or online here. Question 1 A 64-year-old white woman was found on two occasions to have a raised blood pressure of 162/115 mmHg. She was advised to take amlodipine.
Question 2 A 70-year old woman is admitted in cardiogenic shock. She is commenced on an intravenous infusion of dobutamine. What effect at cellular level can be expected from this treatment? A Increased force of contraction of the myofilaments B Decrease in SR calcium release C Decreased inward calcium current (ICa) D Decreased phosphorylation of phospholamban E Decreased calcium uptake into the sarcoplasmic reticulum during diastole
What is the site of action of this drug in the cardiac myocyte? A Voltage-gated L-type calcium channels B Ryanodine receptor C Sarcoplasmic reticulum-Ca2þ ATPase (SERCA) D NaþeCa2þ exchanger E T-type calcium channels
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THE NORMAL HEART
What peptide, present in excess, is likely to be responsible for the hypertrophy seen in this patient? A Angiotensin ll B Insulin C Acetylcholine D Atrial natriuretic peptide E Troponin I
Question 3 A 58-year-old man presented with a 2-month history of breathlessness on exertion and lying flat. On clinical examination, his heart rate was 96 beats/minute, blood pressure 115/78 mmHg, and jugular venous pressure raised 5 cm. Peripheral oedema was noted. The heart was clinically enlarged, but there were no abnormal heart sounds. Investigations Chest X-ray showed cardiomegaly Echocardiogram showed dilation and hypertrophy of the wall of the left ventricle
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