Myocyte Contraction and Relaxation - Neonatal Cardiology, 3rd Ed.

Neonatal Cardiology, 3rd Ed. Michael Artman

Chapter 2. Myocyte Contraction and Relaxation

■ INTRODUCTION

■ GENERAL OVERVIEW OF CELLULAR ASPECTS OF CARDIAC FUNCTION

■ STRUCTURAL COMPONENTS INVOLVED IN CONTRACTION AND RELAXATION

Changes in Myocyte Size and Morphology Sarcolemma and Transverse Tubules

Sarcoplasmic Reticulum

Contractile Elements

Mitochondria

Cytoskeleton and Extracellular Matrix

■ EXCITATION-CONTRACTION COUPLING

Initiation of Contraction in the

Embryonic Heart

Mechanisms of Contraction and Relaxation

Calcium Influx through Calcium Channels

Calcium Fluxes through the Sodium-Calcium

Exchanger

β-Adrenergic Stimulation

Cyclic Nucleotide Phosphodiesterases

Calcium-Calmodulin-Dependent Protein Kinase Phosphatases

■ SUGGESTED READINGS

■ INTRODUCTION

As described in Chapter 1, the developing mammalian heart undergoes a series of complex and tightly regulated processes during structural organogenesis. Considerable understanding of the genetic control of these pathways has been gained in recent years. Of equal importance are the functional changes in cardiac contraction and relaxation that accompany morphological development of the cardiovascular system. However, compared to our understanding of structural organogenesis, much less is known about the genetic, molecular, and cellular processes that control cardiac contractile function during embryonic development and fetal maturation.

Although age-related changes occur in the functional cardiac responses to pharmacological or physiological interventions, understanding of the underlying mechanisms is generally incomplete. In particular, there is relatively little known about fundamental mechanisms of excitation-contraction coupling and regulation of contractile function in the immature human heart. A thorough understanding of the basic molecular and cellular processes governing contractile function is essential for development of rational and age-appropriate pharmacological strategies for fetal and neonatal patients with impaired cardiac contractile function.

It should be noted that current concepts of molecular and cellular aspects of myocyte function during cardiac development and maturation are derived mainly from animal models. Limited information is available regarding these processes in the immature human heart. The list of suggested readings at the end of this chapter refers to several monographs that provide a comprehensive and detailed overview of contractile function in the mature heart. In addition, references are listed that provide additional information regarding developmental changes in cardiac ultrastructure, metabolism, electrophysiology, and responses to pathophysiological states. Integration of these myocellular changes into a larger perspective of developmental physiology and cardiac mechanics is presented in Chapter 3.

■ GENERAL OVERVIEW OF CELLULAR ASPECTS OF CARDIAC FUNCTION

Excitation, contraction, and relaxation of myocardial cells are mediated by complex ion transport processes and coordination of calcium delivery to and from the contractile proteins (Figure 2-1). At rest, active transport processes (mainly the sodium-potassium pump [Na/K- ATPase]) maintain electrochemical gradients across the sarcolemmal membrane. Consequently, a resting membrane potential is established with the cell interior being negative relative to the extracellular space. Depolarization of the cardiac sarcolemmal membrane occurs largely due to opening of sodium channels, which results in a rapid influx of sodium and a brisk rise in membrane potential from negative to positive values. As described in more detail below, this change in membrane potential is ultimately translated into an increase in intracellular cytosolic calcium, binding of calcium to the contractile protein complex in the myofibrils, and cell shortening (contraction). Relaxation occurs as the resting sarcolemmal membrane potential is re-established, intracellular cytosolic calcium decreases, and calcium dissociates from the contractile protein complex.

These processes must be very tightly regulated to maintain calcium homeostasis and control of contraction and relaxation. During the past several years, it has become clear that many of the pathways and proteins involved in these processes undergo developmental regulation. Consequently, significant age-related differences exist in the fundamental mechanisms of cardiac contraction, relaxation, and regulation of contractile function.

As noted above, most of our knowledge regarding developmental aspects of cardiac contractile function is derived from animal models, such as chickens, rats, rabbits, mice, and zebra fish. A recurring problem in developmental cardiology relates to extrapolating animal studies to human physiology. Because cardiac development and maturation are controlled by genetic and epigenetic factors, there may be species-specific events that are unique for a given animal model. It is therefore useful to compare results from more than one animal model. Studies using human myocardium would provide more relevant results, but the availability of tissue for these types of research is limited, especially for fetal and neonatal human hearts.

■ STRUCTURAL COMPONENTS INVOLVED IN CONTRACTION AND RELAXATION

Changes in Myocyte Size and Morphology

In the human embryo, the elements required for rhythmic contraction and relaxation of the heart become functional by approximately 3 weeks after conception. The major functional units of the developing cardiac myocytes are the sarcolemmal membrane, sarcoplasmic reticulum, mitochondria, and contractile proteins. At the same time, supporting structural components (fibroblasts and the extracellular matrix) are also developing. Each of these elements undergoes progressive development and maturation throughout embryonic, fetal, and early postnatal life. In the early embryo and fetus, the ultrastructural appearance and spatial arrangement of cellular structures are quite different compared to those of the fully mature heart.

Ventricular myocytes change considerably with regard to size, shape, and overall appearance during the transition from late fetus to the adult. In general, the perinatal maturation phase is characterized largely by the addition of cellular structures and more precise spatial organization of the elements involved in contraction and relaxation. Newborn myocytes exhibit random orientation of myofibrils with incomplete sarcomeres. Myofibrils are frequently located in the subsarcolemmal region. Regularly scattered throughout the cell are ribosomes, rough endoplasmic reticulum, and mitochondria. Nuclei are round and centrally located. Relative to cell volume, nuclear volume decreases steadily after birth, coincident with progressive cellular hypertrophy. The clusters of ribosomes, rough endoplasmic reticulum, and extensive Golgi apparatus are all consistent with active protein synthesis during rapid cell growth.

Although the general patterns are similar, the precise timing and temporal relationships of the ultrastructural changes vary from species to species. Table 2-1 summarizes comparisons among various species for the timing of appearance and maturation of important elements involved in cardiac contraction and relaxation. The information presented in Table 2-1 is somewhat generalized because maturation is not uniform throughout the same heart within a given species. Furthermore, considerable variation in morphologic appearance of myocytes can be found within the same heart.

FIGURE 2-1. Schematic diagram of the major components involved in calcium transport, excitationcontraction coupling, contraction, and relaxation in mature mammalian ventricular myocytes. At rest, a negative membrane potential is maintained largely by the action of the sodium-potassium pumps. Contraction in adult myocytes (see also Figure 2-5) is triggered by membrane depolarization (opening of sodium channels), which then promotes opening of voltage-dependent L-type calcium channels. The resulting influx of a relatively small amount of calcium causes the release of a large amount of calcium from the junctional sarcoplasmic reticulum by triggering the opening of specific sarcoplasmic reticulum calcium release channels. This process is termed “calcium-induced calcium release.” The central role of T-tubules in providing the proper spatial orientation for close coupling of L-type calcium channel calcium influx to sarcoplasmic reticulum calcium release is depicted. The rise in cytosolic calcium results in calcium binding to troponin C, activation of the myofilaments, and contraction. The predominant mechanism for lowering calcium to promote relaxation (see also Figure 2-7) is the ATP-dependent reuptake of calcium into the longitudinal sarcoplasmic reticulum via the actions of sarcoplasmic reticulum calcium pumps, which are in turn regulated by the phosphorylation state of phospholamban. During steady state, the same amount of calcium that enters the cell is extruded, mainly by the sodium-calcium exchanger. The P-adrenergic receptor/G protein/adenylyl cyclase complex is illustrated to signify the central role of this system in regulating cardiac contraction and relaxation (see also Figure 2-8).

An important change during postnatal development is a progressive decrease in surface area-to-volume ratio. This is due largely to a progressive increase in myocyte volume during postnatal development. Myocyte division (hyperplasia) is commonly observed during fetal growth and in the early newborn period. However, shortly after birth, continued growth of the heart is attributable largely to hypertrophy of existing myocytes, with relatively slow turnover and cell division. The mechanisms involved in this postnatal switch from hyperplastic to hypertrophic growth are incompletely defined at present. A number of laboratories are working to unravel the basis for this fundamental change in myocyte biology since the implications for cardiac repair and regeneration are profound.

Sarcolemma and Transverse Tubules

In cardiac myocytes, the sarcolemmal membrane is well defined throughout fetal and postnatal development, and the glycocalyx can be visualized quite early in cardiac development. Transverse tubules (T-tubules) are invaginations of the sarcolemma into the cell that allow the extracellular environment to extend into the inner cellular structures. In species with small ventricular myocytes (eg, birds and fish), the T-tubular system is generally not present, presumably because it is not required for efficient excitation-contraction coupling. However, in larger cells, a T-tubular system is necessary to allow transarcolemmal fluxes to occur deep within the cell interior. The presence of T-tubules compensates for the increased cell volume by providing a mechanism for overcoming diffusional restrictions by creating a much larger surface for ion fluxes from outside to inside the cell and vice versa. The T-tubular system is therefore an integral component of contraction and relaxation processes in mammalian myocytes. This is the site of highest density of calcium channels (for the influx of calcium) and the area where the sarcolemma is closest to the sarcoplasmic reticulum (the source of activator calcium for myocyte contraction; Figure 2-1).

In mammalian hearts, T-tubules are one of the last organelles to develop and generally do not appear until after birth. The time course of appearance of T-tubules varies among species (Table 2-1). The relative paucity of T-tubules limits the interaction between sarcolemmal calcium channels and sarcoplasmic reticulum calcium release channels, inhibiting the sarcoplasmic reticulum from participating in excitation-contraction coupling. Morphogenesis of T-tubules and the temporal relationship between the postnatal acquisition of T-tubules in the human heart and the emergence of a mature excitation-contraction coupling phenotype remain to be determined.

Sarcoplasmic Reticulum

Coincident with the progressive development and maturation of the T-tubule system is a change in the appearance and function of the sarcoplasmic reticulum. The sarcoplasmic reticulum is a specialized form of endoplasmic reticulum that is an essential component of contraction and relaxation in the mammalian heart. In mature cardiac myocytes, the sarcoplasmic reticulum stores, releases, and reaccumulates the majority of the calcium that is involved in contraction and relaxation.

The sarcoplasmic reticulum is composed of junctional and longitudinal elements (the longitudinal sarcoplasmic reticulum is also referred to as the free sarcoplasmic reticulum). Important maturational changes in the amount, appearance, and function of the sarcoplasmic reticulum are apparent during late fetal and early postnatal maturation. In the adult, sarcoplasmic reticulum membranes are well organized and prominently related to the thick filaments of myofibrils. The junctional sarcoplasmic reticulum is immediately adjacent to the T-tubules in structures termed “dyads.” It is this close physical relationship that is the central element in the transduction of the changes in sarcolemmal membrane potential and calcium influx to the sarcoplasmic reticulum that trigger calcium release and contraction.

In mature myocytes, the junctional sarcoplasmic reticulum contains the sarcoplasmic reticulum calcium release channels. These channels (also known as ryanodine receptors because of their high-affinity binding to this neutral plant alkaloid), open during excitation-contraction coupling to allow discharge of stored calcium into the cytosol for contraction to occur. In addition, the junctional sarcoplasmic reticulum contains high concentrations of calsequestrin, a calcium-binding protein. This is the site of storage of most of the calcium that cycles through the sarcoplasmic reticulum to and from the contractile proteins with each sequence of contraction and relaxation. Triadin and junctin are smaller proteins contained in the junctional sarcoplasmic reticulum that are important in maintaining the proper spatial relations between calsequestrin and the calcium release channel. Other structural proteins include FK binding protein (also known as calstabin), which helps to stabilize the calcium release channels and promote coordinated calcium release.

Longitudinal sarcoplasmic reticulum contains the greatest density of the ATP-dependent calcium pumps involved in calcium reuptake into the sarcoplasmic reticulum. These proteins are termed “sarcoendoplas- mic reticulum calcium ATPases” (SERCA). The cardiacspecific isoform is SERCA2a. The reuptake of calcium into the sarcoplasmic reticulum by SERCA2a is modulated by a regulatory protein, phospholamban, which is in close physical relationship with SERCA2a. In the basal unphosphorylated state, phospholamban acts as a “brake” to inhibit SERCA2a activity and calcium reuptake into the sarcoplasmic reticulum. When phospholamban is phosphorylated (eg, in response to ^-adrenergic stimulation), the inhibition is removed, SERCA2a activity increases, and relaxation is facilitated due to enhanced sarcoplasmic reticulum calcium reuptake.

In the late fetus and early newborn, peripheral subsarcolemmal couplings between the junctional sarcoplasmic reticulum and sarcolemma can be observed prior to the acquisition of a well-organized T-tubular system. However, as the cells enlarge and the T-tubular system develops, deeper internal couplings (dyads) are acquired. The volume and distribution of the sarcoplasmic reticulum increase during late fetal and early postnatal maturation. Each of the components of the mature sarcoplasmic reticulum undergoes developmental regulation and maturation (ryanodine receptors, SERCA2a, phospholamban, etc.).

Contractile Elements

Sarcomeres

The sarcomere is the primary contractile unit of striated muscle (Figure 2-2). Contraction and relaxation of cardiac muscle depends on the structural organization of contractile and modulatory proteins into filaments that repeatedly move back and forth past one another. Anchoring of the structural filaments, combined with sliding of complementary filaments, results in shortening and generation of force. In mature cardiac cells, the myofibrils are organized into sarcomeres, which are delineated at each end by Z-discs. The Z-disc is a complex group of proteins that links the myofilaments from contiguous sarcomeres into a highly ordered network (see below).

The Z-discs contain projections toward the center of the sarcomere that are termed “thin filaments” and contain an abundance of actin. Thick filaments are polymers of myosin and titin, a very large structural protein. The thick filaments are arranged along the same long axis of the sarcomere and are interspersed among the thin filaments (Figure 2-2). I-bands are composed of thin filaments, troponin complexes, and tropomyosin. A-bands are composed of overlapping thin and thick filaments. The dark M-band in the center of the A-band consists of thick filaments cross-linked to titin. Mutations in a variety of sarcomeric and Z-disc proteins are associated with both hypertrophic and dilated forms of familial cardiomyopathy (Chapter 9).

In early fetal life, sarcomeres can be observed in cardiac myocytes. However, the contractile apparatus remains relatively disorganized in the fetal and early newborn heart. Myofibrils initially are irregular and scattered about the interior of the cell. They are arranged in the subsarcolemmal region around a large central mass of nuclei and mitochondria in immature cells. As maturation progresses, the myofibrils come to lie along the long axis of the cell. Progressive organization of the sarcomeres occurs even as contraction and relaxation are occurring.

A progressive increase in myofilament content and maturation of the sarcomeres is consistent across mammalian species, although the timing may vary. A developmental increase in the myofibrillar population with a more orderly arrangement of myofilaments during maturation is often cited as the primary factor responsible for the increase in force generation observed during the late fetal and early newborn period in the mammalian heart. However, developmental changes in sarcomeric protein isoform expression also play an important role in maturational changes in force generation (see below).

Myofilament Proteins

Myofilaments are composed predominantly of myosin and actin (approximately 80% of the total contractile protein content). Other less abundant components, such as troponin and tropomyosin, provide regulatory control and structural support (Figure 2-2).

Giant filament. An essential component of the structural foundation of the sarcomere is titin, the largest protein known in humans. Titin spans half of the sarcomere from the Z-disc at the end to the M-line at the center. The N-terminal ends of titin overlap within the Z-disc, and the C-terminal ends overlap at the M-line to create a continuous filament system (so-called giant filament) that aligns the thick filaments within the myofibril.

FIGURE 2-2. Schematic diagram showing arrangement of thick and thin filaments within the sarcomere. Thick filaments contain myosin, myosin-binding protein C, and titin. The tails of the myosin heavy chains are woven together to form the thick filament. The globular head projects outward to form cross bridges. The thin filaments include actin (which forms the backbone of the thin filament), the troponins, and tropomyosin. Tropomyosin binds to troponin T at multiple sites along the major groove of the actin filament and inhibits actin-myosin interaction. Troponin T binds the troponin complex to tropomyosin, troponin I inhibits interactions between actin and myosin, and troponin C binds calcium. The sarcomere, which lies between two Z-discs, is anchored by interactions between titin and actin with Z-disc proteins, including LIM domain-binding protein 3 (cipher/ZASP), a-actinin, cardiac LIM domain protein (MLP), and telethonin. Adapted from Mahony L. Development of myocardial structure and function. In: Allen HD, Driscoll DJ, Shaddy RE, Feltes TF, eds. Moss and Adams’ Heart Disease in Infants, Children and Adolescents, 7th ed. Philadelphia, PA: Lippincott Williams & Wilkins; 2008:577.

Titin does more than simply connect the myofibrils. The segment of titin at the I-band contains serial spring elements that determine passive tension of the myocytes, thereby impacting late diastolic filling and resting sarcomere length. This likely contributes to the Frank Starling mechanism. In addition, this extensible region provides elastic recoil that drives early diastolic filling.

Thick filaments. The predominant protein in thick filaments is myosin. Individual thick filaments contain several different polypeptides comprising a globular head and a tail section (Figure 2-2). Each of the approximately 300 myosin molecules in each sarcomere is composed of two heavy-chain myosin monomers and a stalk of four light-chain monomers. The heavy chains form a large, bilobed globular head and contain its ATPase activity, essential to release the energy necessary for cross-bridge formation. The light chains (essential and regulatory) form the long stalk of the thick filament, joined to the head by a flexible joint. It is the globular head that forms the cross bridge with the actin molecule, the ultimate step in sarcomere shortening.

An important protein associated with the thick filaments is myosin-binding protein C. This protein helps to stabilize thick filaments by forming transverse fibers that connect adjacent filaments near the center of the sarcomere. Other important structural proteins at the M-line include myomesin, M-protein, and obscurin, all of which appear to act with myosin-binding protein C to facilitate force transmission along the thick filaments.

Thin filaments. Each thin filament contains three major proteins: actin, tropomyosin, and troponin (Figure 2-2). Repetitive association and dissociation of actin with myosin results in contraction and relaxation. Troponin is composed of three major subunits. The inhibitory subunit is troponin I (TnI), the calcium-binding subunit is troponin C (TnC), and the tropomyosin-binding subunit is troponin T (TnT). These proteins are required in the final stages of activation of actin binding with myosin to form the actomyosin complex, which initiates contraction.

Calcium plays an integral role in relaxation and contraction by binding to one of the troponin subunits, troponin C (TnC). Although there are several calcium-binding sites on TnC, attachment to the low- affinity site II is thought to be the trigger for cross-bridge formation. Binding of calcium to this site removes the inhibition that the tropomyosin-troponin complex confers on the actin-myosin interaction and triggers force generation and contraction. The number of cross bridges formed contributes to the amount of force developed by the contracting myocyte and depends on the amount of calcium released by the sarcoplasmic reticulum and intrinsic properties of the contractile proteins, such as calcium sensitivity. At any given calcium concentration, an increase in calcium sensitivity results in greater force and the rate of force generation. Conversely, a decrease in calcium sensitivity reduces force and the rate of force generation. Relaxation occurs as calcium dissociates from TnC and the proteins return to their resting conformational states.

Regulation of contraction and relaxation is very complex and cannot be explained simply by delivery of calcium to and removal from the contractile proteins. There is compelling evidence that cytosolic calcium peaks well before maximum force production is achieved and that calcium is nearly completely removed from the contractile elements before the onset of relaxation. These observations suggest that cooperative signaling and mechanical feedback through thick and thin filaments are critical determinants of contraction, force generation, and relaxation. In addition, a growing body of literature implicates posttranslational modification of sarcomeric proteins (phosphorylation, nitrosylation, and peroxidation) via various signaling pathways as important regulators of calcium sensitivity and cross-bridge cycling rates. The relationships among the various myofilament proteins, cytoskeletal proteins, extracellular matrix, and signal transduction pathways are complex and not fully understood. However, mutations or abnormalities in the function of many of these proteins clearly can have profound effects on cardiac contractile function. Mutations in myofilament proteins are associated with cardiomyopathy, which is more fully described in Chapter 9. As our understanding of intrinsic sarcomere function and regulation continues to expand, it is likely that new therapeutic approaches targeted at improving contractile function will be developed, but the implications for newborns with heart disease are not defined.

Isoform switching. Developmental changes in thick and thin protein isoforms impact the ability of the sarcomere to contract and relax. Important age-related changes in regulatory processes include changes in myofibrillar ATPase activity (determined by differential expression of myosin isoforms), troponin regulatory proteins, and proteins involved in delivery of calcium to and removal from the contractile complex. In addition to developmental regulation, other factors may affect isoform expression. Hormonal changes, nutritional status, workload, and innervation all play an integrated role in regulating protein isoform expression in the developing heart.

Titin isoforms. Titin is encoded by a single gene, but differential splicing generates isoforms with different degrees of extensibility. Titin isoform expression is developmentally regulated. In general, fetal hearts express more compliant titin isoforms than those expressed in mature hearts; the physiologic impact in humans is not known.

Myosin isoforms. The cardiac myosin heavy chain (MHC) exists in two isoforms, a-MHC and β-MHC. Myosin containing two a chains (V1) has the highest ATPase activity. V2 contains an a-chain and a β-chain and exhibits intermediate ATPase activity. Myosin containing two β-chains (V3) has the lowest ATPase activity. ATPase activity of the heavy-chain correlates with velocity of shortening of the myofibril. Myosin heavy-chain isoform expression changes during development in some species, but in the human ventricle, the V3 isoform predominates during fetal, neonatal, and adult life.

Actin isoforms. The backbone of the thin filaments is formed by polymerization of two strands of actin monomers. Actin is a relatively small globular protein encoded by a multigene family. Two isoforms, skeletal a-actin and cardiac a-actin, are present in striated muscle. These isoforms differ by only four amino acids, two of which are in the myosin-binding region. During development, both actin isoforms are expressed in the human ventricle. In fetal and neonatal hearts, skeletal a-actin mRNA constitutes more than 80% of the total actin, but in the mature heart, the cardiac a-actin isoform constitutes more than 60% of the total actin. The functional consequence of this isoform shift in the human heart is unknown.

Troponin isoforms. The troponin complex of the thin filament confers calcium sensitivity to the actin-myosin complex. TnC, the calcium-binding subunit, remains constant and does not exhibit isoform switching. In contrast, both TnI and TnT exist as multiple isoforms with variable expression during development.

Three isoforms of TnI have been identified that are the products of three separate genes. These isoforms are classified as the fast skeletal form (Tnl-f), the slow skeletal form (Tnl-s), and the cardiac muscle form (Tnl-c). A major difference of the cardiac isoform from the other two forms is that TnI-c (but not TnI-s) has a long internal sequence that becomes phosphorylated in response to adrenergic stimulation. Phosphorylation of TnI-c plays an important role in modulating contractile performance by effects on cooperative activation and mechanical feedback at the level of the sarcomeres.

Developmental changes in TnI isoform expression are proposed to at least partly explain changes in contractile function during perinatal maturation of the heart. In the human fetal heart, the predominant isoform is TnI-s, but Tnl-c is detectable. The transition to Tnl-c alone with the disappearance of Tnl-s occurs by approximately 9 months of age in the human heart. This extended postnatal time course of Tnl isoform switching is likely to affect inotropic responsiveness.

TnT plays a role in regulating myofibrillar ATPase activity and the responsiveness to calcium. Troponin T exists as multiple isoforms that appear to be products of a single gene that undergoes developmentally regulated alternative splicing. Expression patterns during development vary among species. Fetal human and rabbit hearts express four cardiac TnT isoforms, TnT_1-4. Expression of TnT₁ is highest in the fetal human heart and neonatal rabbit heart. The dominant isoform in adult rabbit myocardium is TnT4, and in contrast, normal adult human hearts express only TnT₃. Isoform expression is also affected by pathophysiological conditions. Cardiac cTnT₁ and cTnT4 are up-regulated in failing human hearts harvested from transplant patients and in hearts from children with congestive heart failure. The level of expression of cTnT4 is correlated with the severity of heart failure before surgery and with the duration of recovery. Changes in cardiac TnT isoform expression (both during development and in response to pathophysiological states) may contribute to changes in force development and to differences of myocardial sensitivity to acidosis.

Mitochondria

In immature myocardium, mitochondria are irregularly scattered about the cell. As the cells mature, mitochondrial size becomes more regular, and mitochondria become centrally located and surrounded by myofilaments. To meet the high energy requirements of active muscle, the mitochondria become distributed in a highly regular fashion along the myofilaments as myofibrillar organization advances.

In all mammalian species, a large increase in mitochondrial volume occurs during the postnatal period. In addition, the ultrastructural appearance of myocardial mitochondria changes during maturation. In the fetal heart, cristae are sparse and widely spaced. With progressive maturation of the heart during the postnatal period, the cristae become more densely packed.

These changes in mitochondrial appearance, position, and number reflect the increasing energy requirements following birth and parallel age-related changes in substrate utilization by the heart. Long-chain free fatty acids are the primary energy substrate in adult hearts. Activated free fatty acids are transported into the mitochondria and then are metabolized by β-oxidation, producing ATP. The enzyme carnitine palmitoyl coenzyme A transferase transports activated free fatty acids from the cytosol into the mitochondria. In immature hearts, the activity of this enzyme is decreased. As a result of these and other factors, the primary energy substrates in the immature heart are lactate and carbohydrates. It should be noted that these data are derived from animal studies and comparable information from fetal and neonatal human hearts is lacking. Abnormalities in mitochondrial substrate metabolism can result in cardiomyopathy (Chapter 9).

Cytoskeleton and Extracellular Matrix

The cytoskeleton and extracellular matrix supports the various components of the cardiac myocyte during contraction and relaxation. The cytoskeleton determines cell size and organization and allows tension developed by the contractile proteins to be transmitted throughout the myocyte, to adjacent cells, and to the extracellular matrix. The ultrastructural appearance of immature myocytes suggests that the cytoskeletal structure is much less organized compared with adult myocytes. Adult cells are compartmentalized by intermediate filaments that connect Z-discs to one another. The linking of adjacent sarcomeres organizes the cell and compartmentalizes sarcomeres with longitudinal sarcoplasmic reticulum, mitochondria, and microtubules. In contrast, immature myocytes contain central aggregations of nuclei and mitochondria. This results in an internal load against which the immature myofibrils contract. Consequently, the resting sarcomere length is shorter, and sarcomere shortening is slower. Thus, the relative disorganization of the cellular structures due to cytoskeletal immaturity may have an important impact on the rate and amount of tension that an immature myocyte can generate.

The cytoskeleton also fosters the spatial arrangement of subcellular protein complexes that is required for proper intracellular signaling. Cytoskeletal structural proteins allow for communication between internal and external environments of the cell and are integrally involved in signal transduction and cell-to-cell signaling. Cytoskeletal proteins organize the colocalization of ion channels, signaling molecules, and messengers required for transduction of extracellular signals and mechanical stress. Mutations in several cytoskeletal proteins cause various form of cardiomyopathy (Chapter 9).

The cytoskeleton is a much more dynamic structure than what was originally thought. The cytoskeleton remodels during cell growth and in response to pathophysiologic signals, such as excessive systolic stress or diastolic stretch. Normal development and maturation of the heart and vasculature are critically dependent on proteins within the cytoskeleton and extracellular matrix. Developmental changes in the organization and location of intracellular organelles reflect changes in the composition and organization of the cytoskeleton.

Z-Discs

At each end of the sarcomere is the Z-disc, which links the myofilaments from opposing sarcomeres into a tightly arranged compact lattice. A variety of proteins are localized to Z-discs, including a-actinin, telethonin (T-cap), nebulette, muscle LIM protein, filamin, and myotilin. Z-discs are crucial elements in the transmission of tension generated by individual sarcomeres along the length of the myofibril. In addition, Z-disc proteins serve as docking sites for transcription factors, calcium-signaling proteins, and a variety of kinases and phosphatases involved in regulation of contractile function. Increasing evidence indicates that the Z-disc and its associated components sense cellular mechanical signals and transduce them into signals for cell growth, development, and remodeling (both electrical and mechanical).

Extramyofibrillar Cytoskeleton

Microfilaments, intermediate filaments, and microtubules make up the three major extramyofibrillar cytoskeletal components (Figure 2-3). Microfilaments are composed predominantly of actin and are found in the thin filaments of the sarcomere (sarcomeric actin) and in the cytosol (non- sarcomeric or cytoplasmic actin). Cytosolic microfilaments are localized mainly in the subsarcolemmal space and play an important role in linking the intracellular cytoskeleton with the extracellular matrix and adjacent myocytes.

FIGURE 2-3. Cytoskeletal architecture of the cardiac myocyte. The dystrophin-sarcoglycan complex links the plasma membrane to cytoplasmic actin filaments. Integrins bind the cell to the extracellular matrix and are composed of a- and P-subunits that attach through various proteins (fibronectin, laminin, and proteoglycans) to collagen. The intracellular part of the P-subunit binds to cytoplasmic actin filaments through another protein complex that includes a-actinin, talin, vinculin, and paxillin. Ankyrin links membrane proteins such as ion pumps and channels together, thereby organizing interactions among proteins with related functions. Spectrin links ankyrin to the cytoplasmic actin filaments and is also associated with both costameres and intercalated discs at the sarcolemmal membrane. Cadherins mediate connection of cell-to-cell contacts in both desmosomes and fascia adherens. In the fascia adherens, cadherins link the cytoplasmic actin filaments of adjacent cells through vinculin, catenins, plakoglobin, and a-actinin. In addition to contributing to the structural integrity of the cell, many of these proteins are critically involved in cell signaling. Adapted from Mahony L. Development of myocardial structure and function. In: Allen HD, Driscoll DJ, Shaddy RE, Feltes TF, eds. Moss and Adams’ Heart Disease in Infants, Children and Adolescents, 7th ed. Philadelphia, PA: Lippincott Williams & Wilkins; 2008:580.

Intermediate fibers form an intracellular network that helps to maintain the structural integrity of myocytes. These fibers are formed from polymers of desmin and link Z-discs together and to costameres, the sarcoplasmic reticulum, and sarcolemmal and nuclear membranes. Stress-induced alterations of this network of intermediate fibers may mediate changes in gene expression.

Microtubules are formed from polymerized subunits of a and p tubulin that undergo continuous depolymerization and repolymerization. Stiffness of the cytoskeleton is moderated by the total amount of tubulin and the relative proportions in the polymerized state. Microtubules surround the nucleus and spread longitudinally throughout the cell. They serve to stabilize cell structure by anchoring subcellular organelles and are involved in the transmission of signals within and between cells.

Connections to Adjacent Cells and the Extracellular Matrix

The extracellular matrix surrounds myocardial cells and serves both structural and regulatory functions. The extracellular matrix is complex and contains a variety of components, including (1) connective tissue, mainly elastin and various types of collagen; (2) a gel-like substance consisting of proteoglycans; (3) basement membrane proteins, such as collagen, laminin, and fibronectin; and (4) other molecules, such as cytokines, growth factors, and proteases. The extracellular matrix modulates cell migration, proliferation, adhesion, and cell-to-cell signaling, thereby playing a critical role in normal growth and development as well as in pathological remodeling of the ventricles.

Intercalated discs connect myocytes to adjacent myocytes and serve as important sites of force transmission between myocytes. Intercalated discs are complex structures that contain fascia adherens (analogous to adherens junctions in nonmuscle cells), desmosomes, and gap junctions. A variety of proteins are involved in connecting cells both structurally and functionally by weaving together the various cytoskeletal elements.

The many protein complexes that link the myofibrils and membrane systems of the myocyte to the extracellular matrix are essential for stabilizing cell structure during the stresses of contraction and relaxation. Additionally, these protein complexes are involved in organizing and coordinating membrane signaling processes with the contractile systems. The various cytoskeletal networks within the myocyte and the extracellular matrix are linked through highly complex protein networks called costa- meres (Figure 2-4). These structures, which contain proteins including vinculin, talin, tensin, paxillin, and zyxin, encircle the lateral aspects of the myocyte perpendicular to its long axis, forming a transmembrane physical attachment between the peripheral Z-discs and the extracellular matrix. Costameres thus anchor the myofibrils to the sarcolemma and transmit lateral contractile force from the sarcomere to the extracellular matrix and ultimately to neighboring myocytes. It appears that costameres play a role in converting mechanical stimuli to alterations in cell signaling and gene expression that can result in cell growth or hypertrophy.

Integrins are another essential component of the protein network responsible for transmission of force between myocytes and the extracellular matrix. Integrin molecules are heterodimers of various a- and β-subunits and found within the sarcolemmal membrane adjacent to costameres. The intracellular domain of integrin β-subunits binds to actin microfilaments through costamere proteins, and the extracellular portion of integrin interacts with various extracellular matrix proteins. Integrins are involved in signaling pathways mediated by several G-proteins and protein kinases that modify interactions between the integrins and the extracellular matrix. These pathways are involved in both cell growth and apoptosis and play an essential role in pathophysiological conditions and in normal embryonic and fetal cardiovascular development.

The dystrophin glycoprotein complex also plays an important role in linking the intracellular cytoskeleton to the extracellular matrix. This complex binds actin microfilaments to the extracellular matrix, thereby transmitting force to the extracellular matrix and providing another mechanism for signal transduction. Mutations in dystrophin cause muscular dystrophy, and mutations in the components of the dystrophin glycoprotein complex are present in some patients with dilated cardiomyopathy.

Spectrin is another cytoskeletal protein that binds to actin and helps to coordinate membrane signaling systems with the contractile filaments. Spectrin is found in costameres and intercalated discs and is a component of protein complexes related to the sarcoplasmic reticulum at the Z-discs. Ankyrins are adaptor proteins that bind to spectrin and also to a structurally diverse group of membrane proteins, such as ion channels and pumps, calcium- release channels, and cell adhesion molecules. In this manner, various proteins are physically linked together to foster interactions among proteins with related functions. Mutations in ankyrins are known to cause cardiac arrhythmias in humans and in murine models.

■ EXCITATION-CONTRACTION COUPLING

Initiation of Contraction in the Embryonic Heart

A functional circulation must be established for the embryo to survive. This requires the developing heart to begin to function as a rhythmic pump. Mature ventricular myocytes are not spontaneously active and rely on transmission of electrical signals from the pacemaker cells in the sinoatrial node.

FIGURE 2-4. Costameric proteins associated with Z-lines. Dystrophin glycoprotein complex and integrin—vinculin—talin complex are two major costameric protein complexes. Members of the dystrophin glycoprotein complex and integrin were reported to directly interact with protein components at the Z-line, such as filamin-C. Filamin-C (y-filamin and filamin2) physically links the costamere and the sarcomere by interacting with two major costameric protein complexes: the sarcoglycans in the dystrophin glycoprotein complex and integrin. In addition, filamin-C interacts with the Z-line proteins calsarcin-1 and myotilin. Vinculin, the founding member of the costamere, interacts with multiple proteins, including talin, paxillin, and a-actinin. Integrin interacts with the ILK-pinch-parvin complex and FAK. Dystrophin binds to ankyrin B and G, which are essential to organize the dystrophin and dystroglycan complex. Titin is the largest known protein and crosses longitudinally from the Z-line to the M-line. The N-terminus of titin at the Z-line binds to ankyrin. Not all costameric proteins or Z-line proteins are illustrated in this figure for simplicity. Key proteins are shown to highlight the bridges between the sarcomere and the costamere. Reprinted with permission from Peter AK, et al. The costamere bridges sarcomeres to the sarcolemma in striated muscle. Prog Pediatr Cardiol 2011;31:83-88. Copyright © 2011 with permission from Elsevier.

The mechanisms involved in establishing the beating heart have been clarified in recent years. Two distinct mechanisms were proposed in the past. Originally, it was thought that because the sarcoplasmic reticulum is sparse and underdeveloped in embryonic myocytes, spontaneous depolarization of the sarcolemmal membrane triggers calcium influx through voltage-activated calcium channels. Subsequently, it was proposed that spontaneous sarcoplasmic reticulum calcium oscillations drive contractions and electrical activity. Leaking of sarcoplasmic reticulum calcium into the cytosol would activate the sodium-calcium exchanger to generate action potentials. Recent experimental evidence suggests that both pathways may be involved independently to generate action potentials and global calcium transients. During spontaneous sarcoplasmic reticulum calcium oscillations, the sodium-calcium exchanger extrudes calcium and triggers an action potential that then promotes calcium influx and restoration of the sarcoplasmic reticulum calcium store so that the process can be repeated. Inositol 1,4,5 trisphosphate may play an important role in this process. Conversely, if the action potential occurs spontaneously, it can result in a global whole-cell calcium transient as well. Having both mechanisms available allows embryonic cardiomyocytes to contract without requiring physical connections to other myocytes, enabling cell migration.

Although we have a better understanding of how the embryonic heart begins to contract, the question of why the embryonic heart begins to beat rhythmically is perhaps less clear. The mammalian embryonic heart begins to beat before tissues need to be perfused with blood (and, indeed, before the blood cells have formed). Recently, clues have been provided from studies of zebrafish and mice that indicate that a beating heart is necessary for normal vascular development and for development of the hematopoietic system. Shear stress from fluid flowing through the developing vascular bed stimulates the expression of hematopoietic stem cells. Nitric oxide may be a critical signal for this process. The mechanisms responsible for signaling the embryo that it is time to establish the circulation remain elusive.

Mechanisms of Contraction and Relaxation

The main components of the mature excitation-contraction coupling phenotype of calcium-induced calcium release are sarcolemmal L-type calcium channels (predominantly in the T-tubules) in close physical relation to junctional sarcoplasmic reticulum ryanodine receptors in the dyadic junction. These structural components are known to undergo developmental changes that consequently impact significantly on the mechanisms of excitation-contraction coupling in the immature heart.

Figure 2-5 illustrates the general features of excitation-contraction coupling and calcium transport during contraction in mature mammalian ventricular myocytes. Depolarization of the sarcolemmal membrane promotes opening of voltage-dependent L-type calcium channels. This results in the influx of a relatively small amount of calcium, which alone is not sufficient to directly activate the contractile proteins. It is clear that contraction results from the much greater increase in intracellular calcium that results from release of a large amount of calcium from sarcoplasmic reticulum stores. The opening of specific sarcoplasmic reticulum calcium release channels (also known as ryanodine receptors) is triggered by the influx of calcium across the sarcolemma through the L-type calcium channels. This process is known as calcium- induced calcium release.

The structural basis for calcium-induced calcium release is the colocalization of sarcolemmal L-type calcium channels (predominantly in the T-tubules) and junctional sarcoplasmic reticulum ryanodine receptors in the dyadic junction (Figure 2-5). The close physical relationship of L-type calcium channels and ryanodine receptors (~10 nm) in this region allows a single or a small cluster of ryanodine receptors to open in response to a local, spatially restricted microdomain of elevated calcium concentration that results from the nearby influx of calcium through an L-type calcium channel. Localized, nonpropagating sarcoplasmic reticulum calcium release events (either evoked or spontaneously occurring) have been commonly termed “calcium sparks.” It is the summation of these local release events that results in a global rise in intracellular calcium and activation of contraction. Modulation of the force of contraction is achieved primarily by regulation of the magnitude of the L-type calcium current. Evidence suggests that calcium-induced inactivation of ryanodine receptors is the primary mechanism for the beat-to-beat termination of calcium-induced calcium release. Contraction is terminated and relaxation occurs because of (1) a decrease in sarcoplasmic reticulum calcium release channel openings in conjunction with (2) the reuptake of cytosolic calcium back into the sarcoplasmic reticulum through calcium pumps (and, to a much lesser extent, calcium extrusion from the cell via sodiumcalcium exchange and sarcolemmal calcium pumps).

FIGURE 2-5. Schematic diagram of excitation-contraction coupling in mature mammalian ventricular myocytes. The close relationship between L-type calcium channels and sarcoplasmic reticulum calcium release channels is illustrated. Calcium (blue circles) entering the cell through L-type calcium channels (concentrated in the T-tubules) triggers the opening of specific calcium release channels in the junctional sarcoplasmic reticulum. When this occurs, a large amount of calcium is released into the cytosol, resulting in contraction of the myofilaments. Sarcoplasmic reticulum calcium release channels are also known as ryanodine receptors. The major calcium binding protein in the sarcoplasmic reticulum is calsequestrin. FK binding proteins maintain stability of the sarcoplasmic reticulum calcium release channels and promote coordinated interaction among neighboring channels. See text for additional details.

Morphological considerations in immature myocytes support the feasibility of transsarcolemmal calcium influx as a direct source of activator calcium for contractions (Figure 2-6). Despite a lack of T-tubules, fetal and newborn myocytes are relatively small and have a much higher cell surface area-to-volume ratio than adult myocytes. Ryanodine receptors are not in close proximity to the sites of calcium entry across the sarcolemma in immature myocytes. This feature and the subsarcolemmal position of myofibrils in fetal and newborn myocytes favor direct transsarcolemmal calcium delivery to (and from) the contractile proteins for contraction and relaxation in immature ventricular myocytes (Figure 2-6).

In addition to providing the source of calcium for contraction, the sarcoplasmic reticulum plays a primary role in relaxation in the adult heart. After the rise in intracellular calcium that occurs after calcium release from the junctional sarcoplasmic reticulum, specific calcium pumps in the longitudinal sarcoplasmic reticulum sequester calcium back into the sarcoplasmic reticulum (Figure 2-7). As cytosolic calcium declines, calcium dissociates from the contractile protein complex, and relaxation occurs. Sarcoplasmic reticulum calcium pump activity is in turn regulated by the phosphorylation state of phospholam- ban, which provides another key mechanism for modulating contractile function.

During steady-state conditions, the amount of calcium that enters the myocyte from the extracellular space must be extruded during relaxation. This is achieved predominantly through the action of the sarcolemmal sodiumcalcium exchanger. In addition to its role in relaxation, the exchanger can operate bidirectionally and transport calcium into the cell during depolarization. However, in mature myocytes, the major function of the exchanger is to extrude calcium during relaxation. In addition to the sodium-calcium exchanger, sarcolemmal calcium pumps (plasma membrane calcium-ATPase) move calcium out of the cell, but their contribution to relaxation is relatively minor compared to the activities of SERCA2a and the exchanger.

The functional contribution of the sarcoplasmic reticulum to relaxation in the immature heart has received less attention compared to its contribution to contraction. However, animal studies of immature myocytes support the concept that the sarcoplasmic reticulum has a similarly diminished role in calcium reuptake during relaxation as it has in providing calcium for contraction.

FIGURE 2-6. Schematic diagram of the major components involved in calcium regulation in fetal and neonatal ventricular myocytes. In contrast to adults, immature myocytes lack T-tubules, are smaller in size, and have a greater surface area-to-volume ratio. Sarcoplasmic reticulum structures are underdeveloped, and, as depicted, L-type calcium channels and sarcoplasmic reticulum calcium release channels are physically separated. Consequently, calcium-induced calcium release is diminished, and immature myocytes are therefore much more reliant on transsarcolemmal calcium fluxes for contraction and relaxation. The sodium-calcium exchanger is the major transport pathway for providing calcium to and removal from the contractile proteins in fetal and neonatal myocytes. The sub- sarcolemmal location of the myofilaments facilitates these processes. See text for additional details.

This conclusion is based primarily on studies involving pharmacological manipulation of sarcoplasmic reticulum reuptake that show relatively less impact on relaxation of the immature heart compared to adults. Disabling of the sarcoplasmic reticulum in the neonatal rabbit heart has demonstrated that the sodium-calcium exchanger is sufficient to promote normal relaxation. In addition, it appears that the other “slow” pathways (mitochondria and the sarcolemmal calcium ATPase) are relatively more active in neonatal cells compared with adult cells. Taken together, these studies provide compelling evidence for a much lesser role of the sarcoplasmic reticulum in relaxation prior to cardiac maturation.

Calcium Influx through Calcium Channels

During normal excitation-contraction coupling in mature mammalian ventricular myocardium, the role of L-type calcium current is to trigger the opening of specific sarcoplasmic reticulum calcium release channels, thereby initiating calcium-induced calcium release (as described above). L-type calcium channels are functionally mature during fetal stages. However, the physical separation of L-type calcium channels from the junctional sarcoplasmic reticulum in immature hearts is likely responsible for the uncoupling of L-type calcium channels with sarcoplasmic reticulum calcium release channels (Figure 2-6). This notion is supported by the observation that after birth, along with the development of T-tubules, up to threefold more abundant L-type calcium channels are located in T-tubules than in the peripheral sarcolemma, consistent with the postnatal maturation of cardiac excitation-contraction coupling. In developing myocardium, the comparatively greater negative inotropic response to calcium channel blockers in a variety of species suggests a strong dependence on L-type calcium channels for contraction. However, this observation is difficult to reconcile with electrophysiological studies that describe L-type calcium current density (compared with adults) to be lesser in neonatal rabbits, greater in rats, and not different in humans. One possible explanation for the greater sensitivity of immature myocardium to the negative inotropic effects of L-type calcium channel blockers may be the greater depression of contractility resulting from shortening of the action potential with less calcium influx via the sodium-calcium exchanger.

FIGURE 2-7. Schematic diagram of calcium transport pathways during relaxation in mature ventricular myocytes. In order for relaxation to occur, calcium must diffuse away from the myofilaments. This is achieved by removing calcium from the cytosol. Calcium reuptake into the longitudinal sarcoplasmic reticulum is the major pathway involved in relaxation in adult myocytes. The activity of the sarcoplasmic reticulum calcium pump is regulated by the phosphorylation state of phospholamban. In the dephosphorylated state, phospholamban acts to inhibit sarcoplasmic reticulum calcium pump activity. When phospholamban is phosphorylated by cAMP-dependent protein kinase or calcium-calmodulin kinase II, the inhibition is removed, and sarcoplasmic reticulum calcium pump activity increases. During steady state, the small amount of calcium that enters the cell during depolarization is transported back out. This is achieved largely through the action of the sodium-calcium exchanger and, to a lesser extent, the sarcolemmal calcium pump. See text for additional details.

Calcium Fluxes through the Sodium-Calcium Exchanger

There is general agreement that the main functional role of the sodium-calcium exchanger in mature cardiac cells is to extrude calcium during relaxation. However, calcium entry via “reverse” sodium-calcium exchange clearly occurs in a variety of model systems.

Three genes (NCX1, NCX2, and NCX3) encode the mammalian sodium-calcium exchanger. NCX1 is present primarily in the heart. In embryonic mice, NCX1 expression is detected before the first heartbeat and initially appears in a heart-restricted pattern. Recently, it has been shown that targeted ablation of NCX1 is lethal during the embryonic period, apparently due to failure of the forming heart to begin beating. These studies suggest a primary role of the exchanger in supporting electrical excitability, contraction, and relaxation during cardiogenesis.

Sodium-calcium exchanger activity in sarcolemmal vesicles, sarcolemmal exchanger protein content, and steady-state mRNA levels are highest in late fetal and early newborn rabbits and declines postnatally to adult levels by 2 to 3 weeks of age. Immunohistochemical studies in intact myocytes confirm that exchanger protein expression is high at birth in rabbits and that the exchanger is homogeneously distributed over the cell surface. Increased expression of sodium-calcium exchanger message and protein in developing human hearts occurs in a pattern remarkably similar to that observed in rabbits.

The functional consequences of these developmental changes have been confirmed by convincing evidence that sodium-calcium exchange alone is sufficient for generating contractions in newborn rabbit myocytes but not in adult cells. Furthermore, when the sarcoplasmic reticulum is disabled, the sodium-calcium exchanger alone is sufficient for promoting normal relaxation in newborn myocytes but not in adult cells. In addition, sodiumcalcium exchanger current density is high at birth and declines during the first 3 weeks after birth in rabbit ventricular myocytes. Mathematical modeling studies suggested that calcium fluxes via sodium-calcium exchange during an action potential can account for the intracellular calcium transients and subsarcolemmal calcium gradients observed experimentally in newborn rabbit myocytes. These findings lend support to the concept that the sodium-calcium exchanger is a major route for sarcolemmal calcium entry and efflux in the immature heart.

β-Adrenergic Stimulation

Driven by sympathetic neurotransmitters and adrenal hormones, β-adrenergic activation regulates virtually all major constituents of the cardiac excitation-contraction coupling cascade, such as L-type calcium channels, ryanodine receptors, sodium-calcium exchanger, phospholamban, and contractile proteins. These effects are mediated mainly via the classic stimulatory G protein (G_s)-adenylyl cyclase-cAMP-protein kinase A signaling cascade (Figure 2-8) with subsequent phosphorylation of the target proteins by protein kinase A. The overall effects of β-adrenergic stimulation in the mature heart include increases in the rate and force of contraction, the rate of relaxation, heart rate, and conduction velocity. The effects on contractile function are due to increased calcium influx through L-type calcium channels, which provide a greater trigger for sarcoplasmic reticulum calcium release. Enhanced relaxation results from more rapid dissociation of calcium from the contractile proteins (due to phosphorylation of Tnl) and increased reuptake into the sarcoplasmic reticulum resulting from phosphorylation of phospholamban (in the phosphorylated state, the inhibitory effect of phospholamban on SERCA2a activity is reduced).

β-adrenergic agonists exert qualitatively similar responses in immature hearts. However, since these proteins and pathways for calcium transport are less important in the immature heart, alternative cellular and molecular mechanisms may be involved. In addition to the possibility that immature cardiac myocytes might possess qualitatively and quantitatively different sodium-calcium exchanger proteins, β-adrenergic signaling changes dramatically during development. Even though β-adrenergic receptors are detected in early fetal hearts long before postnatal sympathetic innervation, the response of L-type calcium current amplitude, calcium transients, and cell contractions to β-adrenergic stimulation becomes evident only during late gestation. This is due to uncoupling of the receptor from downstream signaling during early gestation. Furthermore, the signal transduction pathways of β-adrenergic receptor in the immature heart might not be identical to that in mature heart. For example, the sensitivity of neonatal cardiac myocytes to β₂-adrenergic receptor agonists is much greater than that of adult cardiac myocytes. The β₂-adrenergic receptor is coupled to the arachidonic acid pathway in fetal chick ventricular myocytes. More surprisingly, repeated β-adrenergic receptor stimulation sensitizes the β-adrenergic signaling pathway in neonatal rat cardiac myocytes, which is in sharp contrast to the agonist-induced desensitization in adult cardiac myocytes.

FIGURE 2-8. Schematic representation of the β-adrenergic signaling pathway. When a β-agonist binds to the β-adrenergic receptor (β adrenoreceptor), the conformational change promotes displacement of GDP by GTP and dissociation of the stimulatory G protein (Gs) complex into the a and βy subunits. The result is activation of adenylyl cyclase and formation of cAMP, which then activates cAMP-dependent protein kinase (protein kinase A). The major substrates for protein kinase A that are involved in regulation of contraction and relaxation include L-type calcium channels, sarcoplasmic reticulum calcium release channel complex, phospholamban, troponin I, and the sodium-potassium pump. See text for additional details.

Cyclic Nucleotide Phosphodiesterases

A family of phosphodiesterase (PDE) enzymes mediates hydrolysis of cAMP and cGMP. Mammalian phosphodiesterases comprise a superfamily of 21 different genes that, with alternative splicing, encode at least 50 different protein products. These various proteins are grouped into 11 isoenzyme categories (PDE1 through PDE11) based on sequence, enzymatic properties, and sensitivity to various inhibitors. These isoenzymes exhibit different specificities for cAMP and cGMP and are differentially expressed in a variety of tissues. Even within cardiac ventricular myocytes, different phosphodiesterases are expressed in various cytosolic or membrane compartments. The isoform that initially commanded most of the interest in the heart was PDE3 because it is localized predominantly to the sarcoplasmic reticulum and plays a major role in regulation phosphorylation state of phospholamban. It is this isoform that is the target of selective phosphodiesterase inhibitors, such as amrinone and milrinone, that are designed to increase contractility. However, PDE3 and other isoforms undergo developmental changes in expression and activity. In most animals species that have been studied, PDE3 activity is absent or markedly diminished at birth. Consequently, PDE3 inhibitors may have relatively little effect on the inotropic or lusitropic state in immature mammalian myocardium. However, in the clinical setting, milrinone improves hemodynamics in human neonates, especially following cardiac surgery. This observation suggests either that PDE3 is expressed at birth in humans or that the favorable responses result from a decrease in afterload in response to milrinone (vasodilation).

Calcium-Calmodulin-Dependent Protein Kinase

Another important mechanism for regulating contractile function involves calcium-calmodulin-dependent protein kinase II (CaMKII). CaMKII is a serine/threonine kinase that is encoded by four separate genes. Alternative splicing produces at least 24 different proteins. Cardiac tissue expresses CaMKIISC, which is involved in regulating contractility, relaxation, and heart rate. In addition, mammalian hearts express CaMKIISB, which regulates nuclear function and gene expression. In cardiac myocytes, CaMKIISC is activated in the presence of calcium and calmodulin. Calmodulin is a ubiquitous calcium-binding protein involved in a variety of cellular functions. CaMKIISC in turn regulates contractile function by phosphorylating phospholamban and the ryanodine receptor complex. The effect of phosphorylating phospholamban is similar to that described above for protein kinase A-mediated phospholamban phosphorylation (relief of the inhibition of SERCA2a-mediated calcium uptake into the sarcoplasmic reticulum), resulting in positive lusitropic and inotropic effects. It is thought that this pathway may predominate in the basal unstimulated state, whereas the protein kinase A pathway may assume a greater role in regulating contractile function during β-adrenergic stimulation.

Phosphatases

As described above, the phosphorylation state of regulatory proteins involved in excitation-contraction coupling plays a major role in modulating contractile function. Once a target protein is phosphorylated by protein kinase A or other protein kinases, the effect will persist until the protein is dephosphorylated. This action is catalyzed by a family of phosphatases that specifically remove the phosphate group and return the protein to the native basal state. Thus, modulation of phosphatase activity or expression has a major influence on the regulation of cardiac contractile function.

Two major groups of serine/threonine phosphatases have been described based on substrate specificity and sensitivity to inhibitors. These are classified as protein phosphatase type 1 (PP1) and type 2 (PP2). There are at least four isoforms of PP1: PP1a, PPiβ, PP1y1, and PP1y2. These isoforms are encoded by distinct genes but share roughly 90% amino acid identity, diverging mainly at the C- and N-terminal ends of the protein. Type 2 phosphatases are classified as PP2A, PP2B, and PP2C, each with different structures, substrate specificities, and regulation. PP2A exists as a heterotrimer in the cytosol with two regulatory subunits and a catalytic subunit. The catalytic subunit has two isoforms, PP2Aa and PP2Aβ.

The predominant phosphatase isoforms in mammalian cardiac tissue are PP1 and PP2A. Relatively less attention has been directed toward the developmental regulation and expression of protein phosphatase isoforms in the heart. However, recent studies in rabbit myocardium showed that steady-state mRNA levels of PP1a, PP1β, and PP2Aa were much higher in newborns compared to adults. In addition, protein levels of PP1 and PP2A were also higher in newborn hearts, and PP1 was membrane bound and PP2A found in soluble fractions. These findings are consistent with previous studies demonstrating a greater effect of inhibitors of PP1 and PP2A on increasing L-type calcium current in newborn myocytes compared to adult cells. Taken together, these results suggest that L-type calcium channels in the newborn heart are relatively dephosphorylated compared to the adult heart. Strategies targeted at moderating the activity of cardiac PP1 and 2A might prove to be useful approaches to increasing contractility in the immature heart, but additional studies are necessary.