Myosin binding protein-C activates thin filaments and inhibits thick filaments in heart muscle cells

Myosin binding protein-C (MyBP-C) is a key regulatory protein in heart muscle, and mutations in the MYBPC3 gene are frequently associated with cardiomyopathy. However, the mechanism of action of MyBP-C remains poorly understood, and both activating and inhibitory effects of MyBP-C on contractility have been reported. To clarify the function of the regulatory N-terminal domains of MyBP-C, we determined their effects on the structure of thick (myosin-containing) and thin (actin-containing) filaments in intact sarcomeres of heart muscle. We used fluorescent probes on troponin C in the thin filaments and on myosin regulatory light chain in the thick filaments to monitor structural changes associated with activation of demembranated trabeculae from rat ventricle by the C1mC2 region of rat MyBP-C. C1mC2 induced larger structural changes in thin filaments than calcium activation, and these were still present when active force was blocked with blebbistatin, showing that C1mC2 directly activates the thin filaments. In contrast, structural changes in thick filaments induced by C1mC2 were smaller than those associated with calcium activation and were abolished or reversed by blebbistatin. Low concentrations of C1mC2 did not affect resting force but increased calcium sensitivity and reduced cooperativity of force and structural changes in both thin and thick filaments. These results show that the N-terminal region of MyBP-C stabilizes the ON state of thin filaments and the OFF state of thick filaments and lead to a novel hypothesis for the physiological role of MyBP-C in the regulation of cardiac contractility.Muscle contraction is driven by the relative sliding of the actin-containing thin filaments along the myosin-containing thick filaments arranged in a parallel array in the muscle sarcomere (Fig. 1A). Filament sliding in turn is driven by a structural change in the myosin head domains (Fig. 1B) while they are bound to actin, coupled to the hydrolysis of ATP (1). Contraction of skeletal and cardiac muscle is triggered by calcium binding to troponin in the thin filaments, accompanied by a change in the structure of the thin filaments that permits myosin head binding (2). However, the strength and dynamics of contraction are modulated by posttranslational modifications in other sarcomeric proteins, including the myosin regulatory light chain (RLC) (3), which is part of the myosin head, and myosin binding protein-C (4–6) (MyBP-C) (Fig. 1B). In an emerging concept of thick filament regulation in striated muscle that is analogous to myosin-linked regulation in smooth muscle (7–11), RLC and MyBP-C are thought to modulate contraction by controlling the conformation of the myosin heads.Open in a separate windowFig. 1.Sarcomere location and domain architecture of MyBP-C. (A) C-zone (green) of the thick filament in relation to its proximal (P) and distal (D) regions and the thin filament (gray). (B) Cartoon representation of MyBP-C (green) anchored to the thick filament backbone (purple) via its C-terminal domains; myosin heads are pink and troponin is yellow. (C) Domain organization and interactions of MyBP-C.According to this concept, the thick filament has an OFF state in which the myosin heads are folded back against its surface (Fig. 1B), rendering them unavailable for interaction with actin, and an ON state in which the heads are released from the thick filament surface and made available for actin binding. The physiological and pathological significance of thick filament regulation and its relationship to the well-studied thin filament mechanisms remain poorly understood, but much recent attention has focused on MyBP-C for two main reasons. First, mutations in the cardiac MYBPC3 gene are commonly associated with hypertrophic cardiomyopathy (12, 13), and this association has driven a wide range of studies at the molecular, cellular, and whole-animal levels aimed at understanding the etiology of MYBPC3-linked disease. Second, although MyBP-C is a constitutive component of the thick filament, there is a large body of evidence that it can also bind the thin filaments (14, 15), raising the possibility that one role of MyBP-C may be to synchronize the regulatory states of the thin and thick filaments (11, 15–17).MyBP-C is localized to the central region or “C-zone” of each half-thick filament (Fig. 1A), appearing in nine transverse stripes with a 43-nm periodicity closely matching that of the myosin heads (Fig. 1B) (10). MyBP-C has 11 Ig-like or fibronectin-like domains (Fig. 1C) denoted C0–C10, with additional linking sequences, notably the MyBP-C “motif” or “m” domain between C1 and C2 and the proline/alanine-rich (P/A) linker between C0 and C1. The m domain has multiple phosphorylation sites (4–6). Constitutive binding to the thick filament is mediated by interactions of domains C8–C10 with myosin and titin. The C1mC2 region binds to the coiled-coil subfragment-2 (S2) domain of myosin adjacent to the myosin heads, and this interaction is abolished by MyBP-C phosphorylation (5); the C0 domain binds to the RLC in the myosin head itself (18). The N-terminal domains of MyBP-C also bind to actin in a phosphorylation-dependent manner (14, 15) (Fig. 1B), and EM and X-ray studies on intact sarcomeres of skeletal muscle suggest that MyBP-C binds to thin filaments under relaxing conditions (10, 11).The function of MyBP-C and the mechanisms underlying its modulation in cardiomyopathy remain poorly understood, however. Ablation of MyBP-C in a knockout mouse model leads to a hypertrophic phenotype associated with impaired contractile function (19), but cardiomyocytes isolated from these mice exhibit increased power output during working contractions (20). A range of studies at the isolated protein and cellular levels have led to the concept that MyBP-C exerts a predominantly inhibitory effect on contractility mediated through two distinct mechanisms (15, 16, 21). MyBP-C may tether myosin heads to the surface of the thick filament, preventing their interaction with actin, and its N terminus may bind to thin filaments, inhibiting interfilament sliding at low load. Other studies, however, have demonstrated an activating effect of MyBP-C mediated by binding of its N-terminal domains to the thin filament. N-terminal fragments of MyBP-C enhance force production in skinned cardiac muscle cells and motility in isolated filament preparations at zero or submaximal calcium concentrations (22–25). The same effect is observed in cardiomyocytes from MyBP-C knockout mice (22), suggesting that the activating effect is not due to competitive removal of an inhibitory effect of native MyBP-C.To resolve these apparently contradictory hypotheses about the physiological function of the N-terminal domains of MyBP-C, we determined the structural changes in the thick and thin filaments of intact sarcomeres in heart muscle cells induced by N-terminal MyBP-C fragments using bifunctional rhodamine probes on RLC and troponin C (TnC) (26). These probes allowed the structural changes in both types of filament to be directly compared with those associated with calcium activation and myosin head binding in the native environment of the cardiac muscle sarcomere. The results lead to a model for the physiological function of MyBP-C that integrates the regulatory roles of the thin and thick filaments and the inhibitory and activating effects of MyBP-C at the level of the intact sarcomere.     

Differential effects of phosphorylation of regions of troponin I in modifying cooperative activation of cardiac thin filaments

Ischemia and heart failure are associated with protein kinase C (PKC) dependent phosphorylation of cardiac troponin I (cTnI). We investigated the effect of phosphorylation of cTnI PKC sites S43, S45 and T144 under normal (pH 7.0) and acidic (pH 6.5) conditions on tension in skinned fiber bundles from a mouse heart. To mimic the PKC phosphorylation, we exchanged troponin (cTn) in these fiber bundles with cTn complex containing either cTnI-(S43E/S45E) or cTnI-(T144E). We determined how pseudo-phosphorylation and acidic pH affect activation of thin filaments by strongly bound crossbridges by use of n-ethyl maleimide (NEM-S1) to mimic rigor. We hypothesized that PKC phosphorylation of cTnI amplifies the effect of ischemic/hypoxic conditions to depress myofilament force and Ca²⁺-responsiveness by reducing the ability of rigor crossbridge to activate force. Pseudo-phosphorylation of cTnI at S43/S45 exacerbated the effect of acidic pH to induce a rightward shift in the Ca²⁺-tension relation. Under acidic conditions, fibers regulated by cTnI-(S43E/S45E) demonstrated a significant reduction in the ability of NEM-S1 to recruit cycling crossbridges, when compared to controls regulated by cTnI. Similar effects of pseudo-phosphorylation of cTnI-(T144) occurred, but to a lesser extent that those of pseudo-phosphorylation of S43/S45. We conclude that under acidic conditions PKC phosphorylation of cTnI residues at S43/S45 and at T144 is likely to have differential, but significant effects in depressing the ability of both Ca²⁺ and rigor crossbridges to activate force generation. Although these effects of PKC dependent phosphorylation may be maladaptive in heart failure, they may also spare ATP consumption and be cardio-protective in ischemia.     

Cardiomyocyte cell cycle activation improves cardiac function after myocardial infarction

AIMS: Cardiomyocyte loss is a major contributor to the decreased cardiac function observed in diseased hearts. Previous studies have shown that cardiomyocyte-restricted cyclin D2 expression resulted in sustained cell cycle activity following myocardial injury in transgenic (MHC-cycD2) mice. Here, we investigated the effects of this cell cycle activation on cardiac function following myocardial infarction (MI). METHODS AND RESULTS: MI was induced in transgenic and non-transgenic mice by left coronary artery occlusion. At 7, 60, and 180 days after MI, left ventricular pressure-volume measurements were recorded and histological analysis was performed. MI had a similar adverse effect on cardiac function in transgenic and non-transgenic mice at 7 days post-injury. No improvement in cardiac function was observed in non-transgenic mice at 60 and 180 days post-MI. In contrast, the transgenic animals exhibited a progressive and marked increase in cardiac function at subsequent time points. Improved cardiac function in the transgenic mice at 60 and 180 days post-MI correlated positively with the presence of newly formed myocardial tissue which was not apparent at 7 days post-MI. Intracellular calcium transient imaging indicated that cardiomyocytes present in the newly formed myocardium participated in a functional syncytium with the remote myocardium. CONCLUSION: These findings indicate that cardiomyocyte cell cycle activation leads to improvement of cardiac function and morphology following MI and may represent an important clinical strategy to promote myocardial regeneration.     

Effects of therapeutic beta blockade on myocardial function and cardiac remodelling in congenital cardiac disease

BACKGROUND: Cardiac remodelling is now recognised as an important aspect of cardiovascular disease progression and is, therefore, emerging as a therapeutic target in cardiac failure due to different etiologies. Little is known about the influence of different therapies for cardiac failure on the remodelling seen in infants with congenital cardiac disease. METHODS: During follow-up of a prospective and randomized trial, we investigated therapeutic effects on neurohormonal activation, ventricular function, and myocardial gene expression. We compared the data from 8 infants with severe congestive heart failure due to left-to-right shunts, who received digoxin and diuretics alone, to 9 infants who received additional treatment with propranolol. RESULTS: In these infants, beta-adrenergic blockade significantly reduced highly elevated levels of renin, from 284 +/- 319 microU/ml compared to 1061 +/- 769 microU/ml. Systolic ventricular function was normal in both groups, but diastolic ventricular function was improved in those receiving propranolol, indicated by significantly lower left atrial pressures, lower end-diastolic pressures, and less pronounced ventricular hypertrophy, the latter estimated by lower ratios of myocardial wall to ventricular cavity areas on average of 42%. Further hemodynamic parameters showed no significant differences between the groups, except for the lower heart rate in infants treated with propranolol. In those treated with digoxin and diuretics, there was a significant downregulation of beta2-receptor and angiotensin-2 receptor genes, and up-regulation of endothelin A receptor and connective tissue growth factor genes, that were partially prevented by additional treatment with propranolol. CONCLUSIONS: Beta-blockade is a new therapeutic approach for congestive heart failure in infants with congenital cardiac disease, producing with significant benefits on neurohormonal activation, diastolic ventricular function, and cardiac remodelling.     

Phosphorylation and function of cardiac myosin binding protein-C in health and disease

During the past 5 years there has been an increasing body of literature describing the roles cardiac myosin binding protein C (cMyBP-C) phosphorylation play in regulating cardiac function and heart failure. cMyBP-C is a sarcomeric thick filament protein that interacts with titin, myosin and actin to regulate sarcomeric assembly, structure and function. Elucidating the function of cMyBP-C is clinically important because mutations in this protein have been linked to cardiomyopathy in more than sixty million people worldwide. One function of cMyBP-C is to regulate cross-bridge formation through dynamic phosphorylation by protein kinase A, protein kinase C and Ca²⁺-calmodulin-activated kinase II, suggesting that cMyBP-C phosphorylation serves as a highly coordinated point of contractile regulation. Moreover, dephosphorylation of cMyBP-C, which accelerates its degradation, has been shown to associate with the development of heart failure in mouse models and in humans. Strikingly, cMyBP-C phosphorylation presents a potential target for therapeutic development as protection against ischemic-reperfusion injury, which has been demonstrated in mouse hearts. Also, emerging evidence suggests that cMyBP-C has the potential to be used as a biomarker for diagnosing myocardial infarction. Although many aspects of cMyBP-C phosphorylation and function remain poorly understood, cMyBP-C and its phosphorylation states have significant promise as a target for therapy and for providing a better understanding of the mechanics of heart function during health and disease. In this review we discuss the most recent findings with respect to cMyBP-C phosphorylation and function and determine potential future directions to better understand the functional role of cMyBP-C and phosphorylation in sarcomeric structure, myocardial contractility and cardioprotection.     

Myosin light chain phosphorylation enhances contraction of heart muscle via structural changes in both thick and thin filaments

Contraction of heart muscle is triggered by calcium binding to the actin-containing thin filaments but modulated by structural changes in the myosin-containing thick filaments. We used phosphorylation of the myosin regulatory light chain (cRLC) by the cardiac isoform of its specific kinase to elucidate mechanisms of thick filament-mediated contractile regulation in demembranated trabeculae from the rat right ventricle. cRLC phosphorylation enhanced active force and its calcium sensitivity and altered thick filament structure as reported by bifunctional rhodamine probes on the cRLC: the myosin head domains became more perpendicular to the filament axis. The effects of cRLC phosphorylation on thick filament structure and its calcium sensitivity were mimicked by increasing sarcomere length or by deleting the N terminus of the cRLC. Changes in thick filament structure were highly cooperative with respect to either calcium concentration or extent of cRLC phosphorylation. Probes on unphosphorylated myosin heads reported similar structural changes when neighboring heads were phosphorylated, directly demonstrating signaling between myosin heads. Moreover probes on troponin showed that calcium sensitization by cRLC phosphorylation is mediated by the thin filament, revealing a signaling pathway between thick and thin filaments that is still present when active force is blocked by Blebbistatin. These results show that coordinated and cooperative structural changes in the thick and thin filaments are fundamental to the physiological regulation of contractility in the heart. This integrated dual-filament concept of contractile regulation may aid understanding of functional effects of mutations in the protein components of both filaments associated with heart disease.Contraction of heart muscle is initiated by an intracellular Ca²⁺ transient followed by binding of Ca²⁺ to troponin in the thin filaments. The resulting change in thin filament structure allows myosin heads or motor domains from the thick filaments to bind to actin in the thin filaments, and a change in the conformation of the actin-attached head domain linked to ATP hydrolysis drives force generation and filament sliding (1–3). During this “working stroke,” small conformational changes in the catalytic domain of the myosin head associated with release of ATP hydrolysis products are amplified by its light chain domain, containing the essential and regulatory light chains (4), to produce a nanometer-scale displacement at the connection of the head to the thick filament backbone.Contractility of heart muscle is also controlled by multiple posttranslational modifications of both thick and thin filament regulatory proteins, and these changes in filament proteins have been widely implicated in the modulation of cardiac output in health and disease (5–7). In the present study, we focused on phosphorylation of the cardiac isoform of the regulatory light chain (cRLC) as a well-characterized and experimentally accessible example of modification of a thick filament component. cRLC is at the thick filament end of the myosin head, where it joins the coiled–coil tail that forms the thick filament backbone. cRLC is partially phosphorylated in vivo under basal conditions (8–12), and changes in its phosphorylation level are linked to heart disease (8, 11, 13, 14). cRLC mutations associated with hypertrophic cardiomyopathy abolish cRLC phosphorylation in vitro (15), and mice expressing nonphosphorylatable cRLCs show severe cardiac dysfunction (10, 16). In the vertebrate heart, cRLCs are phosphorylated almost exclusively by the cardiac isoform of myosin light chain kinase (cMLCK) (17, 18), and cMLCK gene ablation leads to severe cardiac hypertrophy (19).The mechanisms responsible for the regulation of cardiac function by cRLC phosphorylation are poorly understood at the molecular, cellular, and organ levels. Mechanistic hypotheses at the molecular level have been largely based on studies of smooth and skeletal muscle RLCs, which have similar molecular structures and contain phosphorylatable serines analogous to that (Ser15) in cRLC (4, 20–22). Phosphorylation of the analogous serine in smooth muscle RLC (smRLC) is the primary mechanism of contractile regulation in that tissue. Phosphorylation of skRLC plays a similar role in some invertebrate skeletal muscles (23, 24) and enhances contractility in mammalian skeletal muscle (25).Electron microscopy studies of isolated myosins and thick filaments from all these sources (21, 26, 27) suggest a conserved molecular mechanism in which RLC phosphorylation activates or potentiates contractility by disrupting a compact OFF conformation of myosin in which the myosin heads are folded back on the myosin tail (20, 21, 28) (Fig. 1A). In striated muscles, this folded OFF conformation is linked to the formation of an ordered helical lattice of myosin head domains on the surface of the thick filaments, with the long axis of the head roughly parallel to the filament axis. This surface lattice of myosin heads is stabilized by intermolecular interactions between adjacent myosin molecules and possibly between myosin and two other thick filament components, titin, and myosin-binding protein C (MyBP-C). This characteristic folded OFF state of myosin is also present in the C-zone of cardiac thick filaments: the region that contains MyBP-C (26, 27). Phosphorylation of skRLC in isolated thick filaments of skeletal muscle disrupts the lattice, releasing the myosin heads from the filament surface (Fig. 1B). These structural studies on isolated proteins and filaments led to the hypothesis that RLC phosphorylation potentiates contractility in skeletal and cardiac muscle by increasing the number of myosin heads available for actin interaction.Open in a separate windowFig. 1.OFF and ON conformations of the thick and thin filaments in heart muscle. (A) OFF conformations at low [Ca²⁺] in the absence of phosphorylation of the cRLC (blue). (B) ON conformations with calcium (green) bound to troponin C (pink/red) and cRLC phosphorylated (yellow circle, P). Troponin I, troponin T, and tropomyosin are yellow, blue, and sepia, respectively. Essential light chain is brown. BC-cRLC probe dipole is indicated by red double arrows.The aim of the present work was to understand the role of interactions between thick and thin filament-based regulatory mechanisms in a heart muscle cell, in which the normal structure, organization, and interrelationship of the filaments is preserved. We measured the structural changes in the thick filaments of ventricular trabeculae by polarized fluorescence from bifunctional probes attached to the cRLC, exploiting the fact that the native cRLCs in demembranated trabeculae can be efficiently replaced by labeled cRLCs with negligible effect on trabecular function (29, 30). We used in situ phosphorylation of cRLC by an expressed cMLCK to control the regulatory state of the thick filament and further exploited the cRLC exchange protocol to introduce cRLC mutants for mechanistic studies. We studied the relationship between thick and thin filament regulation using calcium titrations with probes on both the cRLC in the thick filaments and troponin in the thin filaments. The results presented below show that integration of thick and thin filament-based signaling pathways is essential for the normal regulation of contractility in the heart.     

Phenotypic variation in myocardial macrophage populations suggests a role for macrophage activation in SIV-associated cardiac disease

Cardiac abnormalities are common in HIV-infected individuals, and have been especially well documented as contributors to mortality in HIV-infected children. Underlying pathogenetic mechanisms responsible for myocardial disease in HIV-infection remain imperfectly understood. SIV-infected rhesus monkeys develop a spectrum of cardiac lesions similar to those seen in HIV-infected people, providing an important model for pathogenesis studies. Retrospective analysis of cardiac tissue collected at necropsy from SIV-infected rhesus monkeys was performed to evaluate myocardial macrophage and dendritic cell populations as a function of previously quantitated lymphocytic inflammatory infiltrates and cardiomyocyte degeneration or necrosis. Variations in the size and phenotype of macrophage and dendritic cell populations were examined as possible contributors to the pathogenesis of SIV-associated inflammatory lesions. Macrophages labeling immunohistochemically for CD163 differed substantially from macrophages labeling for HAM56 in overall number, distribution across groups, involvement in inflammatory clusters, correlation with the DC-SIGN(+) subpopulation of macrophages, and correlation with numbers of SIV-infected cells. CD163(+) macrophages occurred in significantly higher numbers in uninflamed hearts from SIV-infected animals than in hearts from SIV-infected animals with myocarditis or uninfected controls (p < 0.01). Numbers of CD163(+) cells correlated positively with numbers of SIV-infected cells (p < 0.05) suggesting that the CD163(+) population was associated with decreased inflammatory infiltration and reduced control of virus within the heart. As CD163 has been associated with nonclassical macrophage activation and an antiinflammatory phenotype, these results suggest that a balance between classical and nonclassical activation may affect levels of inflammatory infiltration and of myocardial virus burden.     

Pressure-flow relations in the pulmonary artery during myocardial ischaemia: implications for right ventricular function in coronary disease

Because exercise induced pulmonary hypertension may disturb optimal coupling between the right ventricle and pulmonary artery in coronary artery disease, high fidelity pulmonary artery and right ventricular pressure and electromagnetic pulmonary artery flow velocity data were recorded at rest and during supine exercise in 10 control subjects free of detectable cardiovascular disease and in 11 patients with coronary artery disease. The pulmonary artery impedance and power spectra were calculated from Fourier analysis of pressure and flow waveforms. Total hydraulic power expended per unit of forward flow was computed as an index of right ventricular-pulmonary artery coupling. In coronary artery disease exercise produced substantial increases in pulmonary artery pressure, pulmonary artery characteristic impedance, and total power per unit flow. These changes did not occur in control subjects. Despite a significant exercise increase in right ventricular end diastolic pressure and peak right ventricular dP/dt, and independent of the presence of right coronary artery involvement, the right ventricular stroke output response during exercise was significantly blunted in the coronary artery disease patients. Pulmonary vascular resistance was unchanged by exercise in either group. Exercise induced ischaemia presents an increased pulsatile hydraulic load to the right ventricle. Increased pulmonary artery input impedance impairs the hydraulic efficiency of right ventricular-pulmonary artery coupling and may contribute to the limitation of right ventricular ejection performance in coronary artery disease.     

Remote control of A-band cardiac thin filaments by the I-Z-I protein network of cardiac sarcomeres

A conventional view of the role of sarcomeric thin filaments in cardiac function is that they react with cross-bridges that translate them toward the center of the sarcomere in a reaction triggered by Ca(2+) and powered by ATP. However, thin filaments also engage in a complex network of protein-protein interactions in the Z-disc. Thus, in the modern context, understanding of what thin filaments do in the heart must take into account not only A-band regions that react with cross-bridges, but also I-Z-I regions that rarely, if ever, react with cross-bridges and dwell near and within the Z-disc. To highlight these multiplex functions of the thin filament, I discuss the hypothesis that physical and chemical reactions at the interface of the thin filaments with Z-disc proteins control the docking and activity of kinases and phosphatases that control the levels of phosphorylation of thin filament regulatory proteins. Testing this hypothesis has taken on new significance with the identification of multisite phosphorylation of thin filament proteins as a critical element in the control of cardiac contraction and relaxation reserve and in maladaptive mechanisms in heart failure. Moreover, multiple mutations in Z-disc proteins that link to prevalent cardiomyopathies are likely to alter this remote control of A-band thin filament function.     

Role of endothelium-derived nitric oxide in the regulation of cardiac oxygen metabolism: implications in health and disease

Endothelium-derived NO is considered to be primarily an important determinant of vascular tone and platelet activity; however, the modulation of myocardial metabolism by NO may be one of its most important roles. This modulation may be critical for the regulation of tissue metabolism. Several physiological processes act in concert to make endothelial NO synthase-derived NO potentially important in the regulation of mitochondrial respiration in cardiac tissue, including (1) the nature of the capillary network in the myocardium, (2) the diffusion distance for NO, (3) the low toxicity of NO at physiological (nanomolar) concentrations, (4) the fact that low PO(2) in tissue facilitates the action of NO on cytochrome oxidase, and (5) the formation of oxygen free radicals. A decrease in NO production is involved in the pathophysiological modifications that occur in heart failure and diabetes, disease states associated with altered cardiac metabolism that contributes to the evolution of the disease process. In contrast, several drugs (eg, angiotensin-converting enzyme inhibitors, amlodipine, and statins) can restore or maintain endogenous production of NO by endothelial cells, and this mechanism may explain part of their therapeutic efficiency. Thus, the purpose of this review is to critically evaluate the role of NO in the control of mitochondrial respiration, with special emphasis on its effect on cardiac metabolism.     

Acceleration of crossbridge kinetics by protein kinase A phosphorylation of cardiac myosin binding protein C modulates cardiac function

Normal cardiac function requires dynamic modulation of contraction. beta1-adrenergic-induced protein kinase (PK)A phosphorylation of cardiac myosin binding protein (cMyBP)-C may regulate crossbridge kinetics to modulate contraction. We tested this idea with mechanical measurements and echocardiography in a mouse model lacking 3 PKA sites on cMyBP-C, ie, cMyBP-C(t3SA). We developed the model by transgenic expression of mutant cMyBP-C with Ser-to-Ala mutations on the cMyBP-C knockout background. Western blots, immunofluorescence, and in vitro phosphorylation combined to show that non-PKA-phosphorylatable cMyBP-C expressed at 74% compared to normal wild-type (WT) and was correctly positioned in the sarcomeres. Similar expression of WT cMyBP-C at 72% served as control, ie, cMyBP-C(tWT). Skinned myocardium responded to stretch with an immediate increase in force, followed by a transient relaxation of force and finally a delayed development of force, ie, stretch activation. The rate constants of relaxation, k(rel) (s-1), and delayed force development, k(df) (s-1), in the stretch activation response are indicators of crossbridge cycling kinetics. cMyBP-C(t3SA) myocardium had baseline k(rel) and k(df) similar to WT myocardium, but, unlike WT, k(rel) and k(df) were not accelerated by PKA treatment. Reduced dobutamine augmentation of systolic function in cMyBP-C(t3SA) hearts during echocardiography corroborated the stretch activation findings. Furthermore, cMyBP-C(t3SA) hearts exhibited basal echocardiographic findings of systolic dysfunction, diastolic dysfunction, and hypertrophy. Conversely, cMyBP-C(tWT) hearts performed similar to WT. Thus, PKA phosphorylation of cMyBP-C accelerates crossbridge kinetics and loss of this regulation leads to cardiac dysfunction.     

Growth-factor-mediated cardiac stem cell activation in myocardial regeneration

The concept of an intrinsic regenerative capacity of the adult mammalian myocardium owing to the presence of cardiac stem cells (CSCs) in the atria and ventricles is starting to be accepted by the cardiovascular research community. The identification of this cell population has improved the prospects for developing successful clinical protocols for human myocardial regeneration. In the normal adult myocardium, only a small fraction of CSCs undergo amplification and differentiation to replace the parenchymal cells lost by normal wear and tear. Physiological or pathological stimuli cause substantial activation of CSCs, which is mediated by a paracrine feedback loop between myocytes and CSCs. In response to stress, the myocytes produce growth factors and cytokines, for which CSCs have receptors, and autocrine, self-sustaining activation of growth-factor production is simultaneously triggered in the CSCs. These findings from human and animal studies led us to test whether in situ activation of CSCs by growth factors would be as effective as transplantation of CSCs into the regenerating myocardium after ischemia in an animal model that has relevance to humans. In a porcine model, we produced extensive and functionally relevant myocardial regeneration. Here, we discuss the properties of endogenous myocardial stem cells that might be exploited to produce clinical myocardial regeneration without the need for cell transplantation.     

Visualization of cardiac muscle thin filaments and measurement of their lengths by electron tomography

AIMS: An intriguing difference between vertebrate skeletal and cardiac muscles is that the lengths of the thin filaments are constant in the former but variable in the latter. The thick filaments have constant lengths in both types of muscles. The contractile behaviour of a muscle is affected by the lengths of both types of filaments as the tension generated during contraction depends on the amount of filament overlap. To understand the behaviour of cardiac muscle, it is important to know the distribution of the thin filament lengths. The previous detailed analysis by Robinson and Winegrad used serial transverse sections to determine the lengths of the thin filaments. However, the precision, set by the 100 nm section thickness, was low. Here, we have used electron tomography to produce 3D images of rat and mouse cardiac muscles in which we can actually see individual thin filaments up to the free ends and see that these free ends have variable locations. For comparison, we also measure the thin filament lengths in skeletal muscle (frog sartorius). METHODS AND RESULTS: Cardiac papillary muscles were obtained from a rat (Sprague-Dawley) and a mouse (C57/B6). Skeletal muscle (sartorius) was obtained from a frog (Rana pipiens). Longitudinal sections (100 nm thick) were used to produce tilt series and tomograms from which the thin filament paths were traced. Cardiac papillary muscle thin filaments in rat and mouse range from 0.94 to 1.10 microm, with a mean length of 1.04 microm and standard deviation of 0.03 microm. For frog sartorius muscle, the thin filament length was 0.94 microm with standard deviation of 0.01 microm. CONCLUSION: Electron tomography of cardiac and skeletal muscles allows direct visualization and high precision measurement of the lengths of thin filaments.     

Tropomodulin function and thin filament assembly in cardiac myocytes

The regulation of thin filament length is a fundamental property of all striated muscles. Tropomodulin is an actin and tropomyosin binding protein that is exclusively associated with the free (pointed) ends of thin filaments. In vitro and in vivo studies reveal that tropomodulin is an actin filament pointed end capping protein, which is required to maintain the final length of thin filaments and is essential for contractile activity in embryonic chick cardiac myocytes. Understanding the mechanisms of thin filament assembly, as well as determining the roles of proteins modulating actin filament dynamics, is important for future considerations of the molecular bases for myopathies seen in various types of heart disease.     

Potassium control in chronic kidney disease: implications for neuromuscular function

In Australia, approximately 1.7 million adults have evidence of chronic kidney disease (CKD). This complex disease can result in a multitude of complications, including hyperkalaemia, which is common and well recognised. The advent of new therapeutics aimed at lowering serum potassium has raised the possibility of optimising potassium control to enable greater use of renin–angiotensin–aldosterone system inhibitors in the management of CKD. Recent studies suggest that hyperkalaemia also has implications for peripheral neuropathy in CKD, a complication that substantially contributes to patient morbidity. This review examines evidence of the relationship between potassium and peripheral neuropathy, with a discussion of clinical implications. We searched PubMed for original and review articles using pre‐specified key words, clinical guidelines and population data. The major findings were that contemporary CKD cohorts demonstrate a high prevalence of peripheral neuropathy, even in stage 3–4 CKD, including those without diabetes. The severity of the problem has been emphasised by an ominous rise in foot complications and amputation rates in dialysis patients, highlighting the need for increased awareness of the condition in earlier stages of CKD and targeted treatment strategies. It is likely that the pathophysiology of peripheral neuropathy in CKD is multifaceted, with potential influences from potassium, vascular abnormalities, diabetes, inflammation and unknown middle molecules. Despite these complexities, the relationship between potassium and nerve function in dialysis has been well established, and recent research in stage 3–4 CKD suggests that assertive potassium control may improve neuromuscular outcomes in CKD. These small studies should be confirmed in large, multicentre settings.     

Creatine phosphate consumption and the actomyosin crossbridge cycle in cardiac muscles

To investigate the regulation of the actomyosin crossbridge cycle in cardiac muscles, the effects of ATP, ADP, Pi, and creatine phosphate (CP) on the rate of force redevelopment (ktr) were measured. We report that CP is a primary determinant in controlling the actomyosin crossbridge cycling kinetics of cardiac muscles, because a reduction of CP from 25 to 2.5 mmol/L decreased ktr by 51% despite the presence of 5 mmol/L MgATP. The effects of CP on ktr were not a reflection of reduced ATP or accumulated ADP, because lowering ATP to 1 mmol/L or increasing ADP to 1 mmol/L did not significantly decrease ktr. Therefore, the effect of CP on the actomyosin crossbridge cycle is proposed to occur through a functional link between ADP release from myosin and its rephosphorylation by CP-creatine kinase to regenerate ATP. In activated fibers, the functional link influenced the kinetics of activated crossbridges without affecting the aggregate number of force-generating crossbridges. This was demonstrated by the ability of CP to affect ktr in maximally and submaximally activated fibers without altering the force per cross-sectional area. The data also confirm the important contribution of strong binding crossbridges to cardiac muscle activation, likely mediated by cooperative recruitment of adjacent crossbridges to maximize force redevelopment against external load. These data provide additional insight into the role of CP during pathophysiological conditions such as ischemia, suggesting that decreased CP may serve as a primary determinant in the observed decline of dP/dt.