W. Glen Pyle

At the crossroads of myocardial signaling: the role of Z-discs in intracellular signaling and cardiac function.

Understanding the molecular interactions among components of cardiac Z-discs and their role in signaling has become pivotal in explaining long- and short-term regulation of cardiac function. In striated muscle, the ends of the thin filaments from opposing sarcomeres overlap and are cross-linked by an elaborate array of proteins to form a highly ordered, yet dynamic network that is the Z-disc. We review here a current picture of the function and structure of the Z-disc of mammalian cardiac myocytes. We emphasize provocative findings that advance new theories about the place of cardiac Z-discs in myocardial intra- and intercellular signaling in myocardial physiology and pathology. Relatively new approaches, especially yeast two-hybrid screens, immunoprecipitation, and pull down assays, as well as immunohistochemical analysis have significantly altered previous views of the protein content of the Z-disc. These studies have generally defined domain structure and binding partners for Z-disc proteins, but the functional significance of the binding network and of the domains in cardiac cell biology remains an unfolding story. Yet, even at the present level of understanding, perceptions of potential functions of the Z-disc proteins are expanding greatly and leading to new and exciting experimental approaches toward mechanistic understanding. The theme of the following discussion of these Z-disc proteins centers on their potential to function not only as a physical anchor for myofilament and cytoskeletal proteins, but also as a pivot for reception, transduction, and transmission of mechanical and biochemical signals.

Intracellular Localization and Functional Effects of P21-Activated Kinase-1 (Pak1) in Cardiac Myocytes

Abstract— We investigated intracellular localization and substrate specificity of P21-activated kinase-1 (Pak1) in rat cardiac myocytes. Pak1 is a serine/threonine protein kinase that is activated by Rac1/Cdc42 and important in signaling of stress responses. Yet the localization and in vivo function of Pak1 in heart cells is poorly understood. Studies reported here indicate that Pak1 physically interacts with protein phosphatase 2a and localizes to the Z-disk, cell membrane, intercalated disc, and nuclear membrane of adult rat heart myocytes. We compared levels of phosphorylation of cardiac troponin I (cTnI) in control myocytes with phosphorylation of cTnI and myosin binding protein C (C-protein) in myocytes with increased Pak1 activity. The increase in activity was induced by infection of myocytes with a recombinant adenovirus (AdPak1) containing cDNA for a constitutively active Pak1. Control cells were infected with a virus (AdLacZ) containing LacZ. Basal levels of phosphorylation of cTnI and C-protein were relatively high in the myocytes infected with AdLacZ. However, phosphorylation of cTnI and C-protein in cells expressing constitutively active Pak1 was significantly reduced compared with those expressing LacZ. Measurement of Ca2+ tension relations in single myocytes demonstrated that this reduction in phosphorylation of cTnI and C-protein was associated with the predicted increase in sensitivity to Ca2+. Our data provide evidence for a novel pathway of phosphatase regulation in cardiac myocytes.

Actin Capping Protein: An Essential Element in Protein Kinase Signaling to the Myofilaments

Actin capping protein (CapZ) binds the barbed ends of actin at sarcomeric Z-lines. In addition to anchoring actin, Z-discs bind protein kinase C (PKC). Although CapZ is crucial for myofibrillogenesis, its role in muscle function and intracellular signaling is unknown. We hypothesized that CapZ downregulation would impair myocardial function and disrupt PKC-myofilament signaling by impairing PKC–Z-disc interaction. To test these hypotheses, we examined transgenic (TG) mice in which cardiac CapZ protein is reduced. Fiber bundles were dissected from papillary muscles and detergent extracted. Some fiber bundles were treated with PKC activators phenylephrine (PHE) or endothelin (ET) before detergent extraction. We simultaneously measured Ca2+-dependent tension and actomyosin MgATPase activity. CapZ downregulation increased myofilament Ca2+ sensitivity without affecting maximum tension or actomyosin MgATPase activity. Maximum tension and actomyosin MgATPase activity were decreased after PHE or ET treatment of wild-type (WT) muscle. Fiber bundles from TG hearts did not respond to PHE or ET. Immunoblot analysis revealed an increase in myofilament-associated PKC-&egr; after PHE or ET exposure of WT preparations. In contrast, myofilament-associated PKC-&egr; was decreased after PHE or ET treatment in TG myocardium. Protein levels of myofilament-associated PKC-&bgr; were decreased in TG ventricle. C-protein and troponin I phosphorylation was increased after PHE or ET treatment in WT and TG hearts. Basal phosphorylation levels of C-protein and troponin I were higher in TG myocardium. These results indicate that downregulation of CapZ, or other changes associated with CapZ downregulation, increases cardiac myofilament Ca2+ sensitivity, inhibits PKC-mediated control of myofilament activation, and decreases myofilament-associated PKC-&bgr;.

Short-Term Disruption of Diurnal Rhythms After Murine Myocardial Infarction Adversely Affects Long-Term Myocardial Structure and Function

Rationale: Patients in intensive care units are disconnected from their natural environment. Synchrony between environmental diurnal rhythms and intracellular circadian rhythms is essential for normal organ biology; disruption causes pathology. Whether disturbing rhythms after myocardial infarction (MI) exacerbates long-term myocardial dysfunction is not known. Objective: Short-term diurnal rhythm disruption immediately after MI impairs remodeling and adversely affects long-term cardiac structure and function in a murine model. Methods and Results: Mice were infarcted by left anterior descending coronary artery ligation (MI model) within a 3-hour time window, randomized to either a normal diurnal or disrupted environment for 5 days, and then maintained under normal diurnal conditions. Initial infarct size was identical. Short-term diurnal disruption adversely affected body metabolism and altered early innate immune responses. In the first 5 days, crucial for scar formation, there were significant differences in cardiac myeloperoxidase, cytokines, neutrophil, and macrophage infiltration. Homozygous clock mutant mice exhibited altered infiltration after MI, consistent with circadian mechanisms underlying innate immune responses crucial for scar formation. In the proliferative phase, 1 week after MI, this led to significantly less blood vessel formation in the infarct region of disrupted mice; by day 14, echocardiography showed increased left ventricular dilation and infarct expansion. These differences continued to evolve with worse cardiac structure and function by 8 weeks after MI. Conclusions: Diurnal rhythm disruption immediately after MI impaired healing and exacerbated maladaptive cardiac remodeling. These preclinical findings suggest that disrupted diurnal rhythms such as found in modern intensive care unit environments may adversely affect long-term patient outcome.

Short Term Disruption of Diurnal Rhythms Following Murine Myocardial Infarction Adversely Affects Long Term Myocardial Structure and Function

Rationale: Patients in intensive care units are disconnected from their natural environment. Synchrony between environmental diurnal rhythms and intracellular circadian rhythms is essential for normal organ biology; disruption causes pathology. Whether disturbing rhythms after myocardial infarction (MI) exacerbates long-term myocardial dysfunction is not known. Objective: Short-term diurnal rhythm disruption immediately after MI impairs remodeling and adversely affects long-term cardiac structure and function in a murine model. Methods and Results: Mice were infarcted by left anterior descending coronary artery ligation (MI model) within a 3-hour time window, randomized to either a normal diurnal or disrupted environment for 5 days, and then maintained under normal diurnal conditions. Initial infarct size was identical. Short-term diurnal disruption adversely affected body metabolism and altered early innate immune responses. In the first 5 days, crucial for scar formation, there were significant differences in cardiac myeloperoxidase, cytokines, neutrophil, and macrophage infiltration. Homozygous clock mutant mice exhibited altered infiltration after MI, consistent with circadian mechanisms underlying innate immune responses crucial for scar formation. In the proliferative phase, 1 week after MI, this led to significantly less blood vessel formation in the infarct region of disrupted mice; by day 14, echocardiography showed increased left ventricular dilation and infarct expansion. These differences continued to evolve with worse cardiac structure and function by 8 weeks after MI. Conclusions: Diurnal rhythm disruption immediately after MI impaired healing and exacerbated maladaptive cardiac remodeling. These preclinical findings suggest that disrupted diurnal rhythms such as found in modern intensive care unit environments may adversely affect long-term patient outcome.

Consequences of Circadian and Sleep Disturbances for the Cardiovascular System

Circadian rhythms play a crucial role in our cardiovascular system. Importantly, there has been a recent flurry of clinical and experimental studies revealing the profound adverse consequences of disturbing these rhythms on the cardiovascular system. For example, circadian disturbance worsens outcome after myocardial infarction with implications for patients in acute care settings. Moreover, disturbing rhythms exacerbates cardiac remodelling in heart disease models. Also, circadian dyssynchrony is a causal factor in the pathogenesis of heart disease. These discoveries have profound implications for the cardiovascular health of shift workers, individuals with circadian and sleep disorders, or anyone subjected to the 24/7 demands of society. Moreover, these studies give rise to 2 new frontiers for translational research: (1) circadian rhythms and the cardiac sarcomere, which sheds new light on our understanding of myofilament structure, signalling, and electrophysiology; and (2) knowledge translation, which includes biomarker discovery (chronobiomarkers), timing of therapies (chronotherapy), and other new promising approaches to improve the management and treatment of cardiovascular disease. Reconsidering circadian rhythms in the clinical setting benefits repair mechanisms, and offers new promise for patients.

Relaxin alters cardiac myofilament function through a PKC-dependent pathway

The pregnancy hormone relaxin (RLX) is a powerful cardiostimulatory peptide. Despite its well-characterized effects on the heart, the intracellular mechanisms responsible for RLXs positive inotropic effects are unknown. Cardiac myofilaments are the central contractile elements of the heart, and changes in the phosphorylation status of myofilament proteins are known to mediate changes in function. The first objective of this study was to determine whether RLX stimulates myofilament activation and alters the phosphorylation of one or more myofilament proteins. RLX works through a variety of intracellular signaling cascades in different tissue types. Protein kinases A (PKA) and C (PKC) are two common molecules implicated in RLX signaling and are known to affect myofilament function. Thus the second objective of this study was to determine whether RLX mediates its myocardial effects through PKA or PKC activation. Murine myocardium was treated with recombinant H2-RLX, and cardiac myofilaments were isolated. RLX increased cardiac myofilament force development at physiological levels of intracellular Ca(2+) without altering myofilament ATP consumption. Myosin binding protein C, troponin T, and troponin I phosphorylation levels were increased with RLX treatment. Immunoblot analysis revealed an increase in myofilament-associated PKC-delta, decreases in PKC-alpha and -beta(II), but no effect on PKC-epsilon. Inhibition of PKC with chelerythrine chloride or PKC-delta with rottlerin prevented the RLX-dependent changes in myofilament function and protein phosphorylation. PKA antagonism with H-89 had no effect on the myofilament effects of RLX. This study is the first to show that RLX-dependent changes in myofilament-associated PKC alters myofilament activation in a manner consistent with its cardiostimulatory effects.

Cardiac contraction, calcium transients, and myofilament calcium sensitivity fluctuate with the estrous cycle in young adult female mice

This study established conditions to induce regular estrous cycles in female C57BL/6J mice and investigated the impact of the estrous cycle on contractions, Ca2+ transients, and underlying cardiac excitation-contraction (EC)-coupling mechanisms. Daily vaginal smears from group-housed virgin female mice were stained to distinguish estrous stage (proestrus, estrus, metestrus, diestrus). Ventricular myocytes were isolated from anesthetized mice. Contractions and Ca2+ transients were measured simultaneously (4 Hz, 37 °C). Interestingly, mice did not exhibit regular cycles unless they were exposed to male pheromones in bedding added to their cages. Field-stimulated myocytes from mice in estrus had larger contractions (∼2-fold increase), larger Ca2+ transients (∼1.11-fold increase), and longer action potentials (>2-fold increase) compared with other stages. Larger contractions and Ca2+ transients were not observed in estrus myocytes voltage-clamped with shorter action potentials. Voltage-clamp experiments also demonstrated that estrous stage had no effect on Ca2+ current, EC-coupling gain, diastolic Ca2+, sarcoplasmic reticulum (SR) Ca2+ content, or fractional release. Although contractions were largest in estrus, myofilament Ca2+ sensitivity was lowest (EC50 values ∼1.15-fold higher) in conjunction with increased phosphorylation of myosin binding protein C in estrus. Contractions were enhanced in ventricular myocytes from mice in estrus because action potential prolongation increased SR Ca2+ release. These findings demonstrate that cyclical changes in reproductive hormones associated with the estrous cycle can influence myocardial electrical and contractile function and modify Ca2+ homeostasis. However, such changes are unlikely to occur in female mice housed in groups under conventional conditions, since these mice do not exhibit regular estrous cycles.

Rapid Changes in Cardiac Myofilament Function following the Acute Activation of Estrogen Receptor-Alpha

Estrogens have well-recognized and complex cardiovascular effects, including altering myocardial contractility through changes in myofilament function. The presence of multiple estrogen receptor (ER) isoforms in the heart may explain some discrepant findings about the cardiac effects of estrogens. Most studies examining the impact of estrogens on the heart have focused on chronic changes in estrogen levels, and have not investigated rapid, non-genomic pathways. The first objective of this study was to determine how acute activation of ERα impacts cardiac myofilaments. Nongenomic myocardial estrogen signaling is associated with the activation of a variety of signaling pathways. p38 MAPK has been implicated in acute ER signaling in the heart, and is known to affect myofilament function. Thus, the second objective of this study was to determine if acute ERα activation mediates its myofilament effects through p38 MAPK recruitment. Hearts from female C57Bl/6 mice were perfused with the ERα agonist PPT and myofilaments isolated. Activation of ERα depressed actomyosin MgATPase activity and decreased myofilament calcium sensitivity. Inhibition of p38 MAPK attenuated the myofilament effects of ERα activation. ERα stimulation did not affect global myofilament protein phosphorylation, but troponin I phosphorylation at the putative PKA phosphorylation sites was decreased. Changes in myofilament activation did not translate into alterations in whole heart function. The present study provides evidence supporting rapid, non-genomic changes in cardiac myofilament function following acute ERα stimulation mediated by the p38 MAPK pathway.

2-Deoxyadenosine triphosphate restores the contractile function of cardiac myofibril from adult dogs with naturally occurring dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a major type of heart failure resulting from loss of systolic function. Naturally occurring canine DCM is a widely accepted experimental paradigm for studying human DCM. 2-Deoxyadenosine triphosphate (dATP) can be used by myosin and is a superior energy substrate over ATP for cross-bridge formation and increased systolic function. The objective of this study was to evaluate the beneficial effect of dATP on contractile function of cardiac myofibrils from dogs with naturally occurring DCM. We measured actomyosin NTPase activity and contraction/relaxation properties of isolated myofibrils from nonfailing (NF) and DCM canine hearts. NTPase assays indicated replacement of ATP with dATP significantly increased myofilament activity in both NF and DCM samples. dATP significantly improved maximal tension of DCM myofibrils to the NF sample level. dATP also restored Ca(2+) sensitivity of tension that was reduced in DCM samples. Similarly, dATP increased the kinetics of contractile activation (kACT), with no impact on the rate of cross-bridge tension redevelopment (kTR). Thus, the activation kinetics (kACT/kTR) that were reduced in DCM samples were restored for dATP to NF sample levels. dATP had little effect on relaxation. The rate of early slow-phase relaxation was slightly reduced with dATP, but its duration was not, nor was the fast-phase relaxation or times to 50 and 90% relaxation. Our findings suggest that myosin utilization of dATP improves cardiac myofibril contractile properties of naturally occurring DCM canine samples, restoring them to NF levels, without compromising relaxation. This suggests elevation of cardiac dATP is a promising approach for the treatment of DCM.