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.

Cardiac Troponin T (cTnT) is one prominent substrate through which protein kinase C (PKC) exerts its effect on cardiomyocyte function. To determine the specific functional effects of the cTnT PKC-dependent phosphorylation sites (Thr¹⁹⁷, Ser²⁰¹, Thr²⁰⁶, and Thr²⁸⁷) we first mutated these residues to glutamate (E) or alanine (A). cTnT was selectively mutated to generate single, double, triple, and quadruple mutants. Bacterially expressed mutants were evaluated in detergent-treated mouse left ventricular papillary muscle fiber bundles where the endogenous troponin was replaced with a recombinant troponin complex containing either cTnT phosphorylated by PKC-α or a mutant cTnT. We simultaneously determined isometric tension development and actomyosin Mg-ATPase activity of the exchanged fiber bundles as a function of Ca²⁺ concentration. Our systematic analysis of the functional role of the multiple PKC phosphorylation sites on cTnT identified a localized region that controls maximum tension, ATPase activity, and Ca²⁺ sensitivity of the myofilaments. An important and novel finding of our study was that Thr²⁰⁶ is a functionally critical cTnT PKC phosphorylation residue. Its exclusive phosphorylation by PKC-α or replacement by Glu (mimicking phosphorylation) significantly decreased maximum tension, actomyosin Mg-ATPase activity, myofilament Ca²⁺ sensitivity, and cooperativity. On the other hand the charge modification of the other three residues together (T197/S201/T287-E) had no functional effect. Fibers bundles containing phosphorylated cTnT-wt (but not the T197/S201/T206/T287-E) exhibited a significant decrease of tension cost as compared with cTnT-wt.

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.

We investigated intracellular localization and substrate specificity of P ²¹ -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 Ca ²⁺ 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 Ca ²⁺ . Our data provide evidence for a novel pathway of phosphatase regulation in cardiac myocytes.

In cardiovascular research, sex and gender have not typically been considered in research design and reporting until recently. This has resulted in clinical research findings from which not only all women, but also gender-diverse individuals have been excluded. The resulting dearth of data has led to a lack of sex- and gender-specific clinical guidelines and raises serious questions about evidence-based care. Basic research has also excluded considerations of sex. Including sex and/or gender as research variables not only has the potential to improve the health of society overall now, but it also provides a foundation of knowledge on which to build future advances. The goal of this guidelines article is to provide advice on best practices to include sex and gender considerations in study design, as well as data collection, analysis, and interpretation to optimally establish rigor and reproducibility needed to inform clinical decision-making and improve outcomes. In cardiovascular physiology, incorporating sex and gender is a necessary component when optimally designing and executing research plans. The guidelines serve as the first guidance on how to include sex and gender in cardiovascular research. We provide here a beginning path toward achieving this goal and improve the ability of the research community to interpret results through a sex and gender lens to enable comparison across studies and laboratories, resulting in better health for all.

Circadian rhythms are essential to cardiovascular health and disease. Temporal coordination of cardiac structure and function has focused primarily at the physiological and gene expression levels, but these analyses are invariably incomplete, not the least because proteins underlie many biological processes. The purpose of this study was to reveal the diurnal cardiac proteome and important contributions to cardiac function. The 24-h day-night murine cardiac proteome was assessed by two-dimensional difference in gel electrophoresis (2D-DIGE) and liquid chromatography-mass spectrometry. Daily variation was considerable, as ∼7.8% (90/1,147) of spots exhibited statistical changes at paired times across the 24-h light- (L) dark (D) cycle. JTK_CYCLE was used to investigate underlying diurnal rhythms in corresponding mRNA. We next revealed that disruption of the L:D cycle altered protein profiles and diurnal variation in cardiac function in Langendorff-perfused hearts, relative to the L:D cycle. To investigate the role of the circadian clock mechanism, we used cardiomyocyte clock mutant (CCM) mice. CCM myofilaments exhibited a loss of time-of-day-dependent maximal calcium-dependent ATP consumption, and altered phosphorylation rhythms. Moreover, the cardiac proteome was significantly altered in CCM hearts, especially enzymes regulating vital metabolic pathways. Lastly, we used a model of pressure overload cardiac hypertrophy to demonstrate the temporal proteome during heart disease. Our studies demonstrate that time of day plays a direct role in cardiac protein abundance and indicate a novel mechanistic contribution of circadian biology to cardiovascular structure and function.