Function Of Cardiac Muscle Cells Research Articles

Substrate stiffness induces a chronic adaptation in contractile work and electrophysiology of cardiomyocytes.

The mechanical function or failure of the heart is defined by the adaptability of its contractility under different conditions. This, coupled with dynamic changes of heart mechanics, via hemodynamics or pathological stiffening, have driven decades of research to understand how external mechanical cues affect function of cardiac muscle cells (CMs). Despite a growing body of research on the effect of microenvironment mechanics (often stiffness) on contractility of CMs, there has been little appreciation of how this adaptation evolves over time. In vivo, CMs experience a gradual increase in the stiffness of their microenvironment, from ∼1 kPa in neonatal heart, to ∼10 kPa for adult heart to ∼50 kPa in fibrotic myocardium (e.g post myocardial infarction). While such mechanical changes have shown to affect CMs behaviour, the role of chronic adaptation over time is often ignored. Here we quantified the evolution of contractile work of CMs over six days. Primary CMs were cultured on substrates of neonatal, adult, and fibrotic myocardium and we measured their contractility using Traction Force Microsocpy (TFM) on days 6, 8, 10, and 12. We observed a gradual increase in contractility in all conditions, suggesting an adaptation mechanism controlling the acute response over longer time scales. To study their electrical response, we used dye-free diffraction imaging. We observed that monolayers on 1 kPa substrates show higher autonomous beating rates, and while substrates with stiffnesses of 10 and 50 kPa show similar beating rates, the monolayers on the 50 kPa substrate have a smaller contraction magnitude. We verified these results with fluorescent imaging of a calcium indicator (FlouO-4). CMs on neonatal stiffness showed significantly faster action potentials (smaller duration) than their counterparts on stiffer substrates. These results indicate the importance of the time-dependent response of CMs to the mechanics of their microenvironment.

Stem cells from the amniotic fluid: a promising tool to face the cytokine storm associated to SARS‐CoV‐2 infections

Infection with severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), the etiological agent of coronavirus disease 19 (COVID‐19) is associated with significant morbidity and mortality. Despite significant progress since the emergence of this disease, efficacious therapeutics are still urgently required. Infection is associated with a broad spectrum of symptoms and varying disease severity. In severe cases, a multi‐organ disease is apparent, involving the respiratory, coagulation, immune and cardiac systems. A central tenant of severe disease is an excessive immune response to infection leading to a cytokine storm and associated organ and tissue damage. Immunological therapies capable of attenuating the cytokine storm may therefore provide novel treatment options, and the present study explored whether mesenchymal stem cells (MSCs) from the amniotic fluid (AF) and their derivatives, through their immunomodulatory, anti‐oxidant, and regenerative functions, could provide such an option.Next Generation Sequencing analysis of MSCs isolated from human AF identified a plethora of molecules with the potential to positively influence the prognosis in SARS‐Cov‐2 infection patients. These included mRNAs involved in the regulation of several immune pathways that are basis of the impaired immune response and cytokine storm caused seen in SARS‐CoV‐2‐positive patients, such as: HIF‐1, IL‐17, Toll‐like receptors, RAP1, TNF, WNT, PI3K‐Akt and NF‐kappa B signalling. AF‐MSCs also contained bioactive molecules involved in respiratory endothelial protection and repair, such as VEGF, IL‐1, TGF‐β1, EGFR, in extracellular matrix re‐organization or associated with cardiac muscle cell function. Analysis of non‐coding RNAs identified the presence of microRNAs that regulate macrophage polarization (towards an anti‐inflammatory phenotype) and attenuate inflammation, as well as regulate the host response to viral infection. Among those, exosomal miRNAs have been also identified.Building upon these observations, further efforts will be made to elucidate the potential therapeutic use of AF‐MSCs and their derivatives in combating the immune‐related side effects of SARS‐CoV‐2. The successful incorporation of such an MSC‐based therapy into the treatment regimen of SARS‐CoV‐2 infected patients has significant potential to reduce the morbidity and mortality associated with COVID‐19.

Intracoronary Sarcoplasmic Reticulum Calcium-ATPase Gene Therapy in Advanced Heart Failure Patients with reduced Ejection Fraction: A Prospective Cohort Study

OBJECTIVE:Heart failure is a progressive and debilitating disease. Intracoronary sarcoplasmic reticulum calcium-ATPase gene therapy may improve the function of cardiac muscle cells. This study aimed to test the hypothesis that intracoronary sarcoplasmic reticulum calcium-ATPase gene therapy can improve outcomes and reduce the number of recurrent and terminal events in advanced heart failure patients with reduced ejection fraction.METHODS:A total of 768 heart failure patients with reduced ejection fraction and New York Heart Association classification II to IV were included in this prospective cohort study. Patients either underwent intracoronary sarcoplasmic reticulum calcium-ATPase gene therapy (CA group, n=384) or received oral placebo (PA group; n=384). Data regarding recurrent and terminal event(s), treatment-emergent adverse effects, and outcome measures were collected and analyzed.RESULTS:After a follow-up period of 18 months, intracoronary sarcoplasmic reticulum calcium-ATPase gene therapy reduced the number of hospital admissions (p=0.001), ambulatory treatments (p=0.0004), and deaths (p=0.024). Additionally, intracoronary sarcoplasmic reticulum calcium-ATPase gene therapy improved the left ventricular ejection fraction (p<0.0001) and Kansas City Cardiomyopathy Questionnaire score (p<0.0001). The number of recurrent and terminal events/patients were higher in the PA group than in the CA group after the follow-up period of 18 months (p=0.015). The effect of the intracoronary sarcoplasmic reticulum calcium-ATPase gene therapy was independent of the confounding variables. No new arrhythmias were reported in the CA group.CONCLUSIONS:Intracoronary sarcoplasmic reticulum calcium-ATPase gene therapy reduces the number of recurrent and terminal events and improves the clinical course of advanced heart failure patients with reduced ejection fraction.

Genetic and epigenetic regulation of cardiomyocytes in development, regeneration and disease.

Embryonic and postnatal life depend on the uninterrupted function of cardiac muscle cells. These cells, termed cardiomyocytes, display many fascinating behaviors, including complex morphogenic movements, interactions with other cell types of the heart, persistent contractility and quiescence after birth. Each of these behaviors depends on complex interactions between both cardiac-restricted and widely expressed transcription factors, as well as on epigenetic modifications. Here, we review recent advances in our understanding of the genetic and epigenetic control of cardiomyocyte differentiation and proliferation during heart development, regeneration and disease. We focus on those regulators that are required for both heart development and disease, and highlight the regenerative principles that might be manipulated to restore function to the injured adult heart.

Sex differences in distribution of cannabinoid receptors (CB1 and CB2), S100A6 and CacyBP/SIP in human ageing hearts

BackgroundWomen live about 4 years longer due to lower prevalence of cardiovascular complication with ageing. However, the mechanisms involved in the preservation of heart functionality in women have not been fully elucidated.The endocannabinoid system fulfils a significant role in the regulation of cardiovascular system functioning. Cannabinoids, acting through specific receptors (CB1 and CB2), influence on blood pressure, heart rate and myocardial contractility. The function of cardiac muscle cells is strictly dependent on calcium ions. Calcium homeostasis in cardiomyocytes is subjected to complex regulation via calcium-binding proteins. Among them, increasing attention has been paid to the recently discovered S100A6 and CacyBP/SIP.In order to better understand sex differences in the regulation of cardiomyocyte function during ageing, we undertook the present research aimed at immunohistochemical identification and comparative evaluation of cannabinoid receptors, S100A6 and CacyBP/SIP, in the myocardium of ageing men and women.MethodsThe study was conducted on the hearts of 12 men and 10 women (organ donors) without a history of cardiovascular disease. The subjects were divided into two age groups: subjects older than 50 years and subjects under 50 years old. Paraffin heart sections were processed by immunohistochemistry for detection of cannabinoids receptors, S100A6 and CacyBP/SIP. In the heart samples from each study, participant’s expression of genes coding for CB1, CB2, S100A6 and CacyBP/SIP using real-time PCR method was measured.ResultsCB1 and CB2 immunoreactivity in the cytoplasm of cardiomyocytes in the heart of subjects over 50 was weaker than in younger individuals. In the heart of younger men, CB1-immunoreactivity was weaker and CB2-immunoreaction was stronger compared to women. In the hearts of older men, the CB1-immunostaining was more intense and CB2-immunoreactivity was weaker than in women. Immunodetection of CB1 shoved the presence of receptor in the intercalated discs, but only in the hearts of individuals over the 50 years old. In the hearts of older individuals, stronger immunolabelling was observed for S100A6 and CacyBP/SIP. Male hearts had greater S100A6-immunoreactivity (both age groups) but less CacyBP/SIP immunostaining (individuals over 50 years) compared to the age-matched women. The expression of genes coding CB1, CB2, S100A6 and CacyBP/SIP in the human heart was sex and age-dependent. Observed changes between men and women as well as between subject under and over 50 years were consistent with immunohistochemically stated changes in peptide content.ConclusionTogether, the data presented here indicate a close interaction between ageing and sex on the distribution and levels of cannabinoid receptors (CB1, CB2), S100A6 and CacyBP/SIP in the human heart.

Short-term effects of β2-AR blocker ICI 118,551 on sarcoplasmic reticulum SERCA2a and cardiac function of rats with heart failure.

The study was conducted to examine the effects of ICI 118,551 on the systolic function of cardiac muscle cells of rats in heart failure and determine the molecular mechanism of selective β2-adrenergic receptor (β2-AR) antagonist on these cells. The chronic heart failure model for rats was prepared through abdominal aortic constriction and separate cardiac muscle cells using the collagenase digestion method. The rats were then divided into Sham, HF and HF+ICI 50 nM goups and cultivated for 48 h. β2-AR, Gi/Gs and sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) protein expression levels in the cardiac muscle cells were evaluated by western blotting and changes in the systolic function of cardiac muscle cells based on the boundary detection system of contraction dynamics for individual cells was measured. The results showed that compared with the Sham group, the survival rate, percentage of basic contraction and maximum contraction amplitude percentage of cardiac muscle cells with heart failure decreased, Gi protein expression increased while Gs and SERCA2a protein expression decreased. Compared with the HF group, the maximum contraction amplitude percentage of cardiac muscle cells in group HF+ICI 50 nM decreased, the Gi protein expression level increased while the SERCA2a protein expression level decreased. Following the stimulation of Ca2+ and ISO, the maximum contraction amplitude percentage of cardiac muscle cells in the HF+ICI 50 nM group was lower than that in group HF. This indicated that ICI 118,551 has negative inotropic effects on cardiac muscle cells with heart failure, which may be related to Gi protein. Systolic function of cardiac muscle cells with heart failure can therefore be reduced by increasing Gi protein expression and lowering SERCA2a protein expression.

Examination of the Effects of Heterogeneous Organization of RyR Clusters, Myofibrils and Mitochondria on Ca2+ Release Patterns in Cardiomyocytes.

Spatio-temporal dynamics of intracellular calcium, [Ca2+]i, regulate the contractile function of cardiac muscle cells. Measuring [Ca2+]i flux is central to the study of mechanisms that underlie both normal cardiac function and calcium-dependent etiologies in heart disease. However, current imaging techniques are limited in the spatial resolution to which changes in [Ca2+]i can be detected. Using spatial point process statistics techniques we developed a novel method to simulate the spatial distribution of RyR clusters, which act as the major mediators of contractile Ca2+ release, upon a physiologically-realistic cellular landscape composed of tightly-packed mitochondria and myofibrils. We applied this method to computationally combine confocal-scale (~ 200 nm) data of RyR clusters with 3D electron microscopy data (~ 30 nm) of myofibrils and mitochondria, both collected from adult rat left ventricular myocytes. Using this hybrid-scale spatial model, we simulated reaction-diffusion of [Ca2+]i during the rising phase of the transient (first 30 ms after initiation). At 30 ms, the average peak of the simulated [Ca2+]i transient and of the simulated fluorescence intensity signal, F/F0, reached values similar to that found in the literature ([Ca2+]i ≈1 μM; F/F0≈5.5). However, our model predicted the variation in [Ca2+]i to be between 0.3 and 12.7 μM (~3 to 100 fold from resting value of 0.1 μM) and the corresponding F/F0 signal ranging from 3 to 9.5. We demonstrate in this study that: (i) heterogeneities in the [Ca2+]i transient are due not only to heterogeneous distribution and clustering of mitochondria; (ii) but also to heterogeneous local densities of RyR clusters. Further, we show that: (iii) these structure-induced heterogeneities in [Ca2+]i can appear in line scan data. Finally, using our unique method for generating RyR cluster distributions, we demonstrate the robustness in the [Ca2+]i transient to differences in RyR cluster distributions measured between rat and human cardiomyocytes.

Ageing-related cardiomyocyte functional decline is sex and angiotensin II dependent

Clinically, heart failure is an age-dependent pathological phenomenon and displays sex-specific characteristics. The renin-angiotensin system mediates cardiac pathology in heart failure. This study investigated the sexually dimorphic functional effects of ageing combined with angiotensin II (AngII) on cardiac muscle cell function, twitch and Ca(2+)-handling characteristics of isolated cardiomyocytes from young (~13 weeks) and aged (~87 weeks) adult wild type (WT) and AngII-transgenic (TG) mice. We hypothesised that AngII-induced contractile impairment would be exacerbated in aged female cardiomyocytes and linked to Ca(2+)-handling disturbances. AngII-induced cardiomyocyte hypertrophy was evident in young adult mice of both sexes and accentuated by age (aged adult ~21-23 % increases in cell length relative to WT). In female AngII-TG mice, ageing was associated with suppressed cardiomyocyte contractility (% shortening, maximum rate of shortening, maximum rate of relaxation). This was associated with delayed cytosolic Ca(2+) removal during twitch relaxation (Tau ~20 % increase relative to young adult female WT), and myofilament responsiveness to Ca(2+) was maintained. In contrast, aged AngII-TG male cardiomyocytes exhibited peak shortening equivalent to young TG; yet, myofilament Ca(2+) responsiveness was profoundly reduced with ageing. Increased pro-arrhythmogenic spontaneous activity was evident with age and cardiac AngII overexpression in male mice (42-55 % of myocytes) but relatively suppressed in female aged transgenic mice. Female myocytes with elevated AngII appear more susceptible to an age-related contractile deficit, whereas male AngII-TG myocytes preserve contractile function with age but exhibit desensitisation of myofilaments to Ca(2+) and a heightened vulnerability to arrhythmic activity. These findings support the contention that sex-specific therapies are required for the treatment of age-progressive heart failure.

P.11.5 PMO-mediated dystrophin exon 23 skipping restores mitochondrial function in the mdx mouse heart

Approximately 20% of boys with Duchenne Muscular Dystrophy will die of dilated cardiomyopathy. The cardiomyopathy is characterised by disrupted structure and function of cardiac muscle cells and reduced energy production. However, the mechanisms responsible for the altered energy metabolism have been poorly understood. We have previously sought to identify the mechanisms for metabolic inhibition in mdx mouse cardiomyopathy. Calcium influx through the L-type Ca2+ channel (also known as the dihydropyridine receptor) in cardiac myocytes is essential for contraction. Calcium is also important for the regulation of mitochondrial function and production of ATP that is required to meet the energy demands of the heart. We have shown that the L-type Ca2+ channel can regulate mitochondrial function and metabolic activity in cardiac myocytes. In mdx heart, the communication between the L-type Ca2+ channel and the mitochondria is altered as a result of disruption of the cytoskeletal architecture. This contributes to metabolic inhibition in the mdx heart. We demonstrate that treatment of mdx mice with a phosphorodiamidate morpholino oligomer, designed to induce skipping of dystrophin exon 23, “restored” the increase in mitochondrial membrane potential in mdx cardiomyocytes after activation of the L-type Ca2+ channel with the dihydropyridine receptor agonist BayK (−). These results confirm that metabolic inhibition occurs as a result of the absence of dystrophin, and oligomer therapy may be able to normalise metabolic activity and restore contractility in mdx mouse heart.

Overexpression of diacylglycerol kinase ζ inhibits endothelin-1-induced decreases in Ca2+ transients and cell shortening in mouse ventricular myocytes

Endothelin-1 (ET-1) is released in various cardiovascular disorders including congestive heart failure, and may modulate significantly the disease process by its potent action on vascular and cardiac muscle cell function and gene regulation. In adult mouse ventricular cardiomyocytes loaded with indo-1, ET-1 induced a sustained negative inotropic effect (NIE) in association with decreases in Ca2+ transients. The ET-1-induced effects on Ca2+ transients and cell shortening were abolished in diacylglycerol (DAG) kinase ζ-overexpressing mouse ventricular myocytes. A nonselective protein kinase C (PKC) inhibitor, GF109203X, inhibited the ET-1-induced decreases in Ca2+ transients and cell shortening in concentration-dependent manners, whereas a selective Ca2+-dependent PKC inhibitor, Gö6976, did not affect the ET-1-induced effects. A phospholipase Cβ inhibitor, U73122, and an inhibitor of phospholipase D, C2-ceramide, partially, but significantly, attenuated the ET-1-induced effects. Derivatives of the respective inhibitors with no specific effects, U73343 and dihydro-C2-ceramide, did not affect the ET-1-induced effects. Taken together, these results indicate that activation of a Ca2+-independent PKC isozyme by 1,2-DAG, which is generated by phospholipase Cβ and phospholipase D activation and inactivated by phosphorylation via DAG kinase, is responsible for the ET-1-induced decreases in Ca2+ transients and cell shortening in mouse ventricular cardiomyocytes.

Distribution and organization of desmin in cultured adult cardiac muscle cells: reflection on function.

The cell-culture model for the study of desmin in adult cardiac muscle cells has provided insight into the function of desmin based on its distribution and structural organization. Initially, desmin emerged as a filamentous network from the existing amorphous form in the growing adult cardiac myocytes in vitro. Later, desmin became organized in various forms. In addition to the presence of a periodic array of desmin in the Z-line regions as observed in cardiac myocytes in vivo, longitudinally and transversely oriented strands of desmin were observed along the length of myofibrils in cardiac myocytes in vitro. These desmin strands and transverse perodicities formed a complex interwoven network, interlacing myofibrils of cells. Desmin and alpha-actinin were organized in ribbon- or aponeuroses-like structures that appeared as sheet-like, supportive structures for the cell body. The cellular cytoplasmic processes containing myofibrils were supported by desmin bars. The complex desmin network, desmin bars, transverse strands and ribbons or aponeuroses were observed in in vitro cardiac myocytes in contrast to in vivo cardiac myocytes. The functional implication of desmin, as indicated by in vivo studies, required more information concerning the organization of desmin for its supportive function, and is addressed in the present study. The elaborate organization of desmin provides evidence for its supportive function for the maintenance of the structural integrity and function of cardiac muscle cells.