Contraction Of Cardiac Myocytes Research Articles

Serum SERCA2a levels in heart failure patients are associated with adverse events after discharge.

Calcium homeostasis imbalance is one of the important pathological mechanisms in heart failure. Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a), a calcium ATPase on the sarcoplasmic reticulum in cardiac myocytes, is a myocardial systolic-diastolic Ca2 + homeostasis regulating enzyme that is not only involved in cardiac diastole but also indirectly affects cardiac myocyte contraction. SERCA2a expression was found to be decreased in myocardial tissue in heart failure, however, there are few reports on serum SERCA2a expression in patients with heart failure, and this study was designed to investigate whether serum SERCA2a levels are associated with the occurrence of adverse events after discharge in patients hospitalized with heart failure. Patients with heart failure hospitalized in the cardiovascular department of the Second Affiliated Hospital of Guangdong Medical University, China, from July 2018 to July 2019 were included in this study, and serum SERCA2a concentrations were measured; each enrolled patient was followed up by telephone after 6 months (6 ± 1 months) for general post-discharge patient status. The correlation between serum SERCA2a levels and the occurrence of adverse events (death or readmission due to heart failure) after hospital discharge was assessed using multiple analysis and trend analysis. Seventy-one patients with heart failure were finally included in this study, of whom 38 (53.5%) were men and 33 (46.5%) were women (All were postmenopausal women). Multiple analysis revealed no correlation between serum SERCA2a levels and the occurrence of adverse events in the total study population and in male patients, but serum SERCA2a levels were associated with the occurrence of adverse outcome events after hospital discharge in female patients (OR = 1.02, P = .047). Further analysis using a trend analysis yielded a 4.0% increase in the risk of adverse outcomes after hospital discharge for each unit increase in SERCA2a in female patients (OR = 1.04; P = .02), while no significant difference was seen in men. This study suggests that serum SERCA2a levels at admission are associated with the occurrence of post-discharge adverse events in postmenopausal female patients hospitalized with heart failure.

FKBP12 binds to the cardiac ryanodine receptor with negative cooperativity: implications for heart muscle physiology in health and disease.

Cardiac ryanodine receptors (RyR2) release the Ca2+ from intracellular stores that is essential for cardiac myocyte contraction. The ion channel opening is tightly regulated by intracellular factors, including the FK506 binding proteins, FKBP12 and FKBP12.6. The impact of these proteins on RyR2 activity and cardiac contraction is debated, with often apparently contradictory experimental results, particularly for FKBP12. The isoform that regulates RyR2 has generally been considered to be FKBP12.6, despite the fact that FKBP12 is the major isoform associated with RyR2 in some species and is bound in similar proportions to FKBP12.6 in others, including sheep and humans. Here, we show time- and concentration-dependent effects of adding FKBP12 to RyR2 channels that were partly depleted of FKBP12/12.6 during isolation. The added FKBP12 displaced most remaining endogenous FKBP12/12.6. The results suggest that FKBP12 activates RyR2 with high affinity and inhibits RyR2 with lower affinity, consistent with a model of negative cooperativity in FKBP12 binding to each of the four subunits in the RyR tetramer. The easy dissociation of some FKBP12/12.6 could dynamically alter RyR2 activity in response to changes in in vivo regulatory factors, indicating a significant role for FKBP12/12.6 in Ca2+ signalling and cardiac function in healthy and diseased hearts. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

Electrical Remodeling in Right Ventricular Failure Due to Pulmonary Hypertension: Unraveling Novel Therapeutic Targets.

Arrhythmias in the setting of right-ventricular (RV) remodeling contribute to majority of deaths in patients with pulmonary hypertension. However, the underlying mechanism of electrical remodeling remains elusive, especially ventricular arrhythmias. Here, we analyzed the RV transcriptome of pulmonary arterial hypertension (PAH) patients with compensated RV or decompensated RV and identified 8 and 45 differentially expressed genes known to be involved in regulating the electrophysiological properties of excitation and contraction of cardiac myocytes, respectively. Transcripts encoding voltage-gated Ca2+ and Na+ channels were notably decreased in PAH patients with decompensated RV, along with significant dysregulation of KV and Kir channels. We further showed similarity of the RV channelome signature with two well-known animal models of PAH, monocrotaline (MCT)- and Sugen-hypoxia (SuHx)-treated rats. We identified 15 common transcripts among MCT, SuHx, and PAH patients with decompensated RV failure. In addition, data-driven drug repurposing using the channelome signature of PAH patients with decompensated RV failure predicted drug candidates that may reverse the altered gene expression. Comparative analysis provided further insight into clinical relevance and potential preclinical therapeutic studies targeting mechanisms involved in arrhythmogenesis.

Serial block face scanning electron microscopy reveals region-dependent remodelling of transverse tubules post-myocardial infarction.

The highly organized transverse tubule (t-tubule) network facilitates cardiac excitation–contraction coupling and synchronous cardiac myocyte contraction. In cardiac failure secondary to myocardial infarction (MI), changes in the structure and organization of t-tubules result in impaired cardiac contractility. However, there is still little knowledge on the regional variation of t-tubule remodelling in cardiac failure post-MI. Here, we investigate post-MI t-tubule remodelling in infarct border and remote regions, using serial block face scanning electron microscopy (SBF-SEM) applied to a translationally relevant sheep ischaemia reperfusion MI model and matched controls. We performed minimally invasive coronary angioplasty of the left anterior descending artery, followed by reperfusion after 90 min to establish the MI model. Left ventricular tissues obtained from control and MI hearts eight weeks post-MI were imaged using SBF-SEM. Image analysis generated three-dimensional reconstructions of the t-tubular network in control, MI border and remote regions. Quantitative analysis revealed that the MI border region was characterized by t-tubule depletion and fragmentation, dilation of surviving t-tubules and t-tubule elongation. This study highlights region-dependent remodelling of the tubular network post-MI and may provide novel localized therapeutic targets aimed at preservation or restoration of the t-tubules to manage cardiac contractility post-MI.This article is part of the theme issue ‘The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease’.

Raf Kinase Inhibitory Protein regulates the cAMP-dependent protein kinase signaling pathway through a positive feedback loop

Raf Kinase Inhibitory Protein (RKIP) maintains cellular robustness and prevents the progression of diseases such as cancer and heart disease by regulating key kinase cascades including MAP kinase and protein kinase A (PKA). Phosphorylation of RKIP at S153 by Protein Kinase C (PKC) triggers a switch from inhibition of Raf to inhibition of the G protein coupled receptor kinase 2 (GRK2), enhancing signaling by the β-adrenergic receptor (β-AR) that activates PKA. Here we report that PKA-phosphorylated RKIP promotes β-AR-activated PKA signaling. Using biochemical, genetic, and biophysical approaches, we show that PKA phosphorylates RKIP at S51, increasing S153 phosphorylation by PKC and thereby triggering feedback activation of PKA. The S51V mutation blocks the ability of RKIP to activate PKA in prostate cancer cells and to induce contraction in primary cardiac myocytes in response to the β-AR activator isoproterenol, illustrating the functional importance of this positive feedback circuit. As previously shown for other kinases, phosphorylation of RKIP at S51 by PKA is enhanced upon RKIP destabilization by the P74L mutation. These results suggest that PKA phosphorylation at S51 may lead to allosteric changes associated with a higher-energy RKIP state that potentiates phosphorylation of RKIP at other key sites. This allosteric regulatory mechanism may have therapeutic potential for regulating PKA signaling in disease states.

A Stochastic Spatiotemporal Model of Rat Ventricular Myocyte Calcium Dynamics Demonstrated Necessary Features for Calcium Wave Propagation.

Calcium (Ca2+) plays a central role in the excitation and contraction of cardiac myocytes. Experiments have indicated that calcium release is stochastic and regulated locally suggesting the possibility of spatially heterogeneous calcium levels in the cells. This spatial heterogeneity might be important in mediating different signaling pathways. During more than 50 years of computational cell biology, the computational models have been advanced to incorporate more ionic currents, going from deterministic models to stochastic models. While periodic increases in cytoplasmic Ca2+ concentration drive cardiac contraction, aberrant Ca2+ release can underly cardiac arrhythmia. However, the study of the spatial role of calcium ions has been limited due to the computational expense of using a three-dimensional stochastic computational model. In this paper, we introduce a three-dimensional stochastic computational model for rat ventricular myocytes at the whole-cell level that incorporate detailed calcium dynamics, with (1) non-uniform release site placement, (2) non-uniform membrane ionic currents and membrane buffers, (3) stochastic calcium-leak dynamics and (4) non-junctional or rogue ryanodine receptors. The model simulates spark-induced spark activation and spark-induced Ca2+ wave initiation and propagation that occur under conditions of calcium overload at the closed-cell condition, but not when Ca2+ levels are normal. This is considered important since the presence of Ca2+ waves contribute to the activation of arrhythmogenic currents.

Calcium Homeostasis in Ventricular Myocytes of Diabetic Cardiomyopathy.

Diabetes mellitus (DM) is a chronic metabolic disorder commonly characterized by high blood glucose levels, resulting from defects in insulin production or insulin resistance, or both. DM is a leading cause of mortality and morbidity worldwide, with diabetic cardiomyopathy as one of its main complications. It is well established that cardiovascular complications are common in both types of diabetes. Electrical and mechanical problems, resulting in cardiac contractile dysfunction, are considered as the major complications present in diabetic hearts. Inevitably, disturbances in the mechanism(s) of Ca2+ signaling in diabetes have implications for cardiac myocyte contraction. Over the last decade, significant progress has been made in outlining the mechanisms responsible for the diminished cardiac contractile function in diabetes using different animal models of type I diabetes mellitus (TIDM) and type II diabetes mellitus (TIIDM). The aim of this review is to evaluate our current understanding of the disturbances of Ca2+ transport and the role of main cardiac proteins involved in Ca2+ homeostasis in the diabetic rat ventricular cardiomyocytes. Exploring the molecular mechanism(s) of altered Ca2+ signaling in diabetes will provide an insight for the identification of novel therapeutic approaches to improve the heart function in diabetic patients.

Mitochondrial architecture in cardiac myocytes depends on cell shape and matrix rigidity

Contraction of cardiac myocytes depends on energy generated by the mitochondria. During cardiac development and disease, the structure and function of the mitochondrial network in cardiac myocytes is known to remodel in concert with many other factors, including changes in nutrient availability, hemodynamic load, extracellular matrix (ECM) rigidity, cell shape, and maturation of other intracellular structures. However, the independent role of each of these factors on mitochondrial network architecture is poorly understood. In this study, we tested the hypothesis that cell aspect ratio (AR) and ECM rigidity regulate the architecture of the mitochondrial network in cardiac myocytes. To do this, we spin-coated glass coverslips with a soft, moderate, or stiff polymer. Next, we microcontact printed cell-sized rectangles of fibronectin with AR matching cardiac myocytes at various developmental or disease states onto the polymer surface. We then cultured neonatal rat ventricular myocytes on the patterned surfaces and used confocal microscopy and image processing techniques to quantify sarcomeric α-actinin volume, nucleus volume, and mitochondrial volume, surface area, and size distribution. On some substrates, α-actinin volume increased with cell AR but was not affected by ECM rigidity. Nucleus volume was mostly uniform across all conditions. In contrast, mitochondrial volume increased with cell AR on all substrates. Furthermore, mitochondrial surface area to volume ratio decreased as AR increased on all substrates. Large mitochondria were also more prevalent in cardiac myocytes with higher AR. For select AR, mitochondria were also smaller as ECM rigidity increased. Collectively, these results suggest that mitochondrial architecture in cardiac myocytes is strongly influenced by cell shape and moderately influenced by ECM rigidity. These data have important implications for understanding the factors that impact metabolic performance during heart development and disease.

A Dynamic Functional Stem Cell Microtissue Culture System Based on Magnetic Nanoparticles Labeled Endothelial Cells

Living microrobots, in which three-dimensional (3D) multicellular structures are endowed with movement ability through the contraction of cardiac myocytes, may be powerful tools in the future. Inspired by this, we developed a dynamic functional microtissue culture system based on endothelial cells. In contrast to cardiac myocytes or muscle cells, which exhibit rhythmic contraction, we found that cell sheets containing human umbilical vein endothelial cells (HUVECs) could spontaneously and slowly contract and curl into a bowl-like microtissue. In the microtissue, the HUVECs formed tubular structures, which might enhance the angiogenic potential, a function that is beneficial for maintaining the proliferation of microtissue. Dental pulp stem cells (DPSCs) were selected to be incorporated in the dynamic functional microtissue through magnetic controlled technology. With prolonged culture time, the HUVECs/DPSCs composite microtissue containing half endothelial cells still exhibited contraction and curling effects. More importantly, the contraction process significantly enhanced the expression of osteogenesis-related genes and proteins in the composite microtissue. The preliminary research results indicated that this dynamic functional microtissue may hold great promise for future use in many fields, especially in bone regeneration.

Junctional sarcoplasmic reticulum motility in adult mouse ventricular myocytes.

Excitation-contraction (EC) coupling is the coordinated process by which an action potential triggers cardiac myocyte contraction. EC coupling is initiated in dyads where the junctional sarcoplasmic reticulum (jSR) is in tight proximity to the sarcolemma of cardiac myocytes. Existing models of EC coupling critically depend on dyad stability to ensure the fidelity and strength of EC coupling, where even small variations in ryanodine receptor channel and voltage-gated calcium channel-α 1.2 subunit separation dramatically alter EC coupling. However, dyadic motility has never been studied. Here, we developed a novel strategy to track specific jSR units in dissociated adult ventricular myocytes using photoactivatable fluorescent proteins. We found that the jSR is not static. Instead, we observed dynamic formation and dissolution of multiple dyadic junctions regulated by the microtubule-associated molecular motors kinesin-1 and dynein. Our data support a model where reproducibility of EC coupling results from the activation of a temporally averaged number of SR Ca2+ release units forming and dissolving SR-sarcolemmal junctions. These findings challenge the long-held view that the jSR is an immobile structure and provide insights into the mechanisms underlying its motility.

Genome-wide differential expression profiling of lncRNAs and mRNAs associated with early diabetic cardiomyopathy

Diabetic cardiomyopathy is one of the main causes of heart failure and death in patients with diabetes. There are no effective approaches to preventing its development in the clinic. Long noncoding RNAs (lncRNA) are increasingly recognized as important molecular players in cardiovascular disease. Herein we investigated the profiling of cardiac lncRNA and mRNA expression in type 2 diabetic db/db mice with and without early diabetic cardiomyopathy. We found that db/db mice developed cardiac hypertrophy with normal cardiac function at 6 weeks of age but with a decreased diastolic function at 20 weeks of age. LncRNA and mRNA transcripts were remarkably different in 20-week-old db/db mouse hearts compared with both nondiabetic and diabetic controls. Overall 1479 lncRNA transcripts and 1109 mRNA transcripts were aberrantly expressed in 6- and 20-week-old db/db hearts compared with nondiabetic controls. The lncRNA-mRNA co-expression network analysis revealed that 5 deregulated lncRNAs having maximum connections with differentially expressed mRNAs were BC038927, G730013B05Rik, 2700054A10Rik, AK089884, and Daw1. Bioinformatics analysis revealed that these 5 lncRNAs are closely associated with membrane depolarization, action potential conduction, contraction of cardiac myocytes, and actin filament-based movement of cardiac cells. This study profiles differently expressed lncRNAs in type 2 mice with and without early diabetic cardiomyopathy and identifies BC038927, G730013B05Rik, 2700054A10Rik, AK089884, and Daw1 as the core lncRNA with high significance in diabetic cardiomyopathy.

The Cardiovascular Effects of a Meal: J-Tpeak and Tpeak -Tend Assessment and Further Insights Into the Physiological Effects.

Meal intake leads to a significant and prolonged increase in cardiac output to supply the splanchnic vasculature. A meal is associated with sympathetic activation of the cardiovascular system, and food ingestion is correlated with an increase in heart rate, an increase in cardiac stroke volume, and QTc interval shortening for up to 7 hours. Given the complexity of the system, one or several of many mechanisms could explain this observation. The shortening of the QTc interval was correlated with a rise of C‐peptide following food ingestion, but the mechanisms by which C‐peptide may be involved in the modulation of cardiac repolarization are still unknown. This shortening of the myocardial action potential caused by the ingestion of food was further investigated in the present study by measuring the QRS, J‐Tpeak, and Tpeak‐Tend intervals in search of further clues to better understand the underlying mechanisms. A retrospective analysis was conducted based on data collected in a formal thorough QT/QTc study in which 32 subjects received a carbohydrate‐rich “continental” breakfast, moxifloxacin without food, and moxifloxacin with food. We assessed the effect of food on T‐wave morphology using validated algorithms for measurement of J‐Tpeak and Tpeak‐Tend intervals. Our findings demonstrate that a standardized meal significantly shortened J‐Tpeak for 4 hours after a meal and to a much lesser extent and shorter duration (up to 1 hour) prolonged the Tpeak‐Tend and QRS intervals. This suggests that the QTc shortening occurs mainly during phase 2 of the cardiac action potential. As there was no corresponding effect on Tpeak‐Tend beyond the first hour, we conclude that a meal does not interfere with the outward correcting potassium channels but possibly with Ca2+ currents. An effect on mainly Ca2+ aligns well with our understanding of physiology whereby an increase in stroke volume, as observed after a meal, is associated with changes in Ca2+ cycling in and out of the sarcoplasmic reticulum during cardiac myocyte contraction.

Voltage-Gated Sodium Channel β1/β1B Subunits Regulate Cardiac Physiology and Pathophysiology.

Cardiac myocyte contraction is initiated by a set of intricately orchestrated electrical impulses, collectively known as action potentials (APs). Voltage-gated sodium channels (NaVs) are responsible for the upstroke and propagation of APs in excitable cells, including cardiomyocytes. NaVs consist of a single, pore-forming α subunit and two different β subunits. The β subunits are multifunctional cell adhesion molecules and channel modulators that have cell type and subcellular domain specific functional effects. Variants in SCN1B, the gene encoding the Nav-β1 and -β1B subunits, are linked to atrial and ventricular arrhythmias, e.g., Brugada syndrome, as well as to the early infantile epileptic encephalopathy Dravet syndrome, all of which put patients at risk for sudden death. Evidence over the past two decades has demonstrated that Nav-β1/β1B subunits play critical roles in cardiac myocyte physiology, in which they regulate tetrodotoxin-resistant and -sensitive sodium currents, potassium currents, and calcium handling, and that Nav-β1/β1B subunit dysfunction generates substrates for arrhythmias. This review will highlight the role of Nav-β1/β1B subunits in cardiac physiology and pathophysiology.

Microenvironmental Modulation of Calcium Wave Propagation Velocity in Engineered Cardiac Tissues

In the myocardium, rapid propagation of action potentials and subsequent calcium waves is critical for synchronizing the contraction of cardiac myocytes and maximizing cardiac output. In many pathological settings, diverse remodeling of the tissue microenvironment is correlated with arrhythmias and decreased cardiac output, but the precise impact of tissue remodeling on propagation is not completely understood. Our objective was to delineate how multiple features within the cardiac tissue microenvironment modulate propagation velocity. To recapitulate diverse myocardial tissue microenvironments, we engineered substrates with tunable elasticity, patterning, composition, and topography using two formulations of polydimethylsiloxane (PDMS) micropatterned with fibronectin and gelatin hydrogels with flat or micromolded features. We cultured neonatal rat ventricular myocytes on these substrates and quantified cell density, tissue alignment, and cell shape. We used a fluorescent calcium indicator, high-speed microscopy, and newly-developed analysis software to record and quantify calcium wave propagation velocity (CPV). For all substrates, tissue alignment and cell aspect ratio were higher in aligned compared to isotropic tissues. Isotropic CPV and longitudinal CPV were similar across conditions, but transverse CPV was lower on micromolded gelatin hydrogels compared to micropatterned soft and stiff PDMS. In aligned tissues, the anisotropy ratio of CPV (longitudinal CPV/transverse CPV) was lower on micropatterned soft PDMS compared to micropatterned stiff PDMS and micromolded gelatin hydrogels. Propagation velocity in engineered cardiac tissues is sensitive to features in the tissue microenvironment, such as alignment, matrix elasticity, and matrix topography, which may underlie arrhythmias in conditions with pathological tissue remodeling.

Mathematical model for β1-adrenergic regulation of the mouse ventricular myocyte contraction.

The β1-adrenergic regulation of cardiac myocyte contraction plays an important role in regulating heart function. Activation of this system leads to an increased heart rate and stronger myocyte contraction. However, chronic stimulation of the β1-adrenergic signaling system can lead to cardiac hypertrophy and heart failure. To understand the mechanisms of action of β1-adrenoceptors, a mathematical model of cardiac myocyte contraction that includes the β1-adrenergic system was developed and studied. The model was able to simulate major experimental protocols for measurements of steady-state force-calcium relationships, cross-bridge release rate and force development rate, force-velocity relationship, and force redevelopment rate. It also reproduced quite well frequency and isoproterenol dependencies for intracellular Ca2+ concentration ([Ca2+]i) transients, total contraction force, and sarcomere shortening. The mathematical model suggested the mechanisms of increased contraction force and myocyte shortening on stimulation of β1-adrenergic receptors is due to phosphorylation of troponin I and myosin-binding protein C and increased [Ca2+]i transient resulting from activation of the β1-adrenergic signaling system. The model was used to simulate work-loop contractions and estimate the power during the cardiac cycle as well as the effects of 4-aminopyridine and tedisamil on the myocyte contraction. The developed mathematical model can be used further for simulations of contraction of ventricular myocytes from genetically modified mice and myocytes from mice with chronic cardiac diseases.NEW & NOTEWORTHY A new mathematical model of mouse ventricular myocyte contraction that includes the β1-adrenergic system was developed. The model simulated major experimental protocols for myocyte contraction and predicted the effects of 4-aminopyridine and tedisamil on the myocyte contraction. The model also allowed for simulations of work-loop contractions and estimation of the power during the cardiac cycle.

Adrenergic Signaling Strengthens Cardiac Myocyte Cohesion

The sympathetic nervous system is a major mediator of heart function. Intercalated discs composed of desmosomes, adherens junctions, and gap junctions provide the structural backbone for coordinated contraction of cardiac myocytes. Gap junctions dynamically remodel to adapt to sympathetic signaling. However, it is unknown whether such rapid adaption also occurs for the adhesive function provided by desmosomes and adherens junctions. Atomic force microscopy revealed that β-adrenergic signaling enhances both the number of desmoglein 2-specific interactions along cell junctions and the mean desmoglein 2-mediated binding forces, whereas N-cadherin-mediated interactions were not affected. This was accompanied by increased cell cohesion in cardiac myocyte cultures and murine heart slices. Enhanced desmoglein 2-positive contacts and increased junction length as revealed by immunofluorescence and electron microscopy reflected cAMP-induced reorganization of intercellular contacts. The mechanism underlying cAMP-mediated strengthening of desmoglein 2 binding was dependent on expression of the intercalated disc plaque protein plakoglobin (Pg) and direct phosphorylation at S665 by protein kinase A: Pg deficiency as well as overexpression of the phospho-deficient Pg-mutant S665A abrogated both cAMP-mediated junctional remodeling and increase of cohesion. Moreover, Pg knockout hearts failed to functionally adapt to adrenergic stimulation. Taken together, we provide first evidence for positive adhesiotropy as a new cardiac function of sympathetic signaling. Positive adhesiotropy is dependent on Pg phosphorylation at S665 by protein kinase A. This mechanism may be of high medical relevance because loss of junctional Pg is a hallmark of arrhythmogenic cardiomyopathy.

ISH NIA PS 01-04 Does eNOS-palmitoylation involve in palmitic acid-enhanced cardiac inotropy in normal and hypertensive rat cardiac myocyte?

Objective: Palmitic acid (PA) is a predominant metabolic substrate for cardiac β-oxidation. Our recent results have shown that PA increases contraction of cardiac myocytes from normal heart but not from hypertension. S-palmitoylation is an important post-translational modification that affects the translocation in cardiomyocytes. Since endothelial nitric oxide synthase (eNOS) is known to be palmitoylated and the activity of eNOS is essential in fatty acid-dependent β-oxidation in muscle, we aim to identify whether eNOS is palmitoylated by PA and the involvement of eNOS-palmitoylation in PA-regulation of cardiomyocyte contraction. Design and Method: Sarcomere length were measured (field stimulation, 2 Hz, IonOptix, 37 °C). Oxygen consumption rate (OCR) was measured (Instech, 37 °C). NO (nitrite content) was measured by NO assay kit (Griess Reagent System). S-palmitoylation was measured with resin-assisted capture of S-acylated proteins. Results: Our results showed that palmitic acid (PA, 100 μM) significantly increased eNOS palmitoylation but not in hypertension. Pre-treatment of LV myocytes with an inhibitor of palmitoylation, 2-bromopalmitic acid (2BP, 100 μM) significantly reduced eNOS palmitoylation in both groups, suggesting that residual eNOS in hypertension was palmitoylated. Functionally, 2BP induced slight increase of PA-induced myocyte contraction in sham but almost doubled contraction in hypertension. PA increased OCR in cardiomyocytes from both groups. 2BP reduced OCR only in hypertension, suggesting that eNOS-depalmitoylation affects PA-dependent myocyte contraction in hypertension via a mechanism different from mitochondrial function. PA increased nitric oxide (NO) production in LV myocytes from hypertension, whereas tended to reduce it in sham. Inhibition of NOS with Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME, 1 mM, 30min-1hr) or nNOS with S-methyl-l-thiocitrulline (SMTC, 100 nM, 30min-1hr) reduced NO production in hypertension, whereas 2BP increased PA-dependent NO production in both groups in the presence or absence of L-NAME or SMTC. Conclusions: PA induces eNOS-palmitoylation and attenuates eNOS-dependent NO production. eNOS-palmitoylation does not seem to mediate PA-induced cardiac inotropy in normal rat cardiac myocyte.

S-glutathiolation impairs phosphoregulation and function of cardiac myosin-binding protein C in human heart failure.

Cardiac myosin-binding protein C (cMyBP-C) regulates actin-myosin interaction and thereby cardiac myocyte contraction and relaxation. This physiologic function is regulated by cMyBP-C phosphorylation. In our study, reduced site-specific cMyBP-C phosphorylation coincided with increased S-glutathiolation in ventricular tissue from patients with dilated or ischemic cardiomyopathy compared to nonfailing donors. We used redox proteomics, to identify constitutive and disease-specific S-glutathiolation sites in cMyBP-C in donor and patient samples, respectively. Among those, a cysteine cluster in the vicinity of the regulatory phosphorylation sites within the myosin S2 interaction domain C1-M-C2 was identified and showed enhanced S-glutathiolation in patients. In vitro S-glutathiolation of recombinant cMyBP-C C1-M-C2 occurred predominantly at Cys(249), which attenuated phosphorylation by protein kinases. Exposure to glutathione disulfide induced cMyBP-C S-glutathiolation, which functionally decelerated the kinetics of Ca(2+)-activated force development in ventricular myocytes from wild-type, but not those from Mybpc3-targeted knockout mice. These oxidation events abrogate protein kinase-mediated phosphorylation of cMyBP-C and therefore potentially contribute to the reduction of its phosphorylation and the contractile dysfunction observed in human heart failure.-Stathopoulou, K., Wittig, I., Heidler, J., Piasecki, A., Richter, F., Diering, S., van der Velden, J., Buck, F., Donzelli, S., Schröder, E., Wijnker, P. J. M., Voigt, N., Dobrev, D., Sadayappan, S., Eschenhagen, T., Carrier, L., Eaton, P., Cuello, F. S-glutathiolation impairs phosphoregulation and function of cardiac myosin-binding protein C in human heart failure.

Regulation of the Cardiac L-Type Calcium Channel by the Cyclic Nucleotide Cross-Talk Signaling Network

Regulation of L-type Calcium (Ca2+) channel (LCC) gating is critical to shaping the cardiac action potential (AP) and excitation-contraction coupling (ECC), the process by which electrical excitation leads to mechanical contraction of cardiac myocytes. The cyclic nucleotide (cN) cross-talk signaling network, which encompasses the β-adrenergic and the Nitric Oxide (NO)/cGMP/Protein Kinase G (PKG) pathways and their interaction through distinctively-regulated phosphodiesterase isoenzymes (PDEs), regulates LCC current via Protein Kinase A (PKA)- and PKG-mediated phosphorylation. Due to tightly-coupled biochemical reactions, it remains to be clarified how LCC gating is ultimately regulated by this signaling network. In addition, the large number of potential ECC-related phosphorylation targets of PKA and PKG makes it difficult to quantify and isolate the effect of LCC regulation. We have developed a multi-scale, biophysically-detailed computational model of LCC regulation by the cN signaling network based on a wide range of experimental data. Changes in LCC current properties arising from interaction with PKA and PKG are modeled by redistributing the number of channels among four gating modes, corresponding to the four channel phosphorylation states: non-, PKA-, PKG-, and PKA-and-PKG- phosphorylated. The model exhibits experimentally observed single LCC gating characteristics, as well as whole-cell LCC current. Simulations predict how LCC gating modes are redistributed in response to a variety of simulation protocols. Results distinguish LCC regulation exerted between two mechanisms which are difficult to resolve by experimentation alone: 1) LCC interaction with activated PKA and PKG and 2) regulation of PKA and PKG activation as a result of cN cross-talk. Consequently, the model reveals underlying mechanisms that explain LCC current observations under various stimulation scenarios of the signaling network. These results provide insights into how cN signals are integrated via LCC regulation.

Multimodal SHG-2PF Imaging of Microdomain Ca2+-Contraction Coupling in Live Cardiac Myocytes.

Cardiac myocyte contraction is caused by Ca(2+) binding to troponin C, which triggers the cross-bridge power stroke and myofilament sliding in sarcomeres. Synchronized Ca(2+) release causes whole cell contraction and is readily observable with current microscopy techniques. However, it is unknown whether localized Ca(2+) release, such as Ca(2+) sparks and waves, can cause local sarcomere contraction. Contemporary imaging methods fall short of measuring microdomain Ca(2+)-contraction coupling in live cardiac myocytes. To develop a method for imaging sarcomere level Ca(2+)-contraction coupling in healthy and disease model cardiac myocytes. Freshly isolated cardiac myocytes were loaded with the Ca(2+)-indicator fluo-4. A confocal microscope equipped with a femtosecond-pulsed near-infrared laser was used to simultaneously excite second harmonic generation from A-bands of myofibrils and 2-photon fluorescence from fluo-4. Ca(2+) signals and sarcomere strain correlated in space and time with short delays. Furthermore, Ca(2+) sparks and waves caused contractions in subcellular microdomains, revealing a previously underappreciated role for these events in generating subcellular strain during diastole. Ca(2+) activity and sarcomere strain were also imaged in paced cardiac myocytes under mechanical load, revealing spontaneous Ca(2+) waves and correlated local contraction in pressure-overload-induced cardiomyopathy. Multimodal second harmonic generation 2-photon fluorescence microscopy enables the simultaneous observation of Ca(2+) release and mechanical strain at the subsarcomere level in living cardiac myocytes. The method benefits from the label-free nature of second harmonic generation, which allows A-bands to be imaged independently of T-tubule morphology and simultaneously with Ca(2+) indicators. Second harmonic generation 2-photon fluorescence imaging is widely applicable to the study of Ca(2+)-contraction coupling and mechanochemotransduction in both health and disease.