Release In Cardiomyocytes Research Articles

SparkMaster 2: A New Software for Automatic Analysis of Calcium Spark Data.

Calcium (Ca) sparks are elementary units of subcellular Ca release in cardiomyocytes and other cells. Accordingly, Ca spark imaging is an essential tool for understanding the physiology and pathophysiology of Ca handling and is used to identify new drugs targeting Ca-related cellular dysfunction (eg, cardiac arrhythmias). The large volumes of imaging data produced during such experiments require accurate and high-throughput analysis. We developed a new software tool SparkMaster 2 (SM2) for the analysis of Ca sparks imaged by confocal line-scan microscopy, combining high accuracy, flexibility, and user-friendliness. SM2 is distributed as a stand-alone application requiring no installation. It can be controlled using a simple-to-use graphical user interface, or using Python scripting. SM2 is shown to have the following strengths: (1) high accuracy at identifying Ca release events, clearly outperforming previous highly successful software SparkMaster; (2) multiple types of Ca release events can be identified using SM2: Ca sparks, waves, miniwaves, and long sparks; (3) SM2 can accurately split and analyze individual sparks within spark clusters, a capability not handled adequately by prior tools. We demonstrate the practical utility of SM2 in two case studies, investigating how Ca levels affect spontaneous Ca release, and how large-scale release events may promote release refractoriness. SM2 is also useful in atrial and smooth muscle myocytes, across different imaging conditions. SparkMaster 2 is a new, much-improved user-friendly software for accurate high-throughput analysis of line-scan Ca spark imaging data. It is free, easy to use, and provides valuable built-in features to facilitate visualization, analysis, and interpretation of Ca spark data. It should enhance the quality and throughput of Ca spark and wave analysis across cell types, particularly in the study of arrhythmogenic Ca release events in cardiomyocytes.

Abstract P2045: Regulation Of Atrial Natriuretic Peptide Secretion By Palmitoylation And Modulation Of The Rab3a GTPase Cycle In Cardiac Myocytes

Despite the clinical relevance to cardiovascular disease, molecular regulation of atrial natriuretic peptide (ANP) release by cardiomyocytes remains ill-defined. We uncovered novel functions for Rab3gap1 and modulation of Rab3a nucleotide cycling in membrane fusion and atrial natriuretic peptide (ANP) release in cardiomyocytes. We generated transgenic mice overexpressing the Golgi-localized S-acyltransferase, zDHHC9, and identified Rab3gap1 as a novel cardiac substrate of zDHHC9 by S-acyl proteomics. Rab3gap1 palmitoylation and retention at the cardiomyocyte Golgi, Rab3a-GTP levels, and formation of Rab3a-positive post-Golgi vesicles were all enhanced in zDHHC9 overexpressing hearts as were Rab3gap1, Rab3a, and ANP atrial protein levels but circulating ANP levels were reduced, suggesting a defect in ANP secretion. Notably, treatment of cardiomyocytes with the secretagogue phenylephrine (PE) promoted substantial ANP release as expected, but also enhanced Rab3gap1 palmitoylation and Rab3a GTP-loading and peripheral vesicle formation, whereas knockdown of zDHHC9 repressed these PE-induced effects on Rab3gap1 and Rab3a but further enhanced PE-stimulated ANP secretion, suggesting zDHHC9-mediated palmitoylation of Rab3gap1 serves as an intrinsic mechanism to limit ANP release. Consistent with a role for Rab3a nucleotide exchange in modulation of ANP release, overexpression of Rab3gap1 in cardiomyocytes promoted a profound increase in ANP release whereas a GAP-deficient mutant of Rab3gap1 (Rab3gap1 R728A ) was resistant to this effect. Collectively, these data suggest disruption of Rab3a nucleotide cycling perturbs secretory vesicle membrane fusion and ANP release in cardiac myocytes and establish critical roles for Rab3a activity and its modulation by zDHHC9-mediated palmitoylation of Rab3gap1 in governing cardiac homeostasis and endocrine signaling.

Live-cell photoactivated localization microscopy correlates nanoscale ryanodine receptor configuration to calcium sparks in cardiomyocytes

Ca2+ sparks constitute the fundamental units of Ca2+ release in cardiomyocytes. Here we investigate how ryanodine receptors (RyRs) collectively generate these events by employing a transgenic mouse with a photoactivated label on RyR2. This allowed correlative imaging of RyR localization, by super-resolution photoactivated localization microscopy, and Ca2+ sparks, by high-speed imaging. Two populations of Ca2+ sparks were observed: stationary events and ‘traveling’ events that spread between neighboring RyR clusters. Traveling sparks exhibited up to eight distinct releases, sourced from local or distal junctional sarcoplasmic reticulum. Quantitative analyses showed that sparks may be triggered by any number of RyRs within a cluster, and that acute β-adrenergic stimulation augments intracluster RyR recruitment to generate larger events. In contrast, RyR ‘dispersion’ during heart failure facilitates the generation of traveling sparks. Thus, RyRs cooperatively generate Ca2+ sparks in a complex, malleable fashion, and channel organization regulates the propensity for local propagation of Ca2+ release.

Inhibition of TAK1/TAB2 complex formation by costunolide attenuates obesity cardiomyopathy via the NF-κB signaling pathway

BackgroundChronic and persistent obesity can lead to various complications, including obesity cardiomyopathy. Inhibition of the inflammatory response is an effective measure for the intervention of obesity cardiomyopathy. Numerous studies indicate that costunolide (Cos) can reduce inflammation. However, the role of Cos in obesity cardiomyopathy and its molecular targets remains unknown. Hypothesis/PurposeWe aimed to clarify potential cardioprotective effects and mechanism of Cos against obesity cardiomyopathy. MethodsThe model of obesity cardiomyopathy was established by feeding mice with a high-fat diet for 24 weeks. Cos at 10 and 20 mg/kg or vehicle (1% CMCNa solution) was administered once every two days via oral gavage from the 17th to 24th week. Body weight, heart weight/tibia length, cardiac function, myocardial injury markers, pathological morphology of the heart, hypertrophic and fibrotic markers, inflammatory factors were assessed. The targets of Cos were predicted through molecular docking. Pull-down assay and biolayer interferometry were used to confirm the target of Cos. ResultsCos effectively reduces obesity-induced cardiomyocyte inflammation, cardiac hypertrophy and fibrosis, thereby improving cardiac function. We confirmed that Cos can interact with TAK1 and inhibit downstream NF-κB pathway activation by blocking the formation of the TAK1/TAB2 complex, thus inhibiting inflammatory cytokine release in cardiomyocytes. ConclusionOur results demonstrated that Cos significantly improved myocardial remodeling and cardiac dysfunction against obesity cardiomyopathy by reducing myocardial inflammation. Therefore, Cos may serve as a promising therapeutic agent in obesity cardiomyopathy.

Long Non-coding RNA N1LR Protects Against Myocardial Ischemic/Reperfusion Injury Through Regulating the TGF-β Signaling Pathway.

Long non-coding RNAs (lncRNAs) have been shown to play critical roles in various cell biological processes. However, the mechanism of lncRNAs in acute myocardial infarction (AMI) is not fully understood. Previous studies showed that lncRNA N1LR was down-regulated in ischemic cerebral stroke and its up-regulation was protective. The current study was designed to assess the protective effect of N1LR and further to explore potential mechanisms of N1LR in ischemic/reperfusion (I/R) injury after AMI. Male C57BL/6J mice and H9c2 cardiomyocytes were selected to construct in vivo and in vitro pathological models. In H9c2 cell line, N1LR expression was markedly decreased after H2O2 and CoCl2 treatments and N1LR overexpression alleviated apoptosis, inflammation reaction, and LDH release in cardiomyocytes treated with H2O2 and CoCl2. Mouse in vivo study showed that overexpression of N1LR enhanced cardiac function and suppressed inflammatory response and fibrosis. Mechanistically, we found that the expression of transforming growth factor (TGF)-β1 and smads were significantly decreased in the N1LR overexpression group exposed to H2O2. In a summary, our study indicated that N1LR can act as a protective factor against cardiac ischemic-reperfusion injury through regulating the TGF-β/Smads signaling pathway.

RyR2 Gain-of-Function and Not So Sudden Cardiac Death

RyR2 Gain-of-Function and Not So Sudden Cardiac Death

Exercise Training Stabilizes RyR2-Dependent Ca2+ Release in Post-infarction Heart Failure.

Aim: Dysfunction of the cardiac ryanodine receptor (RyR2) is an almost ubiquitous finding in animal models of heart failure (HF) and results in abnormal Ca2+ release in cardiomyocytes that contributes to contractile impairment and arrhythmias. We tested whether exercise training (ET), as recommended by current guidelines, had the potential to stabilize RyR2-dependent Ca2+ release in rats with post-myocardial infarction HF.Materials and Methods: We subjected male Wistar rats to left coronary artery ligation or sham operations. After 1 week, animals were characterized by echocardiography and randomized to high-intensity interval ET on treadmills or to sedentary behavior (SED). Running speed was adjusted based on a weekly VO2max test. We repeated echocardiography after 5 weeks of ET and harvested left ventricular cardiomyocytes for analysis of RyR2-dependent systolic and spontaneous Ca2+ release. Phosphoproteins were analyzed by Western blotting, and beta-adrenoceptor density was quantified by radioligand binding.Results: ET increased VO2max in HF-ET rats to 127% of HF-SED (P < 0.05). This coincided with attenuated spontaneous SR Ca2+ release in left ventricular cardiomyocytes from HF-ET but also reduced Ca2+ transient amplitude and slowed Ca2+ reuptake during adrenoceptor activation. However, ventricular diameter and fractional shortening were unaffected by ET. Analysis of Ca2+ homeostasis and major proteins involved in the regulation of SR Ca2+ release and reuptake could not explain the attenuated spontaneous SR Ca2+ release or reduced Ca2+ transient amplitude. Importantly, measurements of beta-adrenoceptors showed a normalization of beta1-adrenoceptor density and beta1:beta2-adrenoceptor ratio in HF-ET.Conclusion: ET increased aerobic capacity in post-myocardial infarction HF rats and stabilized RyR2-dependent Ca2+ release. Our data show that these effects of ET can be gained without major alterations in SR Ca2+ regulatory proteins and indicate that future studies should include upstream parts of the sympathetic signaling pathway.

Chemical Biology Tools to Elucidate the Solution Conformation and Cellular Interactions of Cyclic Adenosine 5′‐Diphosphate Ribose

The movement of calcium ions (Ca2+) is intimately involved in regulating many physiological processes, and so there is great interest in the structure of molecules, especially second messengers, that regulate this flow. One such messenger is cyclic adenosine 5′‐diphosphate ribose (cADPR), which is suspected to play a role in Ca2+ release in cardiomyocytes. The speculated mechanism of action involves the binding of cADPR to FKBP12.6, releasing FKBP12.6 from RyR2 channels, through which Ca2+ enters the sarcoplasm from the sarcoplasmic reticulum during ventricular systole. Guided by the principle that structure determines function, we aim to advance the investigation of regulatory cADPR interactions by synthesizing and studying cADPR and its analogs using the combined tools of liquid chromatography‐mass spectrometry (LCMS) and nuclear magnetic resonance (NMR) spectroscopy. The endogenous cyclization of nicotinamide adenine dinucleotide (NAD+) to cADPR by CD38 is mimicked in vitro using ADP‐ribosylcyclase (ADP‐RC, sourced from Aplysia californica). The low substrate specificity of ADP‐RC conveniently permits the cyclization of various cADPR analogs. Kinetic assays were prompted by an observed feedback inhibition of ADP‐RC by nicotinamide (IC50 ~ 1mM), and performed using LCMS. Following synthesis and purification, NMR experimental data provided insight into favored structural conformations of cADPR (Saatori et al., 2018) Photoaffinity labeling (PAL) was aimed at identifying the cognate binding partners of cADPR, some of which have been previously described by Walseth et al. (1993). Conformational analyses of cADPR analogs revealed structural trends with correlations to the biological activity of the analogs.Support or Funding InformationDepartment of Chemistry, Saint John’s University

Correlating Calcium Sparks and Ryanodine Receptor Localization in Live Cardiomyocytes

Ca2+ sparks constitute the fundamental units of sarcoplasmic reticulum (SR) Ca2+ release in cardiomyocytes. However, despite more than 25 years of investigation, the precise nature by which ryanodine receptors (RyRs) collaborate to generate these release events remains unclear. This challenge is related to both technical limitations in imaging RyRs and the rapid time frame in which sparks occur. Unfortunately, various imaging techniques capable of resolving RyRs, including super-resolution microscopy (dSTORM, DNA-PAINT) and electron microscopy, require fixed samples. To circumvent this limitation, we developed a transgenic mouse with photo-activated (PA) tagRFP targeted to RyR2. This approach allows correlative pairing of RyR localization, determined by PA Localization Microscopy (PALM), and Ca2+ sparks detected by high-speed imaging with a highly inclined light sheet (HILO). Ca2+ spark recordings showed that a subset of events exhibited slow kinetics, with protracted rise times and durations. Subtracting estimated Ca2+ diffusion revealed that prolonged Ca2+ sparks exhibited multiple distinct releases, numbering between 2 and 8 events. Notably, consecutive releases were associated with displacement of the spark centroid. Paired imaging of RyRs confirmed that these “travelling sparks” moved between nearby RyR clusters, with some sparks exhibiting displacement as far as 500 nm along z-lines. Importantly, spark propagation often proceeded between clusters that were not within the closest proximity. Treatment with isoproterenol exaggerated this phenomenon, as a larger fraction of travelling sparks was observed which included as many as 12 distinct release sites. These data suggest that participation of discrete RyR clusters in Ca2+ spark generation is dependent not only on RyR cluster position, but also other factors such as local post-translational modifications which are critically altered during β-adrenergic stimulation.

Junctophilin-2 is a target of matrix metalloproteinase-2 in myocardial ischemia-reperfusion injury.

Junctophilin-2 is a structural membrane protein that tethers T-tubules to the sarcoplasmic reticulum to allow for coordinated calcium-induced calcium release in cardiomyocytes. Defective excitation-contraction coupling in myocardial ischemia-reperfusion (IR) injury is associated with junctophilin-2 proteolysis. However, it remains unclear whether preventing junctophilin-2 proteolysis improves the recovery of cardiac contractile dysfunction in IR injury. Matrix metalloproteinase-2 (MMP-2) is a zinc and calcium-dependent protease that is activated by oxidative stress in myocardial IR injury and cleaves both intracellular and extracellular substrates. To determine whether junctophilin-2 is targeted by MMP-2, isolated rat hearts were perfused in working mode aerobically or subjected to IR injury with the selective MMP inhibitor ARP-100. IR injury impaired the recovery of cardiac contractile function which was associated with increased degradation of junctophilin-2 and damaged cardiac dyads. In IR hearts, ARP-100 improved the recovery of cardiac contractile function, attenuated junctophilin-2 proteolysis, and prevented ultrastructural damage to the dyad. MMP-2 was co-localized with junctophilin-2 in aerobic and IR hearts by immunoprecipitation and immunohistochemistry. In situ zymography showed that MMP activity was localized to the Z-disc and sarcomere in aerobic hearts and accumulated at sites where the striated JPH-2 staining was disrupted in IR hearts. In vitro proteolysis assays determined that junctophilin-2 is susceptible to proteolysis by MMP-2 and in silico analysis predicted multiple MMP-2 cleavage sites between the membrane occupation and recognition nexus repeats and within the divergent region of junctophilin-2. Degradation of junctophilin-2 by MMP-2 is an early consequence of myocardial IR injury which may initiate a cascade of sequelae leading to impaired contractile function.

Ischemic N-Acetylneuraminic Acid Release Impairs Mitochondrial Hexokinases II and Autophagy in Cardiomyocytes

Background: Elevated levels of circulating N-acetylneuraminic acid (Neu5Ac) is considered as an independent risk factor for coronary artery diseases. This study aims to find the potential impact of free Neu5Ac on cardiomyocyte injury. Methods: The impact of free Neu5Ac on mitochondrial hexokinases II (HK-II) and autophagy was investigated in primary cardiomyocytes and the mouse heart subjected to hypoxic insult. Findings: Hypoxia activated neuraminidase-1(Neu-1) to induce sialic acid release in cardiomyocytes. Free Neu5Ac bound to HK-II and impaired mitochondrial function, leading to cell apoptosis. Nutrient deprivation elicits autophagy for cell salvage. HK-II binds and inactivates mTOR. However, Neu5Ac disrupted the binding and impaired autophagic response by reactivating mTOR, evidenced by impaired autophagic flux with increased p62 expression and reduced ratio of LC3-II/LC3-I. Impaired mitochondrial HK-II and autophagy were also observed in isoproterenol-induced ischemic heart of mice. By inhibiting neuraminidase, anti-influenza drugs oseltamivir and zanamivir reduced sialic acid release with improved mitochondrial HK-II and autophagy, resultantly rescuing cardiomyocytes survival from ischemic injury. Interpretation: Myocardial ischemia activates neuraminidase, and the released Neu5Ac acts as a mediator to impair cardiomyocytes. These findings establish a previously unrecognized role of Neu5Ac in cardiac ischemic injury and suggest the potential of neuraminidase inhibition in cardiomyocytes. Funding: This work was supported in part by the National Science Fund of China for Distinguished Young Scholars, Major Research Plan of the National Science Foundation of China, and National Natural Science Foundation of China. Declaration of Interest: The authors declare no conflicts of interest that pertain to this work. Ethical Approval: All animal experiments were carried out according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the procedures were approved by the Animal Care and Use Committee of China Pharmaceutical University.

Cyclosporin A Protected Cardiomyocytes Against Oxidative Stress Injury by Inhibition of NF-κB Signaling Pathway.

This study aims to investigate the effects and the molecular mechanism of cyclosporin A (CsA) against oxidative stress injury in cultured neonatal rat cardiomyocytes. Bax/Bcl-2, cl-casp-9/casp-9, cl-casp-3/casp-3, and iNOS/β-actin ratios and p-IκB and IκB levels were analyzed by western blot. IL-1β and TNF-α levels were analyzed by ELISA. CsA effectively improved the cell viability and reduced the extracellular lactate dehydrogenase release in cardiomyocytes after H2O2-induced oxidative damage. CsA significantly increased the superoxide dismutase activity, glutathione production, and catalase activity but decreased the malonaldehyde level. CsA treatment considerably reduced the H2O2-induced intracellular generation of reactive oxygen species, mitochondrial dysfunction, and release of cytochrome c. CsA could act against H2O2-induced ATP reduction, TCA cycle enzymes, mitochondrial complex I enzyme, and complex V enzyme in cardiomyocytes. CsA significantly decreased the Bax/Bcl-2 ratio, cl-casp-9/casp-9, and cl-casp-3/casp-3 in a concentration-dependent manner. CsA also remarkably reduced the cleaved PARP level and DNA fragmentation. NF-κB was closely related to oxidative stress injury. CsA inhibited NF-κB activation, thereby preventing the upregulation of IL-1β, TNF-α, iNOS, and intracellular NO release. CsA protected cardiomyocytes against H2O2-induced cell injury. Hence, CsA may be developed as a candidate drug to prevent or treat myocardial ischemia reperfusion injury.

Cystic Fibrosis Transmembrane Conductance Regulator Regulates Pannexin 1 Channel Opening for Acidosis‐induced ATP Release from Cardiomyocytes

ATP is released in the heart under a variety of conditions, including hypoxia, ischaemia and catecholamine stimulation. Under physiological conditions, extracellular ATP, and its breakdown product, adenosine, contribute to local vasodilation and regulation of coronary blood flow. We investigated the mechanism of ATP release in cardiomyocytes isolated from adult rat heart.Treatment with lactic acid produced pH depression and enhanced ATP release from cardiomyocytes. On the other hand, simulated ischaemia (metabolic inhibition plus anoxia) lowered ATP release. The acidosis‐induced ATP release was abolished by the specific CFTR inhibitor, CFTRinh‐172, CFTR pore blocker, GlyH‐101, or CFTR siRNA, suggesting that CFTR was involved. Forskolin and IBMX, agents that activate CFTR by elevating the intracellular cAMP, also increased ATP release, confirming the role of CFTR. However, blockade of Pannexin1 channels by Fast Green FCF, Brilliant Blue FCF, or pannexin1 siRNA, attenuated the ATP release during acidosis or forskolin treatment, and further lowered the ATP release during simulated ischaemia, suggesting that Pannexin1 functions as the ATP release channel. Immunofluorescence imaging suggested that CFTR and Pannexin1 were not co‐localized. Inhibition of either the Na+/H+ exchanger (NHE) with amiloride or the Na+/Ca2+ exchanger (NCX) with SN6 or KB‐R7943 abolished acidosis‐induced ATP release. FAK inhibitor 14, FAK autophosphorylation inhibitor, can attenuate the forskolin or lactic‐acid‐stimulated ATP release. Furthermore, forskolin or lactic‐acid‐stimulated ATP release was abolished by bicarbonate free medium or caspase inhibitor. Thus, we have demonstrated that the acidosis‐induced ATP release was involved in calcium induced CFTR open. The cyclic AMP, which activates mitochondrial PKA, is generated within mitochondria by the bicarbonate‐regulated soluble adenylyl cyclase (sAC). Hence, we propose that the CFTR Cl channel opens during acidosis, allowing the movement of bicarbonate into the cell, which activate the HCO3‐sAC‐cAMP‐PKA (mito‐sAC) signaling cascade and indirectly modulates Pannexin1 gating by activation of caspase.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

To Be Or Not to Be: the “Smoker’s Paradox” – An in-Vitro Study

Background/Aims: Clinical studies have reported a better outcome of smokers after myocardial infarction compared to non-smokers. The data are controversial, as some clinical studies did not observe this effect. The cell biological processes involved, which might account for a ‘Smoker’s Paradox’, have not been investigated yet. Therefore, the aim was to elucidate the effect of cigarette smoke on the viability of cardiomyocytes in the context of hypoxia and reperfusion. Methods: HL-1 cells were incubated with different concentrations of cigarette smoke extract (CSE) and subjected to hypoxia/reperfusion to further evaluate influence of CSE on viability of HL-1 cells using flow cytometry analyses, Western Blot and immunofluorescence staining. Results: Incubation with CSE led to a concentration-dependent reduction in HL-1 viability. Adding hypoxia as a stressor enhanced cell death. Caspase-independent apoptosis was the observed type of cell death partly induced by P53 and apoptosis-inducing-factor. Yet a significant increase in LDH release in cardiomyocytes incubated with 4%, 8% and 16% CSE suggests necrosis with rapid DNA depletion. Interestingly, after hypoxia a decreased LDH release under lower CSE concentrations was observed. Moreover, a concentration-dependent increase in proliferation and a trend for increased ATP availability under hypoxic conditions was shown. Conclusions: The trend for less LDH release in hypoxia after low-level CSE incubation might represent a switch from necrosis to apoptosis, which in combination with the increase in metabolic activity and ATP availability might account for the ‘Smoker’s Paradox’. These findings could partly explain inconsistent results of previous clinical studies as the data showed strong evidence for the crucial relevance of the amount of cigarettes smoked. We are in need of future studies distinguishing between different types of smokers to finally verify or falsify the ‘Smoker’s Paradox’.

Wnt5a is associated with right ventricular dysfunction and adverse outcome in dilated cardiomyopathy

The Wingless (Wnt) pathway has been implicated in the pathogenesis of dilated cardiomyopathy (DCM). To explore the role of Wnt modulators Wnt5a and sFRP3 in DCM patients we analyzed the expression of Wnt5a and sFRP3 in plasma and myocardium of DCM patients and evaluated their effects on NFAT luciferase activity in neonatal mouse cardiomyocytes. Elevated circulating Wnt5a (n = 102) was associated with increased pulmonary artery pressures, decreased right ventricular function and adverse outcome, with a stronger association in more severely affected patients. A higher Wnt5a/sFRP3 ratio (n = 25) was found in the right ventricle vs. the left ventricle and was correlated with NFAT activation as well as pulmonary artery pressures. Wnt5a induced NFAT activation and sFRP3 release in cardiomyocytes in vitro, while sFRP3 antagonized Wnt5a. Wnt5a is associated with right ventricular dysfunction and adverse outcome in DCM patients and may promote the progression of DCM through NFAT signaling.

Modeling the Effects of Volatile Anesthetics on L-Type Ca2+ Channels and Ca2+ Induced Ca2+ Release in Cardiomyocytes

Modeling the Effects of Volatile Anesthetics on L-Type Ca2+ Channels and Ca2+ Induced Ca2+ Release in Cardiomyocytes

Regulation of sarcoplasmic reticulum Ca2+ release by serine-threonine phosphatases in the heart

The amount and timing of Ca2+ release from the sarcoplasmic reticulum (SR) during cardiac cycle are the main determinants of cardiac contractility. Reversible phosphorylation of the SR Ca2+ release channel, ryanodine receptor type 2 (RyR2) is the central mechanism of regulation of Ca2+ release in cardiomyocytes. Three major serine-threonine phosphatases including PP1, PP2A and PP2B (calcineurin) have been implicated in modulation of RyR2 function. Changes in expression levels of these phosphatases, their activity and targeting to the RyR2 macromolecular complex were demonstrated in many animal models of cardiac disease and humans and are implicated in cardiac arrhythmia and heart failure. Here we review evidence in support of regulation of RyR2-mediated SR Ca2+ release by serine-threonine phosphatases and the role and mechanisms of dysregulation of phosphatases in various disease states.

Abstract 372: MicroRNA-9 Inhibits Hyperglycemia-induced Cardiac Pyroptosis by Targeting ELAVL1

Diabetic cardiomyopathy is a common complication in patients with diabetes and is associated with underlying chronic inflammation and cardiac cell death, subsequently leading to left ventricular dysfunction and heart failure. ELAV-like protein 1 (ELAVL1, mRNA stabilizing protein) and NLRP3 activation (inflammasome complex protein)-mediated IL-1beta synthesis play a critical role in the progression of heart failure. However, ELAVL1 regulation of pyroptosis (caspase-1-mediated programmed apoptosis) and associated IL-1beta release in cardiomyocytes, especially under diabetic condition, remains elusive. Human diabetic, non-diabetic heart tissues, human ventricular cardiomyocytes and rat cardiomyoblasts exposed to high glucose (HG) were used for our studies. Our data demonstrates that human ventricular cardiomyocytes exposed to high glucose condition showed significant increase in ELAVL1 and NLRP3 expression with a concomitant increase in caspase-1 and IL-1 beta expression. Furthermore, human cardiac tissue from diabetic patients showed increased ELAVL1, caspase-1 and NLRP3 expression as compared to non-diabetic hearts. Intriguingly, ELAVL1 knockdown abrogates TNF-α induced canonical pyroptosis via NLRP3, caspase-1 and IL-1beta suppression. Interestingly, miRNA-9 expression significantly reduces in high glucose treated cardiomyocytes and in human diabetic hearts. Bioinformatics analysis and target validation assays showed that miR-9 directly targets ELAVL1. Inhibition of miR-9 up regulates ELAVL1 expression and activates caspase-1. Alternatively, miR-9 mimics attenuate hyperglycemia-induced ELAVL1 and inhibit cardiomyocyte pyroptosis. To our knowledge, this is the first report to demonstrate the role of miR-9/ELAVL1 in hyperglycemia-induced cardiac pyroptosis. Taken together our study highlights the potential therapeutic implications in preventing cardiomyocyte cell loss in human diabetic failing heart.

Dysregulation of RBFOX2 Is an Early Event in Cardiac Pathogenesis of Diabetes.

Alternative splicing (AS) defects that adversely affect gene expression and function have been identified in diabetic hearts; however, the mechanisms responsible are largely unknown. Here, we show that the RNA-binding protein RBFOX2 contributes to transcriptome changes under diabetic conditions. RBFOX2 controls AS of genes with important roles in heart function relevant to diabetic cardiomyopathy. RBFOX2 protein levels are elevated in diabetic hearts despite low RBFOX2 AS activity. A dominant-negative (DN) isoform of RBFOX2 that blocks RBFOX2-mediated AS is generated in diabetic hearts. DN RBFOX2 interacts with wild-type (WT) RBFOX2, and ectopic expression of DN RBFOX2 inhibits AS of RBFOX2 targets. Notably, DN RBFOX2 expression is specific to diabetes and occurs at early stages before cardiomyopathy symptoms appear. Importantly, DN RBFOX2 expression impairs intracellular calcium release in cardiomyocytes. Our results demonstrate that RBFOX2 dysregulation by DN RBFOX2 is an early pathogenic event in diabetic hearts.

Elevated ventricular wall stress disrupts cardiomyocyte t-tubule structure and calcium homeostasis.

AimsInvaginations of the cellular membrane called t-tubules are essential for maintaining efficient excitation–contraction coupling in ventricular cardiomyocytes. Disruption of t-tubule structure during heart failure has been linked to dyssynchronous, slowed Ca2+ release and reduced power of the heartbeat. The underlying mechanism is, however, unknown. We presently investigated whether elevated ventricular wall stress triggers remodelling of t-tubule structure and function.Methods and resultsMRI and blood pressure measurements were employed to examine regional wall stress across the left ventricle of sham-operated and failing, post-infarction rat hearts. In failing hearts, elevated left ventricular diastolic pressure and ventricular dilation resulted in markedly increased wall stress, particularly in the thin-walled region proximal to the infarct. High wall stress in this proximal zone was associated with reduced expression of the dyadic anchor junctophilin-2 and disrupted cardiomyocyte t-tubular structure. Indeed, local wall stress measurements predicted t-tubule density across sham and failing hearts. Elevated wall stress and disrupted cardiomyocyte structure in the proximal zone were also associated with desynchronized Ca2+ release in cardiomyocytes and markedly reduced local contractility in vivo. A causative role of wall stress in promoting t-tubule remodelling was established by applying stretch to papillary muscles ex vivo under culture conditions. Loads comparable to wall stress levels observed in vivo in the proximal zone reduced expression of junctophilin-2 and promoted t-tubule loss.ConclusionElevated wall stress reduces junctophilin-2 expression and disrupts t-tubule integrity, Ca2+ release, and contractile function. These findings provide new insight into the role of wall stress in promoting heart failure progression.