Calcium Handling In Cardiomyocytes Research Articles

Distinguishing pathophysiological features of heart failure with reduced and preserved ejection fraction: A comparative analysis of two mouse models.

Heart failure (HF) is a heterogeneous condition that can be categorized according to the left ventricular ejection fraction (EF) into HF with reduced (HFrEF) or preserved (HFpEF) EF. Although HFrEF and HFpEF share some common clinical manifestations, the mechanisms underlying each phenotype are often found to be distinct. Identifying shared and divergent pathophysiological features might expand our insights on HF pathophysiology and assist the search for therapies for each HF subtype. In this study, we evaluated and contrasted two new murine models of non-ischaemic HFrEF and cardiometabolic HFpEF in terms of myocardial structure, left ventricular function, gene expression, cardiomyocyte calcium handling, mitochondrial polarization and protein acetylation in a head-to-head fashion. We found that in conditions of similar haemodynamic stress, the HFrEF myocardium underwent a more pronounced hypertrophic and fibrotic remodelling, whereas inflammation was greater in the HFpEF myocardium. We observed opposing features on calcium release, which was diminished in the HFrEF cardiomyocyte but enhanced in the HFpEF cardiomyocyte. Mitochondria were less polarized in both HFrEF and HFpEF cardiomyocytes, reflecting similarly impaired metabolic capacity. Hyperacetylation of cardiac proteins was observed in both models, but it was more accentuated in the HFpEF heart. Despite shared features, unique triggering mechanisms (neurohormonal overactivation in HFrEF vs. inflammation in HFpEF) appear to determine the distinct phenotypes of HF. The findings of the present research stress the need for further exploration of the differential mechanisms underlying each HF subtype, because they might require specific therapeutic interventions. KEY POINTS: The mechanisms underlying heart failure with either reduced (HFrEF) or preserved (HFpEF) ejection fraction are often found to be different. Previous studies comparing pathophysiological traits between HFrEF and HFpEF have been conducted on animals of different ages and strains. The present research contrasted two age-matched mouse models of non-ischaemic HFrEF and cardiometabolic HFpEF to uncover divergent and shared features. We found that upon similar haemodynamic stress, the HFrEF heart experienced a more pronounced hypertrophic and fibrotic remodelling, whereas inflammation appeared to be greater in the HFpEF myocardium. Calcium release was diminished in the HFrEF cardiomyocyte and enhanced in the HFpEF cardiomyocyte. Mitochondria were comparably less polarized in both HFrEF and HFpEF myocytes. Hyperacetylation of proteins was common to both models, but stronger in the HFpEF heart. Casting light on common and distinguishing features might ease the quest for phenotype-specific therapies for heart failure patients.

A Role for Septin GTPases in regulating cytoplasmic calcium handling in Drosophila cardiomyocytes

Proper calcium handling in cardiomyocytes is paramount to maintaining cardiomyocyte contractility and heart function. Data from our lab and others have established that store operated calcium entry (SOCE) is an essential component of cardiomyocyte calcium regulation. SOCE refers to calcium influx that is activated by depletion of endo/sarcoplasmic reticulum (E/SR) calcium stores. The pathway is mediated by STIM proteins, which act as calcium sensors in the E/SR, and ORAI calcium selective influx channels in the plasma membrane. Both upregulation and suppression of SOCE in cardiomyocytes result in cardiomyopathy, demonstrating that proper regulation of cardiomyocyte SOCE is essential. However, the mechanisms responsible for SOCE regulation in cardiomyocytes are not well understood. To this end, septin GTPases have emerged as regulators of SOCE in non-cardiac tissues, with septin 1, 2, or 4 depletion resulting in SOCE suppression and septin 7 depletion resulting in SOCE upregulation. The role of septins in cardiomyocytes is nearly completely unknown. Through intravital imaging analysis of Drosophila heart contractility, we show that cardiomyocyte specific RNAi-based depletion of septins 1, 2, or 4 results in dilated cardiomyopathy nearly identical to that caused by SOCE suppression. Overexpression of SERCA, which pumps Ca2+ into SR stores, reversed the septin 2 phenotype, suggesting a mechanism whereby septin depletion reduces SR Ca2+ store refilling due to suppressed SOCE. In further support of SR Ca2+ store depletion, we found through jRGECO-based Ca2+ imaging of contracting hearts that contractile Ca2+ transients were significantly reduced in septin 2-depleted cardiomyocytes compared to controls. Interestingly, septin 7 suppression resulted in hypertrophic cardiomyopathy like that caused by SOCE upregulation, and micro-computerized tomography (microCT) analysis of heart size further confirmed the hypertrophic phenotype of septin 7 depleted hearts. This septin 7 phenotype was reversed by Orai depletion, supporting a role for SOCE dysregulation in the septin 7 phenotype. These results demonstrate for the first time an essential role for septins in cardiomyocyte physiology involving regulation of Ca2+ homeostasis and cycling. National institutes of Health. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

OTUD1 promotes isoprenaline- and myocardial infarction-induced heart failure by targeting PDE5A in cardiomyocytes

Heart failure represents a major cause of death worldwide. Recent research has emphasized the potential role of protein ubiquitination/deubiquitination protein modification in cardiac pathology. Here, we investigate the role of the ovarian tumor deubiquitinase 1 (OTUD1) in isoprenaline (ISO)- and myocardial infarction (MI)-induced heart failure and its molecular mechanism. OTUD1 protein levels were raised markedly in murine cardiomyocytes after MI and ISO treatment. OTUD1 deficiency attenuated myocardial hypertrophy and cardiac dysfunction induced by ISO infusion or MI operation. In vitro, OTUD1 knockdown in neonatal rat ventricular myocytes (NRVMs) attenuated ISO-induced injuries, while OTUD1 overexpression aggravated the pathological changes. Mechanistically, LC-MS/MS and Co-IP studies showed that OTUD1 bound directly to the GAF1 and PDEase domains of PDE5A. OTUD1 was found to reverse K48 ubiquitin chain in PDE5A through cysteine at position 320 of OTUD1, preventing its proteasomal degradation. PDE5A could inactivates the cGMP-PKG-SERCA2a signaling axis which dysregulate the calcium handling in cardiomyocytes, and leading to the cardiomyocyte injuries. In conclusion, OTUD1 promotes heart failure by deubiquitinating and stabilizing PDE5A in cardiomyocytes. These findings have identified PDE5A as a new target of OTUD1 and emphasize the potential of OTUD1 as a target for treating heart failure.

A Novel Ryr2 Mutation in an Adult Patient with Cardiac Arrest Caused by Ventricular Arrhythmia Suspected to be Catecholaminergic Polymorphic Ventricular Tachycardia: A Case Report

Inherited arrhythmia syndromes remain an important and increasingly recognized cause of ventricular tachycar dia (VT) and sudden cardiac death. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a rare but potentially lethal, heritable cardiac arrhythmia that occurs in response to physical or emotional stress, it often manifests as exercise-induced ventricular arrhythmias, syncope, or sudden death. If untreated, catecholaminergic polymorphic ventricular tachycardia is a highly lethal condition: About 80% of affected individuals experience recurrent syncope, and 30% experience cardiac arrest. CPVT is caused by mutations in genes encoding proteins involved in calcium handling in cardiomyocytes, such as the cardiac ryanodine receptor (RyR2). RyR2 mutations impair the regulation of calcium release from the sarcoplasmic reticulum, leading to delayed afterdepolarizations and triggered arrhythmias. Here, we report a case of CPVT in a patient with RyR2 gene mutation, causing sudden cardiac arrest.

BS-452758-1 SUPPRESSION-REPLACEMENT GENE THERAPY REVERSE REMODELS THE INTRACELLULAR CALCIUM HANDLING DISEASE PHENOTYPE OF TNNT2-MEDIATED HYPERTROPHIC CARDIOMYOPATHY

Hypertrophic cardiomyopathy (HCM), characterized by otherwise unexplained left ventricular hypertrophy, has a prevalence of 1:500. Pathogenic variants in TNNT2-encoded cardiac troponin T causes a thin filament subtype of sarcomeric HCM. A key cellular feature of the pathological remodeling in TNNT2-mediated HCM involves dysregulation of intracellular calcium handling in ventricular cardiomyocytes (CMs). To develop a TNNT2 “suppression-replacement (SupRep)” gene therapy to reverse the intracellular calcium perturbations associated with thin filament, sarcomeric HCM using induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) derived from a patient with TNNT2-R288P-mediated HCM. Custom-designed TNNT2 targeting shRNAs were tested for knockdown (KD) efficiency using TSA201 cells and RT-qPCR. A dual-component SupRep TNNT2 gene therapy was created by cloning into a single construct a custom-designed TNNT2 shRNA that produced greatest KD (suppression) and a “shRNA-immune” (shIMM) TNNT2 cDNA (replacement). Patient-specific TNNT2-R288P and CRISPR/Cas9 variant-corrected isogenic control (IC) iPSC-CMs were generated from a patient with HCM. The intracellular calcium indicator, Fluo-4, was used to detect intracellular free calcium. Our lead TNNT2-shRNA had an 80% KD efficiency of TNNT2. TNNT2-R288P iPSC-CMs treated with TNNT2-SupRep showed a similar KD efficiency. Compared to the IC iPSC-CMs, the TNNT2-R288P iPSC-CMs showed increased irregular events (30.4% vs. 10.9%, p<0.0001), prolonged calcium transient duration 90 (CTD90, 1.01 ± 0.03 s vs. 0.89 ± 0.02 s, p<0.0001) and prolonged peak to 90% decay time (0.57 ± 0.02 s vs. 0.49 ± 0.01 s, p=0.0012). Compared to untreated cells, transfection with TNNT2-SupRep gene therapy, the TNNT2-R288P iPSC-CMs showed a decrease in irregular events (30.4% vs. 14%, p<0.0001), shortened CTD90 (1.01 ± 0.03 s vs. 0.91 ± 0.01 s, p=0.003), and shortened peak to 90% decay time (0.57 ± 0.02 s vs. 0.50 ± 0.02 s, p=0.02). There was no significant difference in irregular events, CTD90, or peak to 90% decay time between TNNT2-R288P iPSC-CMs treated with TNNT2-SupRep and IC iPSC-CMs. Here, we provide the first proof-of-principle SupRep gene therapy for TNNT2-mediated HCM. TNNT2-SupRep gene therapy almost fully restored the impaired intracellular calcium handling properties of TNNT2-R288P patient-derived iPSC-CMs back to the normal state achieved by the CRISPR/Cas9 gene-edited cure.

USP25 Ameliorates Pathological Cardiac Hypertrophy by Stabilizing SERCA2a in Cardiomyocytes.

Pathological cardiac hypertrophy can lead to heart failure and is one of the leading causes of death globally. Understanding the molecular mechanism of pathological cardiac hypertrophy will contribute to the treatment of heart failure. DUBs (deubiquitinating enzymes) are essential to cardiac pathophysiology by precisely controlling protein function, localization, and degradation. This study set out to investigate the role and molecular mechanism of a DUB, USP25 (ubiquitin-specific peptidase 25), in pathological cardiac hypertrophy. The role of USP25 in myocardial hypertrophy was evaluated in murine cardiomyocytes in response to Ang II (angiotensin II) and transverse aortic constriction stimulation and in hypertrophic myocardium tissues of heart failure patients. Liquid chromotography with mass spectrometry/mass spectrometry analysis combined with Co-IP was used to identify SERCA2a (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 2A), an antihypertrophy protein, as an interacting protein of USP25. To clarify the molecular mechanism of USP25 in the regulation of SERCA2a, we constructed a series of mutant plasmids of USP25. In addition, we overexpressed USP25 and SERCA2a in the heart with adenoassociated virus serotype 9 vectors to validate the biological function of USP25 and SERCA2a interaction. We revealed increased protein level of USP25 in murine cardiomyocytes subject to Ang II and transverse aortic constriction stimulation and in hypertrophic myocardium tissues of patients with heart failure. USP25 deficiency aggravated cardiac hypertrophy and cardiac dysfunction under Ang II and transverse aortic constriction treatment. Mechanistically, USP25 bound to SERCA2a directly via its USP (ubiquitin-specific protease) domain and cysteine at position 178 of USP25 exerts deubiquitination to maintain the stability of the SERCA2a protein by removing the K48 ubiquitin chain and preventing proteasomal pathway degradation, thereby maintaining calcium handling in cardiomyocytes. Moreover, restoration of USP25 expression via adenoassociated virus serotype 9 vectors in USP25-/- mice attenuated Ang II-induced cardiac hypertrophy and cardiac dysfunction, whereas myocardial overexpression of SERCA2a could mimic the effect of USP25. We confirmed that USP25 inhibited cardiac hypertrophy by deubiquitinating and stabilizing SERCA2a.

The interplay between cardiac dyads and mitochondria regulated the calcium handling in cardiomyocytes.

Calcium mishandling and mitochondrial dysfunction have been increasingly recognized as significant factors involved in the progression procedure of cardiomyopathy. Ca2+ mishandling could cause calcium-triggered arrhythmias, which could enhance force development and ATP consumption. Mitochondrial disorganization and dysfunction in cardiomyopathy could disturb the balance of energy catabolic and anabolic procedure. Close spatial localization and arrangement of structural among T-tubule, sarcoplasmic reticulum, mitochondria are important for Ca2+ handling. So that, we illustrate the regulating network between calcium handling and mitochondrial homeostasis, as well as its intracellular mechanisms in this review, which would be worthy to develop novel therapeutic strategy and restore the function of injured cardiomyocytes.

Structural basis for the severe adverse interaction of sofosbuvir and amiodarone on L-type Cav channels

Drug-drug interaction of the antiviral sofosbuvir and the antiarrhythmics amiodarone has been reported to cause fatal heartbeat slowing. Sofosbuvir and its analog, MNI-1, were reported to potentiate the inhibition of cardiomyocyte calcium handling by amiodarone, which functions as a multi-channel antagonist, and implicate its inhibitory effect on L-type Cav channels, but the molecular mechanism has remained unclear. Here we present systematic cryo-EM structural analysis of Cav1.1 and Cav1.3 treated with amiodarone or sofosbuvir alone, or sofosbuvir/MNI-1 combined with amiodarone. Whereas amiodarone alone occupies the dihydropyridine binding site, sofosbuvir is not found in the channel when applied on its own. In the presence of amiodarone, sofosbuvir/MNI-1 is anchored in the central cavity of the pore domain through specific interaction with amiodarone and directly obstructs the ion permeation path. Our study reveals the molecular basis for the physical, pharmacodynamic interaction of two drugs on the scaffold of Cav channels.

RBPMS2 Is a Myocardial-Enriched Splicing Regulator Required for Cardiac Function.

RBPs (RNA-binding proteins) perform indispensable functions in the post-transcriptional regulation of gene expression. Numerous RBPs have been implicated in cardiac development or physiology based on gene knockout studies and the identification of pathogenic RBP gene mutations in monogenic heart disorders. The discovery and characterization of additional RBPs performing indispensable functions in the heart will advance basic and translational cardiovascular research. We performed a differential expression screen in zebrafish embryos to identify genes enriched in nkx2.5-positive cardiomyocytes or cardiopharyngeal progenitors compared to nkx2.5-negative cells from the same embryos. We investigated the myocardial-enriched gene RNA-binding protein with multiple splicing (variants) 2 [RBPMS2)] by generating and characterizing rbpms2 knockout zebrafish and human cardiomyocytes derived from RBPMS2-deficient induced pluripotent stem cells. We identified 1848 genes enriched in the nkx2.5-positive population. Among the most highly enriched genes, most with well-established functions in the heart, we discovered the ohnologs rbpms2a and rbpms2b, which encode an evolutionarily conserved RBP. Rbpms2 localizes selectively to cardiomyocytes during zebrafish heart development and strong cardiomyocyte expression persists into adulthood. Rbpms2-deficient embryos suffer from early cardiac dysfunction characterized by reduced ejection fraction. The functional deficit is accompanied by myofibril disarray, altered calcium handling, and differential alternative splicing events in mutant cardiomyocytes. These phenotypes are also observed in RBPMS2-deficient human cardiomyocytes, indicative of conserved molecular and cellular function. RNA-sequencing and comparative analysis of genes mis-spliced in RBPMS2-deficient zebrafish and human cardiomyocytes uncovered a conserved network of 29 ortholog pairs that require RBPMS2 for alternative splicing regulation, including RBFOX2, SLC8A1, and MYBPC3. Our study identifies RBPMS2 as a conserved regulator of alternative splicing, myofibrillar organization, and calcium handling in zebrafish and human cardiomyocytes.

The Role of Epicardial Adipose Tissue in the Development of Atrial Fibrillation, Coronary Artery Disease and Chronic Heart Failure in the Context of Obesity and Type 2 Diabetes Mellitus: A Narrative Review

Cardiovascular diseases (CVDs) are a significant burden globally and are especially prevalent in obese and/or diabetic populations. Epicardial adipose tissue (EAT) surrounding the heart has been implicated in the development of CVDs as EAT can shift from a protective to a maladaptive phenotype in diseased states. In diabetic and obese patients, an elevated EAT mass both secretes pro-fibrotic/pro-inflammatory adipokines and forms intramyocardial fibrofatty infiltrates. This narrative review considers the proposed pathophysiological roles of EAT in CVDs. Diabetes is associated with a disordered energy utilization in the heart, which promotes intramyocardial fat and structural remodeling. Fibrofatty infiltrates are associated with abnormal cardiomyocyte calcium handling and repolarization, increasing the probability of afterdepolarizations. The inflammatory phenotype also promotes lateralization of connexin (Cx) proteins, undermining unidirectional conduction. These changes are associated with conduction heterogeneity, together creating a substrate for atrial fibrillation (AF). EAT is also strongly implicated in coronary artery disease (CAD); inflammatory adipokines from peri-vascular fat can modulate intra-luminal homeostasis through an “outside-to-inside” mechanism. EAT is also a significant source of sympathetic neurotransmitters, which promote progressive diastolic dysfunction with eventual cardiac failure. Further investigations on the behavior of EAT in diabetic/obese patients with CVD could help elucidate the pathogenesis and uncover potential therapeutic targets.

Dual loss of regulator of G protein signaling 2 and 5 exacerbates ventricular myocyte arrhythmias and disrupts the fine-tuning of Gi/o signaling

AimsCardiac contractility, essential to maintaining proper cardiac output and circulation, is regulated by G protein-coupled receptor (GPCR) signaling. Previously, the absence of regulator of G protein signaling (RGS) 2 and 5, separately, was shown to cause G protein dysregulation, contributing to modest blood pressure elevation and exaggerated cardiac hypertrophic response to pressure-overload. Whether RGS2 and 5 redundantly control G protein signaling to maintain cardiovascular homeostasis is unknown. Here we examined how the dual absence of RGS2 and 5 (Rgs2/5 dbKO) affects blood pressure and cardiac structure and function. Methods and resultsWe found that Rgs2/5 dbKO mice showed left ventricular dilatation at baseline by echocardiography. Cardiac contractile response to dobutamine stress test was sex-dependently reduced in male Rgs2/5 dbKO relative to WT mice. When subjected to surgery-induced stress, male Rgs2/5 dbKO mice had 75% mortality within 72–96 h after surgery, accompanied by elevated baseline blood pressure and decreased cardiac contractile function. At the cellular level, cardiomyocytes (CM) from Rgs2/5 dbKO mice showed augmented Ca2+ transients and increased incidence of arrhythmia without augmented contractile response to electrical field stimulation (EFS) and activation of β-adrenergic receptors (βAR) with isoproterenol. Dual loss of Rgs2 and 5 suppressed forskolin-induced cAMP production, which was restored by Gi/o inactivation with pertussis toxin that also reduced arrhythmogenesis during EFS or βAR stimulation. Cardiomyocyte NCX and PMCA mRNA expression was unaffected in Rgs2/5 dbKO male mice. However, there was an exaggerated elevation of EFS-induced cytoplasmic Ca2+ in the presence of SERCA blockade with thapsigargin. ConclusionsWe conclude that RGS2 and 5 promote normal ventricular rhythm by coordinating their regulatory activity towards Gi/o signaling and facilitating cardiomyocyte calcium handling.

Cas9/AAV9-Mediated Somatic Mutagenesis Uncovered the Cell-Autonomous Role of Sarcoplasmic/Endoplasmic Reticulum Calcium ATPase 2 in Murine Cardiomyocyte Maturation.

Sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) is a key player in cardiomyocyte calcium handling and also a classic target in the gene therapy for heart failure. SERCA2 expression dramatically increases during cardiomyocyte maturation in the postnatal phase of heart development, which is essential for the heart to acquire its full function in adults. However, whether and how SERCA2 regulates cardiomyocyte maturation remains unclear. Here, we performed Cas9/AAV9-mediated somatic mutagenesis (CASAAV) in mice and achieved cardiomyocyte-specific knockout of Atp2a2, the gene coding SERCA2. Through a cardiac genetic mosaic analysis, we demonstrated the cell-autonomous role of SERCA2 in building key ultrastructures of mature ventricular cardiomyocytes, including transverse-tubules and sarcomeres. SERCA2 also exerts a profound impact on oxidative respiration gene expression and sarcomere isoform switching from Myh7/Tnni1 to Myh6/Tnni3, which are transcriptional hallmarks of cardiomyocyte maturation. Together, this study uncovered a pivotal role of SERCA2 in heart development and provided new insights about SERCA2-based cardiac gene therapy.

Integrative Computational Modeling of Cardiomyocyte Calcium Handling and Cardiac Arrhythmias: Current Status and Future Challenges.

Cardiomyocyte calcium-handling is the key mediator of cardiac excitation-contraction coupling. In the healthy heart, calcium controls both electrical impulse propagation and myofilament cross-bridge cycling, providing synchronous and adequate contraction of cardiac muscles. However, calcium-handling abnormalities are increasingly implicated as a cause of cardiac arrhythmias. Due to the complex, dynamic and localized interactions between calcium and other molecules within a cardiomyocyte, it remains experimentally challenging to study the exact contributions of calcium-handling abnormalities to arrhythmogenesis. Therefore, multiscale computational modeling is increasingly being used together with laboratory experiments to unravel the exact mechanisms of calcium-mediated arrhythmogenesis. This article describes various examples of how integrative computational modeling makes it possible to unravel the arrhythmogenic consequences of alterations to cardiac calcium handling at subcellular, cellular and tissue levels, and discusses the future challenges on the integration and interpretation of such computational data.

Abstract 12201: Dissecting the Protective Effect of Phosphodiesterase 3A Hyperactivity on the Heart Using Single Nuclei Sequencing and Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Mutations causing hyperactivity of phosphodiesterase 3A (PDE3A) lead to autosomal dominant hypertension with brachydactyly type E. Affected patients suffer from profoundly increased blood pressure and, if untreated, die from stroke at the age of 50 years. Surprisingly, the hearts of affected individuals show little signs of hypertrophic responses to these conditions, indicating a beneficial effect of PDE3A hyperactivity on the damaging mechanisms of the heart. PDE3A inhibition was previously tested as a treatment for heart failure. While PDE3A inhibitors lead to short term positive inotropy, treatment with these drugs decreases overall survival of heart failure patients, underlining the importance of PDE3A in the heart. As arterial hypertension is a major risk factor for cardiovascular disease and proves to be a major burden on the healthcare system, dissecting the mechanisms underlying the protective effect of PDE3A can open new paths to preventing and treating heart failure. We hypothesize that PDE3A activation protects the heart from the detrimental effects of high blood pressure via spatially distinct regulation of cAMP levels through its different isoforms, leading to an altered calcium handling of cardiomyocytes. We are focusing on two mutations causing PDE3A hyperactivity, one in a regulatory hotspot the other in the catalytic domain. Using single nuclei sequencing of left ventricles of rats, genetically engineered to recapitulate the human mutations, we are dissecting the differences in the cell type-specific gene regulatory response to high blood pressure compared to the wildtype. To follow up in a human model system, we used genome editing to introduce the mutations of interest into human induced pluripotent stem cells (hiPSC). hiPSC lines containing PDE3A mutations as well as isogenic matched control hiPSCs were differentiated to generate hiPSC-derived cardiomyocytes (hiPSC-CMs). Preliminary data from a well-established 2D system show significantly altered calcium handling of PDE3A-mutant hiPSC-CMs. Performing RNA-sequencing, calcium imaging and cAMP measurements in isogenic hiPSC-CMs, we are functionally characterizing the effect of PDE3A activity on cardiomyocyte profile and physiology to a high resolution.

Non‐negative blind deconvolution for signal processing in a CRISPR‐edited iPSC‐cardiomyocyte model of dilated cardiomyopathy

We developed an integrated platform for analysis of parameterized data from human disease models. We report a non-negative blind deconvolution (NNBD) approach to quantify calcium (Ca2+ ) handling, beating force and contractility in human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) at the single-cell level. We employed CRISPR/Cas gene editing to introduce a dilated cardiomyopathy (DCM)-causing mutation in troponin T (TnT), TnT-R141W, into wild-type control iPSCs (MUT). The NNDB-based method enabled data parametrization, fitting and analysis in wild-type controls versus isogenic MUT iPSC-CMs. Of note, Cas9-edited TnT-R141W iPSC-CMs revealed significantly reduced beating force and prolonged contractile event duration. The NNBD-based platform provides an alternative framework for improved quantitation of molecular disease phenotypes and may contribute to the development of novel diagnostic tools.

Association of coronary microvascular dysfunction and cardiac bridge integrator 1, a cardiomyocyte dysfunction biomarker.

BackgroundCoronary microvascular dysfunction (CMD) is associated with heart failure with preserved ejection fraction (HFpEF); however, pathophysiology is not well described.HypothesisWe hypothesized that CMD in women with suspected ischemia with no obstructive coronary artery disease (INOCA) is associated with cardiomyocyte dysfunction reflected by plasma levels of a cardiomyocyte calcium handling protein, cardiac bridge integrator 1 (cBIN1).MethodsWomen with suspected INOCA undergoing coronary function testing were included. Coronary flow reserve, vasodilation to nitroglycerin, change in coronary blood flow (ΔCBF), and vasodilation to acetylcholine (ΔAch) were evaluated. cBIN1 score (CS) levels in these women (n = 39) were compared to women with HFpEF (n = 20), heart failure with reduced ejection fraction (HFrEF) (n = 36), and reference controls (RC) (n = 50). Higher CS indicates cardiomyocyte tubule dysfunction.ResultsINOCA, HFpEF, and HFrEF women were older than RC (p < .05). Higher CS was associated with vasoconstriction to acetylcholine (r = −0.43, p = .011) with a trend towards lower ΔCBF (r = 0.30, p = .086). Higher CS was specific for ΔAch and ΔCBF but had limited sensitivity. INOCA women had higher CS than RC, but lower CS than HFpEF/HFrEF groups (p < .001).ConclusionsCS, a plasma biomarker indicating poor cardiomyocyte health, was higher in women with suspected INOCA as compared to RC, but lower than in women with HFpEF. Elevated CS in suspected INOCA patients represents an intermediate group between health and disease, supporting the hypothesis that CMD may progress to HFpEF. Larger prospective cohort studies are needed to confirm the pathophysiological relationship between cBIN1, CMD, and HFpEF.

Influence of miR-221/222 on cardiomyocyte calcium handling and function

BackgroundCardiovascular disease is the leading cause of death worldwide. Cardiac electrical remodeling including altered ion channel expression and imbalance of calcium homeostasis can have detrimental effects on cardiac function. While it has been extensively reported that miR-221/222 are involved in structural remodeling, their role in electrical remodeling still has to be evaluated. We previously reported that subunits of the L-type Ca2+ channel (LTCC) are direct targets of miR-221/222. Furthermore, HL-1 cells transfected with miR-221 or -222 mimics showed a reduction in LTCC current density while the voltage-dependence of activation was not altered. The aim of the present study was to determine the influence of miR-221/222 on cardiomyocyte calcium handling and function.ResultsTransient transfection of HL-1 cells with miR-221/222 mimics led to slower depolarization-dependent Ca2+ entry and increased proportion of non-responding cells. Angiotensin II-induced Ca2+ release from the SR was not affected by miR-221/222. In miR-222-transfected neonatal cardiomyocytes the isoprenaline-induced positive inotropic effect on the intracellular Ca2+ transient was lost and the positive chronotropic effect on spontaneous beating activity was strongly reduced. This could have severe consequences for cardiomyocytes and could lead to a reduced contractility and systolic dysfunction of the whole heart.ConclusionsThis study adds a new role of miR-221/222 in cardiomyocytes by showing the impact on β-adrenergic regulation of LTCC function, calcium handling and beating frequency. Together with the previous report that miR-221/222 reduce GIRK1/4 function and LTCC current density, it expands our knowledge about the role of these miRs on cardiac ion channel regulation.

Inhibition of myocardial cathepsin-L release during reperfusion following myocardial infarction improves cardiac function and reduces infarct size.

AimsIdentifying novel mediators of lethal myocardial reperfusion injury that can be targeted during primary percutaneous coronary intervention (PPCI) is key to limiting the progression of patients with ST-elevation myocardial infarction (STEMI) to heart failure. Here, we show through parallel clinical and integrative preclinical studies the significance of the protease cathepsin-L on cardiac function during reperfusion injury.Methods and resultsWe found that direct cardiac release of cathepsin-L in STEMI patients (n = 76) immediately post-PPCI leads to elevated serum cathepsin-L levels and that serum levels of cathepsin-L in the first 24 h post-reperfusion are associated with reduced cardiac contractile function and increased infarct size. Preclinical studies demonstrate that inhibition of cathepsin-L release following reperfusion injury with CAA0225 reduces infarct size and improves cardiac contractile function by limiting abnormal cardiomyocyte calcium handling and apoptosis.ConclusionOur findings suggest that cathepsin-L is a novel therapeutic target that could be exploited clinically to counteract the deleterious effects of acute reperfusion injury after an acute STEMI.

Exploring Preoperative Hypocalcemia as a Biomarker for Post Left Ventricular Assist Device Survival and Adverse Events

<h3>Purpose</h3> Abnormal calcium handling in cardiomyocytes plays a cardinal role in the initiation and progression of heart failure. However, the effect of serum calcium levels has not been studied in chronic heart failure patients undergoing left ventricular assist device (LVAD) placement. We investigate the impact of preoperative hypocalcemia on adverse events and all-cause mortality in this population over a 24-month period post durable LVAD implantation. <h3>Methods</h3> At a single center, 177 patients received LVAD therapy between June 2011 and October 2018. Patients were classified into either hypocalcemia [(serum calcium < 8.7 mg/dL) n=40] or normocalcemia [(8.7- 10.2 mg/dL) n=108] based on preoperative levels. 29 patients were excluded due to missing data. Unadjusted and adjusted cox models were used to examine for association between hypocalcemia and 2-year all-cause mortality. Survival was compared using Kaplan-Meir plot and log-rank statistics. <h3>Results</h3> The majority of patients in both groups were undergoing LVAD placement for destination therapy (77 vs. 73%, p=0.009) and had a similar history of chronic kidney disease across groups (21 vs. 28%, p=0.36). However, there was a significantly greater number of Caucasian patients in the hypocalcemia group (85 vs. 63%, =0.004). 24-month survival was equal across groups (Log-Rank p=0.13). Likewise on Cox-modelling, there was no difference in survival seen on either the unadjusted [HR= 1.42 (0.89 - 2.27) (p=0.14)] or adjusted survival [1.39 (0.86 - 2.24) (p=0.18)] when adjusted for age, gender, INTERMACS category, and eGFR. Perioperative renal failure and need for hemodialysis were similar between groups. However, the hypocalcemia group required prolonged intubation (46 vs 28%, p=0.03), tracheostomy (28 vs 11%, p=0.001) and increased right ventricular assist device use (23 vs 8%, p=0.02). <h3>Conclusion</h3> Despite no significant impact on survival at 24-months, hypocalcemia may be a poor prognostic marker for some adverse events.

Calcium Dobesilate (CaD) Attenuates High Glucose and High Lipid-Induced Impairment of Sarcoplasmic Reticulum Calcium Handling in Cardiomyocytes.

Calcium dobesilate (CaD) is used effectively in patients with diabetic microvascular disorder, retinopathy, and nephropathy. Here we sought to determine whether it has an effect on cardiomyocytes calcium mishandling that is characteristic of diabetic cardiomyopathy. Cardiomyocytes were sterile isolated and cultured from 1 to 3 days neonatal rats and treated with vehicle (Control), 25 mM glucose+300 μM Palmitic acid (HG+PA), 100 μM CaD (CaD), or HG+PA+CaD to test the effects on calcium signaling (Ca2+ sparks, transients, and SR loads) and reactive oxygen species (ROS) production by confocal imaging. Compared to Control, HG+PA treatment significantly reduced field stimulation-induced calcium transient amplitudes (2.22 ± 0.19 vs. 3.56 ± 0.21, p < 0.01) and the levels of caffeine-induced calcium transients (3.19 ± 0.14 vs. 3.72 ± 0.15, p < 0.01), however significantly increased spontaneous Ca2+ sparks firing levels in single cardiomyocytes (spontaneous frequency 2.65 ± 0.23 vs. 1.72 ± 0.12, p < 0.01) and ROS production (67.12 ± 4.4 vs. 47.65 ± 2.12, p < 0.05), which suggest that HG+PA treatment increases the Spontaneity Ca2+ spark frequency, and then induced partial reduction of SR Ca2+ content and subsequently weaken systolic Ca2+ transient in cardiomyocyte. Remarkably, these impairments in calcium signaling and ROS production were largely prevented by pre-treatment of the cells with CaD. Therefore, CaD may contribute to a good protective effect on patients with calcium mishandling and contractile dysfunction in cardiomyocytes associated with diabetic cardiomyopathy.