Cardiomyocyte Relaxation Research Articles

Human induced pluripotent stem cell-derived cardiomyocytes to study inflammation-induced aberrant calcium transient.

Heart failure with preserved ejection fraction (HFpEF) is commonly found in persons living with HIV (PLWH) even when antiretroviral therapy suppresses HIV viremia. However, studying this condition has been challenging because an appropriate animal model is not available. In this article, we studied calcium transient in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in culture to simulate the cardiomyocyte relaxation defect noted in PLWH and HFpEF and assess whether various drugs have an effect. We show that treatment of hiPSC-CMs with inflammatory cytokines (such as interferon-γ or TNF-α) impairs their Ca2+ uptake into sarcoplasmic reticulum and that SGLT2 inhibitors, clinically proven as effective for HFpEF, reverse this effect. Additionally, treatment with mitochondrial antioxidants (like mito-Tempo) and certain antiretrovirals resulted in the reversal of the effects of these cytokines on calcium transient. Finally, incubation of hiPSC-CMs with serum from HIV patients with and without diastolic dysfunction did not alter their Ca2+-decay time, indicating that the exposure to the serum of these patients is not sufficient to induce the decrease in Ca2+ uptake in vitro. Together, our results indicate that hiPSC-CMs can be used as a model to study molecular mechanisms of inflammation-mediated abnormal cardiomyocyte relaxation and screen for potential new interventions.

Genetic excision of the regulatory cardiac troponin I extension in high-heart rate mammal clades.

Mammalian cardiac troponin I (cTnI) contains a highly conserved amino-terminal extension harboring protein kinase A targets [serine-23 and -24 (Ser23/24)] that are phosphorylated during β-adrenergic stimulation to defend diastolic filling by means of an increased cardiomyocyte relaxation rate. In this work, we show that the Ser23/24-encoding exon 3 of TNNI3 was pseudoexonized multiple times in shrews and moles to mimic Ser23/24 phosphorylation without adrenergic stimulation, facilitating the evolution of exceptionally high resting heart rates (~1000 beats per minute). We further reveal alternative exon 3 splicing in distantly related bat families and confirm that both cTnI splice variants are incorporated into cardiac myofibrils. Because exon 3 of human TNNI3 exhibits a relatively low splice strength score, our findings offer an evolutionarily informed strategy to excise this exon to improve diastolic function during heart failure.

Hsa-miR-548v controls the viscoelastic properties of human cardiomyocytes and improves their relaxation rates.

The impairment of left ventricular (LV) diastolic function with an inadequate increase in myocardial relaxation velocity directly results in lower LV compliance, increased LV filling pressures, and heart failure symptoms. The development of agents facilitating the relaxation of human cardiomyocytes requires a better understanding of the underlying regulatory mechanisms. We performed a high-content microscopy-based screening in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) using a library of 2,565 human miRNA mimics and measured relaxation kinetics via high-computing analyses of motion movies. We identified hsa-miR-548v, a primate-specific miRNA, as the miRNA producing the largest increase in relaxation velocities. This positive lusitropic effect was reproduced in engineered cardiac tissues generated with healthy and BRAF T599R mutant hiPSC-CMs and was independent of changes in calcium transients. Consistent with improvements in viscoelastic responses to mechanical stretch, RNA-Seq showed that hsa-miR-548v downregulated multiple targets, especially components of the mechanosensing machinery. The exogenous administration of hsa-miR-548v in hiPSC-CMs notably resulted in a significant reduction of ANKRD1/CARP1 expression and localization at the sarcomeric I-band. This study suggests that the sarcomere I-band is a critical control center regulating the ability of cardiomyocytes to relax and is a target for improving relaxation and diastolic dysfunction.

Novel endothelial-enriched long non-coding RNA AL590004.3 (IRENE) is dysregulated in HFpEF and regulates endothelial enhancement of cardiomyocyte function

Abstract Background Heart failure with preserved ejection fraction (HFpEF) is the most prevalent form of HF to date. However, there is a lack of medication which stages HFpEF as an unmet clinical need. HFpEF is characterized by cardiac microvascular dysfunction, promoted by co-morbidities-associated low-grade pro-inflammatory state. We have shown that cardiac endothelial cells regulate cardiomyocyte relaxation, which is impaired when endothelial cells are inflamed. Long non-coding RNAs (lncRNAs) have been implicated in various cardiovascular diseases. However, their role in HFpEF is unknown. Here, we discovered a novel lncRNA AL590004.3 which is significantly reduced in the heart of patients with HFpEF. We hypothesized that AL590004.3 is important for cardiac endothelial function, and dysregulation of endothelial AL590004.3 impairs cardiomyocyte relaxation. Methods and results We demonstrated that AL590004.3 is significantly downregulated (∼7 fold, p<0.001) in HFpEF as assessed by in silico analysis of RNA sequencing of HFpEF patient cardiac biopsies. To assess the tissue distribution, we performed RNA sequencing comparing cells from various tissues and found the highest expression of AL590004.3 in the cardiac microvascular endothelial cells (CMECs), indicating its importance in cardiac endothelial cell biology. The expression of AL590004.3 was reduced when CMECs were exposed to pro-inflammatory cytokine TNFα and IL1β, indicating the detrimental effect of inflammation, as displayed by patients with HFpEF. Further, replicative senescent of CMECs led to reduced AL590004.3 expression (2 fold, p<0.005), showing the deleterious contribution of ageing, similar to what occurs in HFpEF patients. We showed that AL590004.3 is mostly expressed in the chromatin fraction (∼90%) of CMECs, suggesting its function in chromatin modification. To assess the function of AL590004.3, we performed loss-of-function. Using endothelial-cardiomyocyte co-culture set-up, we showed silencing of AL590004.3 in CMECs impaired endothelial function to enhance cardiomyocyte relaxation (∼1.5 fold, p<0.01). To dissect the molecular mechanism, we performed RNA sequencing of CMECs devoid of AL590004.3 and revealed four protein-coding genes (PXDC1, FAM50, ECI2, PRPF4B) that are differentially co-regulated, indicating their potential as protein-binding partners, which warrant further investigation. Conclusion We showed that AL590004.3 is a cardiac endothelial cell-specific lncRNA that is significantly downregulated in cardiac biopsy from HFpEF patients. AL590004.3 is important for cardiac endothelial cell function as loss-of-function in CMECs impaired endothelial enhancement of cardiomyocyte relaxation. Herewith, we name this lncRNA Important for the Regulation of ENdothelial function in HFpEF (IRENE).

EGFR/IGF1R Signaling Modulates Relaxation in Hypertrophic Cardiomyopathy.

Diastolic dysfunction is central to diseases such as heart failure with preserved ejection fraction and hypertrophic cardiomyopathy (HCM). However, therapies that improve cardiac relaxation are scarce, partly due to a limited understanding of modulators of cardiomyocyte relaxation. We hypothesized that cardiac relaxation is regulated by multiple unidentified proteins and that dysregulation of kinases contributes to impaired relaxation in patients with HCM. We optimized and increased the throughput of unloaded shortening measurements and screened a kinase inhibitor library in isolated adult cardiomyocytes from wild-type mice. One hundred fifty-seven kinase inhibitors were screened. To assess which kinases are dysregulated in patients with HCM and could contribute to impaired relaxation, we performed a tyrosine and global phosphoproteomics screen and integrative inferred kinase activity analysis using HCM patient myocardium. Identified hits from these 2 data sets were validated in cardiomyocytes from a homozygous MYBPC3c.2373insG HCM mouse model. Screening of 157 kinase inhibitors in wild-type (n=33) cardiomyocytes (n=24 563) resulted in the identification of 17 positive inotropes and 21 positive lusitropes, almost all of them novel. The positive lusitropes formed 3 clusters: cell cycle, EGFR (epidermal growth factor receptor)/IGF1R (insulin-like growth factor 1 receptor), and a small Akt (α-serine/threonine protein kinase) signaling cluster. By performing phosphoproteomic profiling of HCM patient myocardium (n=24 HCM and n=8 donors), we demonstrated increased activation of 6 of 8 proteins from the EGFR/IGFR1 cluster in HCM. We validated compounds from this cluster in mouse HCM (n=12) cardiomyocytes (n=2023). Three compounds from this cluster were able to improve relaxation in HCM cardiomyocytes. We showed the feasibility of screening for functional modulators of cardiomyocyte relaxation and contraction, parameters that we observed to be modulated by kinases involved in EGFR/IGF1R, Akt, cell cycle signaling, and FoxO (forkhead box class O) signaling, respectively. Integrating the screening data with phosphoproteomics analysis in HCM patient tissue indicated that inhibition of EGFR/IGF1R signaling is a promising target for treating impaired relaxation in HCM.

Abstract P2078: Vasohibin Inhibition Improves Myocardial Relaxation In A Rodent Model Of Heart Failure With Preserved Ejection Fraction

Heart failure with preserved ejection (HFpEF) is a complex syndrome with few therapies available to patients. Diastolic dysfunction is a common clinical feature among HFpEF patients, and strategies to mitigate impaired relaxation may have a therapeutic benefit. Recognizing that increased stabilization of the microtubule network induced by detyrosination of α-tubulin contributes to increased cardiomyocyte stiffness ex vivo , we explored if inhibition of detyrosination with a small molecule vasohibin inhibitor (VASHi) could improve in vivo diastolic function in obese/hypertensive (ob) ZSF1 rats, compared to lean/hypertensive ZSF1 and non-hypertensive Wistar Kyoto (Wky) rats as controls. Animals received two intravenous injections of VASHi (6.6mg/kg) or vehicle (veh) at 30 weeks old, when ob have a HFpEF phenotype. Echocardiography and invasive hemodynamics were performed after the first and second injection, respectively. Compared to ob veh-treated animals, ob VASHi-treated animals had a significant improvement in invasively measured time constant of early LV relaxation, tau (veh: 14.73ms ± 0.73 vs. VASHi: 11.02ms ± 0.45; P < 0.05) and echo-derived deceleration time of early LV filling, indicative of improved early relaxation (Fig. A, B). VASHi improved early relaxation in cardiomyocytes isolated from non-failing (NF, N=3) and failing (HF, N=4) human hearts (Fig. C), providing the first evidence of small molecule VASHi improving human myocyte function. These studies indicate that excess detyrosination contributes to impaired early myocyte and myocardial relaxation in a clinically relevant HFpEF model and supports a potential therapeutic role for VASHi in HFpEF.

Cardiac contraction and relaxation are regulated by distinct subcellular cAMP pools

Cells interpret a variety of signals through G-protein-coupled receptors (GPCRs) and stimulate the generation of second messengers such as cyclic adenosine monophosphate (cAMP). A long-standing puzzle is deciphering how GPCRs elicit different physiological responses despite generating similar levels of cAMP. We previously showed that some GPCRs generate cAMP from both the plasma membrane and the Golgi apparatus. Here we demonstrate that cardiomyocytes distinguish between subcellular cAMP inputs to elicit different physiological outputs. We show that generating cAMP from the Golgi leads to the regulation of a specific protein kinase A (PKA) target that increases the rate of cardiomyocyte relaxation. In contrast, cAMP generation from the plasma membrane activates a different PKA target that increases contractile force. We further validated the physiological consequences of these observations in intact zebrafish and mice. Thus, we demonstrate that the same GPCR acting through the same second messenger regulates cardiac contraction and relaxation dependent on its subcellular location.

Machine learning-based classification of cardiac relaxation impairment using sarcomere length and intracellular calcium transients

Impaired relaxation of cardiomyocytes leads to diastolic dysfunction in the left ventricle. Relaxation velocity is regulated in part by intracellular calcium (Ca2+) cycling, and slower outflux of Ca2+ during diastole translates to reduced relaxation velocity of sarcomeres. Sarcomere length transient and intracellular calcium kinetics are integral parts of characterizing the relaxation behavior of the myocardium. However, a classifier tool that can separate normal cells from cells with impaired relaxation using sarcomere length transient and/or calcium kinetics remains to be developed. In this work, we employed nine different classifiers to classify normal and impaired cells, using ex-vivo measurements of sarcomere kinematics and intracellular calcium kinetics data. The cells were isolated from wild-type mice (referred to as normal) and transgenic mice expressing impaired left ventricular relaxation (referred to as impaired). We utilized sarcomere length transient data with a total of n = 126 cells (n = 60 normal cells and n = 66 impaired cells) and intracellular calcium cycling measurements with a total of n = 116 cells (n = 57 normal cells and n = 59 impaired cells) from normal and impaired cardiomyocytes as inputs to machine learning (ML) models for classification. We trained all ML classifiers with cross-validation method separately using both sets of input features, and compared their performance metrics. The performance of classifiers on test data showed that our soft voting classifier outperformed all other individual classifiers on both sets of input features, with 0.94 and 0.95 area under the receiver operating characteristic curves for sarcomere length transient and calcium transient, respectively, while multilayer perceptron achieved comparable scores of 0.93 and 0.95, respectively. However, the performance of decision tree, and extreme gradient boosting was found to be dependent on the set of input features used for training. Our findings highlight the importance of selecting appropriate input features and classifiers for the accurate classification of normal and impaired cells. Layer-wise relevance propagation (LRP) analysis demonstrated that the time to 50% contraction of the sarcomere had the highest relevance score for sarcomere length transient, whereas time to 50% decay of calcium had the highest relevance score for calcium transient input features. Despite the limited dataset, our study demonstrated satisfactory accuracy, suggesting that the algorithm can be used to classify relaxation behavior in cardiomyocytes when the potential relaxation impairment of the cells is unknown.

Evaluating the efficacy and cardiotoxicity of EGFR-TKI AC0010 with a novel multifunctional biosensor

Non-small cell lung cancer (NSCLC) is a leading cause of cancer mortality worldwide. Although epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have dramatically improved the life expectancy of patients with NSCLC, concerns about TKI-induced cardiotoxicities have increased. AC0010, a novel third-generation TKI, was developed to overcome drug resistance induced by EGFR-T790M mutation. However, the cardiotoxicity of AC0010 remains unclear. To evaluate the efficacy and cardiotoxicity of AC0010, we designed a novel multifunctional biosensor by integrating microelectrodes (MEs) and interdigital electrodes (IDEs) to comprehensively evaluate cell viability, electrophysiological activity, and morphological changes (beating of cardiomyocytes). The multifunctional biosensor can monitor AC0010-induced NSCLC inhibition and cardiotoxicity in a quantitative, label-free, noninvasive, and real-time manner. AC0010 was found to significantly inhibit NCI-H1975 (EGFR-L858R/T790M mutation), while weak inhibition was found for A549 (wild-type EGFR). Negligible inhibition was found in the viabilities of HFF-1 (normal fibroblasts) and cardiomyocytes. With the multifunctional biosensor, we found that 10 μM AC0010 significantly affected the extracellular field potential (EFP) and mechanical beating of cardiomyocytes. The amplitude of EFP continuously decreased after AC0010 treatment, while the interval decreased first and then increased. We analyzed the change in the systole time (ST) and diastole time (DT) within a beating interval and found that the DT and DT/beating interval rate decreased within 1 h after AC0010 treatment. This result probably indicated that the relaxation of cardiomyocytes was insufficient, which may further aggravate the dysfunction. Here, we found that AC0010 significantly inhibited EGFR-mutant NSCLC cells and impaired cardiomyocyte function at low concentrations (10 μM). This is the first study in which the risk of AC0010-induced cardiotoxicity was evaluated. In addition, novel multifunctional biosensors can comprehensively evaluate the antitumor efficacy and cardiotoxicity of drugs and candidate compounds.

Diastolic heart failure: 20 years later. Сurrent issues of pathoge-nesis, diagnosis and treatment of heart failure with preserved LVEF

Relevant aspects of the pathogenesis, diagnosis, And treatment of heart failure with preserved LV EFThis review analyzes results of studies of the recent decade that focus on epidemiology, mechanisms of development, diagnostic methods, and treatments of heart failure with preserved ejection fraction (HFpEF). As expected, the prevalence of HFpEF continues to increase due to the growing contribution of comorbidities to the structure of causes for chronic heart failure (CHF), such as arterial hypertension with left ventricular hypertrophy, obesity, chronic kidney disease, as well as due to ageing of the population and decreased contributions of ischemic heart disease and myocardial infarction. Concomitant diseases are a source of low-intensity microvascular inflammation, which is currently assigned a role of a trigger mechanism eventually provoking energy deficiency, disorders of cardiomyocyte relaxation, and diffuse myocardial fibrosis. Both these processes lead to increased heart muscle rigidity and abnormally high left ventricular filling pressure (LVFP). High LVFP is associated with the development of pulmonary venous congestion and impairment of alveolar blood oxygenation, which form the clinical picture of HFpEF. Detecting high LVEF with tissue Doppler echocardiography by the E / e' value became the instrumental basis for the HFpEF diagnostics. Recognition of inflammation and fibrosis as the key pathogenetic factors marked the main vector of modern therapy for HFpEF (anti-inflammatory and antifibrotic). The best implementation of this vector became possible with the advent of drugs from the class of angiotensin receptor and neprilysin inhibitors (ARNI), sodium-glucose cotransporter type 2 (SGLT2) inhibitors, and aldosterone antagonists. However, the efficacy of such treatments is evident only with the LV EF <60-65% while at higher values, the efficacy substantially decreases. This limitation may result from the heterogenous nature of the disease and requires more advanced methods for verification of HFpEF clinical phenotypes. Among such methods, transcriptomic, metabolomic, and proteomic approaches are considered. With the use of capabilities of the "machine learning" and the artificial intelligence, these approaches can become a new frontier in research to represent an important step towards personalized medicine for patients with HFpEF.

Engineered cardiac tissue model of restrictive cardiomyopathy for drug discovery

Restrictive cardiomyopathy (RCM) is defined as increased myocardial stiffness and impaired diastolic relaxation leading to elevated ventricular filling pressures. Human variants in filamin C (FLNC) are linked to a variety of cardiomyopathies, and in this study, we investigate an in-frame deletion (c.7416_7418delGAA, p.Glu2472_Asn2473delinAsp) in a patient with RCM. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) with this variant display impaired relaxation and reduced calcium kinetics in 2D culture when compared with a CRISPR-Cas9-corrected isogenic control line. Similarly, mutant engineered cardiac tissues (ECTs) demonstrate increased passive tension and impaired relaxation velocity compared with isogenic controls. High-throughput small-molecule screening identifies phosphodiesterase 3 (PDE3) inhibition by trequinsin as a potential therapy to improve cardiomyocyte relaxation in this genotype. Together, these data demonstrate an engineered cardiac tissue model of RCM and establish the translational potential of this precision medicine approach to identify therapeutics targeting myocardial relaxation.

A visual detection method of cardiomyocyte relaxation and contraction

Contraction and relaxation are important functions of cardiomyocytes, and measuring their characteristics provides an evaluation index to explore the effects of drugs on cardiomyocytes. In addition, cardiomyocytes have an innate advantage in acting as a biopower by virtue of their ability to contract and relax, which also requires the detection of cardiomyocyte actions. However, existing measurement methods, such as mechanosensor measurements and calcium concentration measurements, have high requirements for experimental equipment and operation and are challenging to perform simultaneously with other cellular manipulations. Here, we propose a simple visual detection method for cardiomyocyte contraction and relaxation. We first recorded the contraction and relaxation of cardiomyocytes under a bright-field microscope, then used the optical flow method to track the sampling points on the cardiomyocytes in the video, and obtained the frequency of cardiomyocyte contraction and relaxation by analyzing the optical flow matrices. This method does not require the use of additional equipment or additional processing of cardiomyocytes, which significantly reduces the operational difficulty of detection and provides a method to achieve real-time detection of cardiomyocyte contraction and relaxation.

Thyroid hormone disorder and the heart: The role of cardiolipin in calcium handling.

What is the central question of this study? Do alterations in thyroid status affect haemodynamic parameters and echocardiographic measurements in the rat postnatal heart, and calcium handling, contractility, relaxation and cardiolipin content in isolated rat cardiomyocytes? What is the main finding and its importance? An imbalance in phospholipids of the mitochondrial membrane such as cardiolipin is related to defects in mitochondrial function. T3 -dependent cardiolipin signals contribute to the maintenance of mitochondrial homeostasis and involve Ca2+ handling, this pathway being more important in hypothyroidism. The objective of this study was to evaluate whether alterations in thyroid status affect (1) haemodynamic parameters and echocardiographic measurements in the rat postnatal heart, and (2) calcium handling, contractility, relaxation and cardiolipin content in isolated rat cardiomyocytes. Sprague-Dawley rats aged 2months treated with T3 (hyperthyroid, 20μg/100g body weight) or 0.02% methimazole (hypothyroid, w/v) for 28 days. Heart function was evaluated by echocardiography. Measurements of mean arterial pressure (MAP), heart rate, Ca2+ transients, cardiomyocyte shortening, number of spontaneous contractions per minute and cardiolipin (CL) content were performed. Thyroid disorders were associated with changes in pacemaker activity without modifications of MAP. Thyroid disorder induced changes in left ventricular diameter which were correlated with modifications of cardiac contractility (altered cell shortening and sarcoplasmic reticulum Ca2+ content). Endocrine disorders altered cardiomyocyte relaxation (reduction in the time to 50% re-lengthening and the time to 50% Ca2+ decay). Thyroid disorder increased the number of spontaneous contractions per minute (an index of pro-arrhythmogenic behaviour). CL content was increased only in hypothyroid rats. Changes in CL content, CL composition and CL-protein interaction in mitochondria from hypothyroid animals are responsible for alterations of contractile and relaxation cardiac function. This mechanism may be not be involved in T3 -treated rats. Maintenance of euthyroidism is of crucial importance to preserve cardiac performance. An imbalance in relation to phospholipids of the mitochondrial membrane such as CL is related to defects in mitochondrial function. T3 -dependent CL signals contribute to the maintenance of mitochondrial homeostasis and involve Ca2+ handling, this pathway being more important in hypothyroidism.

Abstract 10925: A Small Molecule Inhibitor of Sarcomere Contractility Acutely Relieves Prolonged Relaxation in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes With Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is one of the most common genetic cardiac diseases primarily caused by mutations in sarcomeric genes. However, HCM is also associated with mutations in non-sarcomeric proteins and a Finnish founder mutation for HCM in non-sarcomeric protein junctophilin-2 (JPH2) has been identified. MYK-461 is a recently described mechanistically novel small molecule that acts at the sarcomere to specifically inhibit contractility that has been proposed as a treatment for HCM. Here, we use MYK-461 to test whether direct reduction in contractility is sufficient to relieve phenotypic characteristics in human-induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) carrying JPH2 mutation T161K. Hypothesis: Currently all HCM patients are treated the same regardless of the underlying genetic defect. Current medication does not prevent or stop the thickening of the heart muscle; only the symptoms and arrhythmias can be controlled with beta-blockers or calcium blockers. MYK-461 decreases adenosine triphosphatase activity of the cardiac myosin heavy chain to reduce contractility, thus modifying the characteristic pathology of HCM. Methods: Skin fibroblasts from a Finnish patient with HCM causing JPH2 p.(Thr161Lys) were reprogrammed into hiPSCs and further differentiated into CMs. As a control line, an isogenic counterpart was generated using the CRISPR/Cas9 genome editing method. We evaluated the effect of MYK-461 treatment to T161K-hiPSC-CMs by their functional characteristics associated with JPH2 mutation. Results: By video microscopy, we identified increased contractility (BPM; 41±6 SEM, n=9) and prolonged diastolic phase (ms; 394.8±29.7 SEM, n=9) in T161K-hiPSC-CMs. Treatment with MYK-461 reduced contractility (BPM; 36±3 SEM, n=10) and relaxation phase (ms; 293.1±33.6 SEM, n=10) in a dose-dependent manner, displaying functional characteristics closer to isogenic-hiPSC-CMs control group. Conclusions: In conclusion, the results demonstrate that acute reduction in contractility is sufficient to relieve prolonged relaxation. Further, these studies suggest that T161K-hiPSC-CMs provide adequate in vitro platform for modelling the functional effect of MYK-461.

Abstract 15247: A Novel Patient-Derived Stem Cell Model of Restrictive Cardiomyopathy Exhibits Impaired Cardiomyocyte Relaxation Secondary to Increased Myocardial Calcium Sensitivity

Introduction: Restrictive Cardiomyopathy (RCM) is a rare disease characterized by profound, isolated defects in myocardial relaxation, normal or near-normal myocardial wall thickness, and preserved contractile function. Outcomes for patients with RCM are poor, with increased morbidity and mortality compared to other forms of cardiomyopathy. In some patients, RCM can be caused by genetic mutations, notably in genes encoding for components of the sarcomere. While many mutations have been described in RCM patients, the molecular and cellular mechanisms leading from these mutations to the patient’s physiological phenotype are poorly described. Hypothesis: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) derived from RCM patients will recapitulate the myocardial relaxation defects seen in RCM and provide a novel in vitro model for investigating the molecular mechanisms underpinning this disease. Methods: Induced pluripotent stem cells (iPSCs) were derived from a pediatric patient with RCM caused by a TNNT2 R94C mutation. CRISPR was used to correct the mutation, and cellular mechanics and calcium handling properties were compared to the patient line using a novel high-throughput traction-force imaging platform. Transcriptional profiles were compared using RNA-seq. Results: RCM cells carrying the TNNT2 R94C mutation show increased calcium sensitivity leading to increased resting tension at baseline, as well as prolonged calcium kinetics leading to prolonged relaxation times. Gene expression profiling reveals changes in expression of ECM genes, which may further contribute to the impaired myocardial relaxation seen in RCM. Conclusions: Patient-derived hiPSC-CMs from an RCM patient recapitulate the relaxation defects seen in RCM and highlight the central role of increased calcium sensitivity in impaired myocardial relaxation.

Dysregulation of cardiac endothelial lncRNA H19 in COVID19 patients induces endothelial dysfunction, which impairs cardiomyocyte function

Abstract Background COVID19 is accompanied by cardiac complications. Long non-coding RNAs (lncRNAs) have been implicated in the pathogenesis of cardiovascular diseases. However, their contribution to the cardiac manifestation of COVID19 is unknown. Methods and results We discovered that endothelial-enriched lncRNA H19 is downregulated in the heart of patients with COVID19 (∼2 fold, p<0.01). H19 was highly expressed in cardiac microvascular endothelial cells (CMEC) as compared to the other endothelial cell types (∼10 fold, p<0.05), suggesting its cardiac enrichment. H19 silencing in CMEC induced endothelial stress phenotype and a reduction in endothelial markers VE-cadherin and eNOS (∼1.5 fold, p<0.01), indicating its importance in endothelial physiology. Using the endothelial-cardiomyocyte co-culture system we previously developed, we showed that H19 silencing in CMEC reduced cardiomyocyte (CM) relaxation and contraction (∼1.5 fold, p<0.01). Interestingly, exposure to plasma from COVID19 patients decreased endothelial H19 level and impaired endothelial enhancement of CM function. Mechanistically, reduced level of H19 increased endothelial IL6 expression (∼1.5 fold, p<0.01). Further, exposure of CMs to IL6 also impaired CM relaxation and contraction, suggesting that endothelial cells devoid of H19 release IL6 which represses CM function. Interestingly, we found increased IL6 levels in the heart of COVID19 patients (∼2 fold, p<0.05). Indeed, the impairment of endothelial enhancement of CM function upon H19 silencing in CMEC was restored in the presence of tocilizumab, an IL6 receptor antagonist (∼1.5 fold, p<0.01). Furthermore, the impairment of the endothelial control on CM function upon exposure to COVID19 plasma was mitigated when the patients were treated with tocilizumab (∼1.5 fold, p<0.01). Conclusion COVID19 reduces cardiac endothelial H19 level and induces impairment of endothelial enhancement of CM function via increased release of endothelial-derived IL6, the effect that can be rescued in the presence of IL6 receptor blocker tocilizumab. Funding Acknowledgement Type of funding sources: Public grant(s) – EU funding. Main funding source(s): ERC

Low expression of the K280N TNNT2 mutation is sufficient to increase basal myofilament activation in human hypertrophy cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is an autosomal dominant genetic disorder with patients typically showing heterozygous inheritance of a pathogenic variant in a gene encoding a contractile protein. Here, we study the contractile effects of a rare homozygous mutation using explanted tissue and human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to gain insight into how the balance between mutant and WT protein expression affects cardiomyocyte function. Force measurements were performed in cardiomyocytes isolated from a HCM patient carrying a homozygous troponin T mutation (cTnT-K280N) and healthy donors. To discriminate between mutation-mediated and phosphorylation-related effects on Ca2+-sensitivity, cardiomyocytes were treated with alkaline phosphatase (AP) or protein kinase A (PKA). Troponin exchange experiments characterized the relation between mutant levels and myofilament function. To define mutation-mediated effects on Ca2+-dynamics we used CRISPR/Cas9 to generate hiPSC-CMs harbouring heterozygous and homozygous TnT-K280N mutations. Ca2+-transient and cell shortening experiments compared these lines against isogenic controls. Myofilament Ca2+-sensitivity was higher in homozygous cTnT-K280N cardiomyocytes and was not corrected by AP- and PKA-treatment. In cTnT-K280N cells exchanged with cTnT-WT, a low level (14%) of cTnT-K280N mutation elevated Ca2+-sensitivity. Similarly, exchange of donor cells with 45 ± 2% cTnT-K280N increased Ca2+-sensitivity and was not corrected by PKA. cTnT-K280N hiPSC-CMs show elevated diastolic Ca2+ and increases in cell shortening. Impaired cardiomyocyte relaxation was only evident in homozygous cTnT-K280N hiPSC-CMs. The cTnT-K280N mutation increases myofilament Ca2+-sensitivity, elevates diastolic Ca2+, enhances contractility and impairs cellular relaxation. A low level (14%) of the cTnT-K280N sensitizes myofilaments to Ca2+, a universal finding of human HCM.

Resistance Exercise Training Mitigates Left Ventricular Dysfunctions in Pulmonary Artery Hypertension Model.

ResumoFundamentoA hipertrofia e a dilatação do ventrículo direito observadas na hipertensão arterial pulmonar (HAP) prejudicam a dinâmica do ventrículo esquerdo (VE) achatando o septo interventricular.ObjetivoInvestigar se o treinamento físico resistido (TFR) de intensidade baixa a moderada é benéfico para funções contráteis do VE e de cardiomiócitos em ratos durante o desenvolvimento de HAP induzida por monocrotalina (MCT).MétodosForam usados ratos Wistar machos (Peso corporal: ~ 200 g). Para avaliar o tempo até o possível surgimento de insuficiência cardíaca (ou seja, ponto de desfecho), os ratos foram divididos em dois grupos, hipertensão com sedentarismo até a insuficiência (HSI, n=6) e hipertensão com treinamento até a insuficiência (HTI, n=6). Para testar os efeitos do TFR, os ratos foram divididos entre grupos de controle sedentários (CS, n=7), hipertensão com sedentarismo (HS, n=7) e hipertensão com treinamento (HT, n=7). A HAP foi induzida por duas injeções de MCT (20 mg/kg, com um intervalo de 7 dias). Os grupos com treinamento foram submetidos a um protocolo de TFR (subir escadas; 55-65% da máxima carga carregada), 5 dias por semana. A significância estatística foi definida em p <0,05.ResultadosO TFR prolongou o ponto de desfecho (~25%), melhorou a tolerância ao esforço físico (~55%) e atenuou as disfunções de contratilidade de VE e de cardiomiócitos promovidas pela MCT preservando a fração de ejeção e o encurtamento fracional, a amplitude do encurtamento, e as velocidades de contração e relaxamento nos cardiomiócitos. O TFR também preveniu os aumentos de fibrose e colágeno tipo I no ventrículo esquerdo causados pela MCT, além de manter as dimensões de miócitos e colágeno tipo III reduzidas por MCT.ConclusãoO TFR de intensidade baixa a moderada é benéfico para funções contráteis de VE e cardiomiócitos em ratos durante o desenvolvimento de HAP induzida por MCT.

Warm, but not hypoxic acclimation, prolongs ventricular diastole and decreases the protein level of Na+/Ca2+ exchanger to enhance cardiac thermal tolerance in European sea bass

One of the physiological mechanisms that can limit the fish's ability to face hypoxia or elevated temperature, is maximal cardiac performance. Yet, few studies have measured how cardiac electrical activity and associated calcium cycling proteins change with acclimation to those environmental stressors. To examine this, we acclimated European sea bass for 6 weeks to three experimental conditions: a seasonal average temperature in normoxia (16 °C; 100% air sat.), an elevated temperature in normoxia (25 °C; 100% air sat.) and a seasonal average temperature in hypoxia (16 °C; 50% air sat.). Following each acclimation, the electrocardiogram was measured to assess how acclimation affected the different phases of cardiac cycle, the maximal heart rate (fHmax) and cardiac thermal performance during an acute increase of temperature. Whereas warm acclimation prolonged especially the diastolic phase of the ventricular contraction, reduced the fHmax and increased the cardiac arrhythmia temperature (TARR), hypoxic acclimation was without effect on these functional indices. We measured the level of two key proteins involved with cellular relaxation of cardiomyocytes, i.e. sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) and Na+/Ca2+ exchanger (NCX). Warm acclimation reduced protein level of both NCX and SERCA and hypoxic acclimation reduced SERCA protein levels without affecting NCX. The changes in ventricular NCX level correlated with the observed changes in diastole duration and fHmax as well as TARR. Our results shed new light on mechanisms of cardiac plasticity to environmental stressors and suggest that NCX might be involved with the observed functional changes, yet future studies should also measure its electrophysiological activity.

Synthetic growth hormone-releasing hormone agonist ameliorates the myocardial pathophysiology characteristic of heart failure with preserved ejection fraction.

To test the hypothesis that the activation of the growth hormone-releasing hormone (GHRH) receptor signalling pathway within the myocardium both prevents and reverses diastolic dysfunction and pathophysiologic features consistent with heart failure with preserved ejection fraction (HFpEF). Impaired myocardial relaxation, fibrosis, and ventricular stiffness, among other multi-organ morbidities, characterize the phenotype underlying the HFpEF syndrome. Despite the rapidly increasing prevalence of HFpEF, few effective therapies have emerged. Synthetic agonists of the GHRH receptors reduce myocardial fibrosis, cardiomyocyte hypertrophy, and improve performance in animal models of ischaemic cardiomyopathy, independently of the growth hormone axis. CD1 mice received 4- or 8-week continuous infusion of angiotensin-II (Ang-II) to generate a phenotype with several features consistent with HFpEF. Mice were administered either vehicle or a potent synthetic agonist of GHRH, MR-356 for 4-weeks beginning concurrently or 4-weeks following the initiation of Ang-II infusion. Ang-II-treated animals exhibited diastolic dysfunction, ventricular hypertrophy, interstitial fibrosis, and normal ejection fraction. Cardiomyocytes isolated from these animals exhibited incomplete relaxation, depressed contractile responses, altered myofibrillar protein phosphorylation, and disturbed calcium handling mechanisms (ex vivo). MR-356 both prevented and reversed the development of the pathological phenotype in vivo and ex vivo. Activation of the GHRH receptors increased cAMP and cGMP in cardiomyocytes isolated from control animals but only cAMP in cardiac fibroblasts, suggesting that GHRH-A exert differential effects on cardiomyocytes and fibroblasts. These findings indicate that the GHRH receptor signalling pathway(s) represents a new molecular target to counteract dysfunctional cardiomyocyte relaxation by targeting myofilament phosphorylation and fibrosis. Accordingly, activation of GHRH receptors with potent, synthetic GHRH agonists may provide a novel therapeutic approach to management of the myocardial alterations associated with the HFpEF syndrome.