Impaired Contractile Function Research Articles

Integrative Single-Cell Analysis of Cardiomyopathy Identifies Differences in Cell Stemness and Transcriptional Regulatory Networks among Fibroblast Subpopulations.

Cardiomyopathy encompasses a broad spectrum of diseases affecting myocardial tissue, characterized clinically by abnormalities in cardiac structure, heart failure, and/or arrhythmias. Clinically heterogeneous, major types include dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM), restrictive cardiomyopathy (RM), ischemic cardiomyopathy (ICM), among which DCM is more prevalent, while ICM exhibits higher incidence and mortality rates. Myocardial injury during cardiomyopathy progression may lead to myocardial fibrosis. Failure to intervene early and inhibit the process of myocardial fibrosis may culminate in heart failure. Cardiac fibroblasts constitute crucial cellular components determining the extent and quality of myocardial fibrosis, with various subpopulations exerting diverse roles in cardiomyopathy progression. Despite this, understanding of the cellular plasticity and transcriptional regulatory networks of cardiac fibroblasts in cardiomyopathy remains limited. Therefore, in this study, we conducted comprehensive single-cell analysis of cardiac fibroblasts in cardiomyopathy to explore differences in cellular plasticity and transcriptional regulatory networks among fibroblast subpopulations, with the aim of providing as many useful references as possible for the diagnosis, prognosis, and treatment of cardiomyopathy. Cells with mitochondrial gene expression comprising >20% of total expressed genes were excluded. Differential expression genes (DEGs) and stemness genes within cardiac fibroblast subpopulations were subjected to Gene Ontology (GO) analysis of biological processes (BP) and AUCell analysis. Monocle software was employed to analyze the pseudo-temporal trajectory of cardiac fibroblasts in cardiomyopathy. Additionally, the Python package SCENIC was utilized to assess enrichment of transcription factors and activity of regulators within cardiac fibroblast subpopulations in cardiomyopathy. Following batch effect correction, 179,927 cells were clustered into 32 clusters, designated as T_NK cells, endothelial cells, myeloid cells, fibroblasts, pericytes, SMCs, CMs, proliferating cells, EndoCs, and EPCs. Among them, 8148 fibroblasts were further subdivided into 4 subpopulations, namely C0 THBS4+ Fibroblasts, C1 LINC01133+ Fibroblasts, C2 FGF7+ Fibroblasts, and C3 AGT + Fibroblasts. Results from GO_BP and AUCell analyses suggest that C3 AGT + Fibroblasts may be associated with immune response activation, protein transport, and myocardial contractile function, correlating with disease progression in cardiomyopathy. Transcription factor enrichment analysis indicates that FOS is the most significant TF in C3 AGT + Fibroblasts, also associated with the M1 module, possibly implicated in protein hydrolysis, intracellular DNA replication, and cell proliferation. Moreover, correlation analysis of transcriptional regulatory activity between fibroblast subpopulations reveals a more pronounced heterogeneity within C3 AGT + Fibroblasts in cardiomyopathy. C3 AGT + Fibroblasts exhibit increased sensitivity towards adverse outcomes in cardiomyopathy, such as myocardial fibrosis and impaired cardiac contractile function, compared to other cardiac fibroblast subpopulations. The differential cellular plasticity and transcriptional regulatory activity between C3 AGT + Fibroblasts and other subgroups offer new perspectives for targeting fibroblast subpopulation activity to treat cardiomyopathy. Additionally, stemness genes EPAS1 and MYC, along with the regulator FOS, may play roles in modulating the biological processes of cardiac fibroblasts in cardiomyopathy.

Localized translation and sarcomere maintenance requires ribosomal protein SA in mice.

Cardiomyocyte sarcomeres contain localized ribosomes, but the factors responsible for their localization and the significance of localized translation are unknown. Using proximity labeling, we identified Ribosomal Protein SA (RPSA) as a Z-line protein. In cultured cardiomyocytes, the loss of RPSA led to impaired local protein translation and reduced sarcomere integrity. By employing CAS9 expressing mice along with adeno-associated viruses expressing CRE recombinase and single-guide RNAs targeting Rpsa, we knocked out Rpsa in vivo and observed mis-localization of ribosomes and diminished local translation. These genetic mosaic mice with Rpsa knockout in a subset of cardiomyocytes developed dilated cardiomyopathy, featuring atrophy of RPSA-deficient cardiomyocytes, compensatory hypertrophy of unaffected cardiomyocytes, left ventricular dilation, and impaired contractile function. We demonstrate that RPSA C-terminal domain is sufficient for localization to the Z-lines and that if the microtubule network is disrupted RPSA loses its sarcomeric localization. These findings highlight RPSA as a ribosomal factor essential for ribosome localization to the Z-line, facilitating local translation and sarcomere maintenance.

Cardiac tissue model of immune-induced dysfunction reveals the role of free mitochondrial DNA and the therapeutic effects of exosomes.

Despite tremendous progress in the development of mature heart-on-a-chip models, human cell-based models of myocardial inflammation are lacking. Here, we bioengineered a vascularized heart-on-a-chip with circulating immune cells to model severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced acute myocarditis. We observed hallmarks of coronavirus disease (COVID-19)-induced myocardial inflammation, as the presence of immune cells augmented the secretion of proinflammatory cytokines, triggered progressive impairment of contractile function, and altered intracellular calcium transients. An elevation of circulating cell-free mitochondrial DNA (ccf-mtDNA) was measured first in the heart-on-a-chip and then validated in COVID-19 patients with low left ventricular ejection fraction, demonstrating that mitochondrial damage is an important pathophysiological hallmark of inflammation-induced cardiac dysfunction. Leveraging this platform in the context of SARS-CoV-2-induced myocardial inflammation, we established that administration of endothelial cell-derived exosomes effectively rescued the contractile deficit, normalized calcium handling, elevated the contraction force, and reduced the ccf-mtDNA and cytokine release via Toll-like receptor-nuclear factor κB signaling axis.

Pyruvate kinase M2 sustains cardiac mitochondrial integrity in septic cardiomyopathy by regulating PHB2-dependent mitochondrial biogenesis.

Previous studies have highlighted the protective effects of pyruvate kinase M2 (PKM2) overexpression in septic cardiomyopathy. In our study, we utilized cardiomyocyte-specific PKM2 knockout mice to further investigate the role of PKM2 in attenuating LPS-induced myocardial dysfunction, focusing on mitochondrial biogenesis and prohibitin 2 (PHB2). Our findings confirmed that the deletion of PKM2 in cardiomyocytes significantly exacerbated LPS-induced myocardial dysfunction, as evidenced by impaired contractile function and relaxation. Additionally, the deletion of PKM2 intensified LPS-induced myocardial inflammation. At the molecular level, LPS triggered mitochondrial dysfunction, characterized by reduced ATP production, compromised mitochondrial respiratory complex I/III activities, and increased ROS production. Intriguingly, the absence of PKM2 further worsened LPS-induced mitochondrial damage. Our molecular investigations revealed that LPS disrupted mitochondrial biogenesis in cardiomyocytes, a disruption that was exacerbated by the absence of PKM2. Given that PHB2 is known as a downstream effector of PKM2, we employed PHB2 adenovirus to restore PHB2 levels. The overexpression of PHB2 normalized mitochondrial biogenesis, restored mitochondrial integrity, and promoted mitochondrial function. Overall, our results underscore the critical role of PKM2 in regulating the progression of septic cardiomyopathy. PKM2 deficiency impeded mitochondrial biogenesis, leading to compromised mitochondrial integrity, increased myocardial inflammation, and impaired cardiac function. The overexpression of PHB2 mitigated the deleterious effects of PKM2 deletion. This discovery offers a novel insight into the molecular mechanisms underlying septic cardiomyopathy and suggests potential therapeutic targets for intervention.

Effects of chemically induced ovarian failure on single muscle fiber contractility in a mouse model of menopause

ObjectiveMenopause is associated with impaired skeletal muscle contractile function. The temporal and mechanistic bases of this dysfunction are unknown. Using a mouse model of menopause, we identified how gradual ovarian failure affects single muscle fiber contractility. Study designOvarian failure was chemically induced over 120 days, representing the perimenopausal transition. Mice were sacrificed and soleus and extensor digitorum longus muscles were dissected and chemically permeabilized for single fiber mechanical testing. Main outcome measuresMuscle fiber contractility was assessed via force, rate of force redevelopment, instantaneous stiffness, and calcium sensitivity. ResultsPeak force and cross-sectional area of the soleus were, respectively, ~33 % and ~24 % greater following ovarian failure compared with controls (p < 0.05) with no differences in force produced by the extensor digitorum longus across groups (p > 0.05). Upon normalizing force to cross-sectional area there were no differences across groups (p > 0.05). Following ovarian failure, rate of force redevelopment of single fibers from the soleus was ~33 % faster compared with controls. There was no shift in the midpoint of the force‑calcium curve between groups or muscles (p > 0.05). However, following ovarian failure, Type I fibers from the soleus had a higher calcium sensitivity between pCa values of 4.5 and 6.2 compared with controls (p < 0.05), with no differences for Type II fibers or the extensor digitorum longus (p > 0.05). ConclusionsIn our model of menopause, alterations to muscle contractility were less evident than in ovariectomized models. This divergence across models highlights the importance of better approximating the natural trajectory of menopause during and after the transitional phase of ovarian failure on neuromuscular function.

Atrial fibrillation-associated electrical remodelling in human induced pluripotent stem cell-derived atrial cardiomyocytes: a novel pathway for antiarrhythmic therapy development.

Atrial fibrillation (AF) is associated with tachycardia-induced cellular electrophysiology alterations which promote AF chronification and treatment resistance. Development of novel antiarrhythmic therapies is hampered by the absence of scalable experimental human models that reflect AF-associated electrical remodelling. Therefore, we aimed to assess if AF-associated remodelling of cellular electrophysiology can be simulated in human atrial-like cardiomyocytes derived from induced pluripotent stem cells in the presence of retinoic acid (iPSC-aCM), and atrial-engineered human myocardium (aEHM) under short term (24 h) and chronic (7 days) tachypacing (TP). First, 24-h electrical pacing at 3 Hz was used to investigate whether AF-associated remodelling in iPSC-aCM and aEHM would ensue. Compared to controls (24 h, 1 Hz pacing) TP-stimulated iPSC-aCM presented classical hallmarks of AF-associated remodelling: (i) decreased L-type Ca2+ current (ICa,L) and (ii) impaired activation of acetylcholine-activated inward-rectifier K+ current (IK,ACh). This resulted in action potential shortening and an absent response to the M-receptor agonist carbachol in both iPSC-aCM and aEHM subjected to TP. Accordingly, mRNA expression of the channel-subunit Kir3.4 was reduced. Selective IK,ACh blockade with tertiapin reduced basal inward-rectifier K+ current only in iPSC-aCM subjected to TP, thereby unmasking an agonist-independent constitutively active IK,ACh. To allow for long-term TP, we developed iPSC-aCM and aEHM expressing the light-gated ion-channel f-Chrimson. The same hallmarks of AF-associated remodelling were observed after optical-TP. In addition, continuous TP (7 days) led to (i) increased amplitude of inward-rectifier K+ current (IK1), (ii) hyperpolarization of the resting membrane potential, (iii) increased action potential-amplitude and upstroke velocity as well as (iv) reversibly impaired contractile function in aEHM. Classical hallmarks of AF-associated remodelling were mimicked through TP of iPSC-aCM and aEHM. The use of the ultrafast f-Chrimson depolarizing ion channel allowed us to model the time-dependence of AF-associated remodelling in vitro for the first time. The observation of electrical remodelling with associated reversible contractile dysfunction offers a novel platform for human-centric discovery of antiarrhythmic therapies.

Features of drug therapy for functional gastrointestinal disorders in children of preschool and primary school age

The article explores modern concepts about functional gastrointestinal disorders in children, considers features of the classification according to the Rome criteria and Russian clinical guidelines, and presents the evolution of views in the context of understanding the general pathophysiological mechanism as impaired “gut-brain” interactions, which leads to dysmotility in different parts of the gastrointestinal tract, visceral hypersensitivity, changes in the local immune system functions, dysbiosis of the gut microbiota and a failure of the central nervous system to process signals. Dysregulation of the central nervous system and the enteric nervous system causes functional gastrointestinal disorders with pain syndrome: irritable bowel syndrome, functional dyspepsia and centrally mediated abdominal pain syndrome. In addition, the article discusses the Russian version of the guidelines due to differences in the diagnostic instrumental and laboratory facilities, as well as national peculiarities of marketing authorization, indications and contraindications for drug use in children. Subject to understanding of the mechanisms of functional gastrointestinal disorders in children, the authors presented information on the role of trimebutine, a universal motility regulator of different parts of the gastrointestinal tract, in the development of functional gastrointestinal disorders, as well as modern approaches to their prevention and correction using a drug of proven efficacy. Numerous national and foreign studies proved the efficacy of trimebutine in hyperkinetic and hypokinetic movement disorders, decreased gastric evacuation function, and impaired gallbladder contractile function in patients with both hypomotor and hypermotor types of functional disorders. The accumulated experience determines the prospects for its use in pediatric practice.

Matrix metalloproteinase-2 proteolyzes mitofusin-2 and impairs mitochondrial function during myocardial ischemia-reperfusion injury.

During myocardial ischemia and reperfusion (IR) injury matrix metalloproteinase-2 (MMP-2) is rapidly activated in response to oxidative stress. MMP-2 is a multifunctional protease that cleaves both extracellular and intracellular proteins. Oxidative stress also impairs mitochondrial function which is regulated by different proteins, including mitofusin-2 (Mfn-2), which is lost in IR injury. Oxidative stress and mitochondrial dysfunction trigger the NLRP3 inflammasome and the innate immune response which invokes the de novo expression of an N-terminal truncated isoform of MMP-2 (NTT-MMP-2) at or near mitochondria. We hypothesized that MMP-2 proteolyzes Mfn-2 during myocardial IR injury, impairing mitochondrial function and enhancing the inflammasome response. Isolated hearts from mice subjected to IR injury (30min ischemia/40min reperfusion) showed a significant reduction in left ventricular developed pressure (LVDP) compared to aerobically perfused hearts. IR injury increased MMP-2 activity as observed by gelatin zymography and increased degradation of troponin I, an intracellular MMP-2 target. MMP-2 preferring inhibitors, ARP-100 or ONO-4817, improved post-ischemic recovery of LVDP compared to vehicle perfused IR hearts. In muscle fibers isolated from IR hearts the rates of mitochondrial oxygen consumption and ATP production were impaired compared to those from aerobic hearts, whereas ARP-100 or ONO-4817 attenuated these reductions. IR hearts showed higher levels of NLRP3, cleaved caspase-1 and interleukin-1β in the cytosolic fraction, while the mitochondria-enriched fraction showed reduced levels of Mfn-2, compared to aerobic hearts. ARP-100 or ONO-4817 attenuated these changes. Co-immunoprecipitation showed that MMP-2 is associated with Mfn-2 in aerobic and IR hearts. ARP-100 or ONO-4817 also reduced infarct size and cell death in hearts subjected to 45min ischemia/120min reperfusion. Following myocardial IR injury, impaired contractile function and mitochondrial respiration and elevated inflammasome response could be attributed, at least in part, to MMP-2 activation, which targets and cleaves mitochondrial Mfn-2. Inhibition of MMP-2 activity protects against cardiac contractile dysfunction in IR injury in part by preserving Mfn-2 and suppressing inflammation.

Antisenescence Therapy Improves Function in a Human Model of Cardiac Fibrosis-on-a-Chip

Cardiac fibrosis is a significant contributor to heart failure and is characterized by abnormal ECM deposition and impaired contractile function. We have previously developed a model of cardiac fibrosis via TGF-β treatment of engineered microtissues using heart-on-a-chip technology which incorporates human induced pluripotent stem cell-derived cardiomyocytes and cardiac fibroblasts. Here, we describe that these cardiac fibrotic tissues expressed markers associated with cellular senescence via transcriptomic analysis. Treatment of fibrotic tissues with the senolytic drugs dasatinib and quercetin (D+Q) led to an improvement of contractile function, reduced passive tension, and downregulated senescence-related gene expression, an outcome we were previously unable to achieve using standard-of-care drugs. The improvement in functional parameters was also associated with a reduction in fibroblast density, though no changes in absolute collagen deposition were observed. This study demonstrates the benefit of senolytic treatment for cardiac fibrosis in a human-relevant model, supporting data in animal models, and will enable the further elucidation of cell-specific effects of senolytics and how they impact cardiac fibrosis and senescence.

Role of Coxsackievirus B3-Induced Immune Responses in the Transition from Myocarditis to Dilated Cardiomyopathy and Heart Failure.

Dilated cardiomyopathy (DCM) is a cardiac disease marked by the stretching and thinning of the heart muscle and impaired left ventricular contractile function. While most patients do not develop significant cardiac diseases from myocarditis, disparate immune responses can affect pathological outcomes, including DCM progression. These altered immune responses, which may be caused by genetic variance, can prolong cytotoxicity, induce direct cleavage of host protein, or encourage atypical wound healing responses that result in tissue scarring and impaired mechanical and electrical heart function. However, it is unclear which alterations within host immune profiles are crucial to dictating the outcomes of myocarditis. Coxsackievirus B3 (CVB3) is a well-studied virus that has been identified as a causal agent of myocarditis in various models, along with other viruses such as adenovirus, parvovirus B19, and SARS-CoV-2. This paper takes CVB3 as a pathogenic example to review the recent advances in understanding virus-induced immune responses and differential gene expression that regulates iron, lipid, and glucose metabolic remodeling, the severity of cardiac tissue damage, and the development of DCM and heart failure.

Melatonin and metformin ameliorated trastuzumab-induced cardiotoxicity through the modulation of mitochondrial function and dynamics without reducing its anticancer efficacy

Trastuzumab has an impressive level of efficacy as regards antineoplasticity, however it can cause serious cardiotoxic side effects manifested by impaired cardiac contractile function. Although several pharmacological interventions, including melatonin and metformin, have been reported to protect against various cardiovascular diseases, their potential roles in trastuzumab-induced cardiotoxicity remain elusive. We hypothesized that either melatonin or metformin co-treatment effectively attenuates trastuzumab-mediated cardiotoxicity through attenuating the impaired mitochondrial function and mitochondrial dynamics. Male Wistar rats were divided into control (normal saline, n = 8) and trastuzumab group (4 mg/kg/day for 7 days, n = 24). Rats in the trastuzumab group were subdivided into 3 interventional groups (n = 8/group), and normal saline, or melatonin (10 mg/kg/day), or metformin (250 mg/kg/day) were orally administered for 7 consecutive days. Cardiac parameters were determined, and biochemical investigations were carried out on blood and heart tissues. Trastuzumab induced left ventricular (LV) dysfunction by increasing oxidative stress, inflammation, and apoptosis. It also impaired cardiac mitochondrial function, dynamics, and autophagy. Treatment with either melatonin or metformin equally attenuated trastuzumab-induced cardiac injury, indicated by a marked reduction in inflammation, oxidative damage, cardiac mitochondrial injury, mitochondrial dynamic imbalance, autophagy dysregulation, and apoptosis, leading to improved LV function, as demonstrated by increased LV ejection fraction. Melatonin and metformin conferred equal levels of cardioprotection against trastuzumab-induced cardiotoxicity, which may provide novel and promising approaches for management of cardiotoxicity induced by trastuzumab.

Abstract 13676: Sublingual Nitroglycerin Lowers Aortic Augmentation Pressure at Rest and During Exercise in Patients With HFpEF

Infusion or inhalation of organic nitrates lower ascending aortic pressures, and reduce mid-late systolic afterload via a reduction in augmentation pressure (aAP, the difference between the first [aP1] and second [aP2] peaks of the aortic pressure waveform) at rest and during exercise in patients with heart failure and preserved ejection fraction (HFpEF). Sublingual nitroglycerin (NTG) is more accessible for patients as compared to the infusion or inhalation of nitrates. It is unknown whether NTG spray (400μg) elicits these same effects as organic nitrates on aortic hemodynamics at rest and during exercise. Therefore, in 15 patients with HFpEF (age, 71 ± 6 years; 10 women; BMI 39.2 ± 6.2 kg/m 2 ), as part of our ongoing clinical trial, we measured beat-by-beat radial artery blood pressure and estimated aortic pressure in real-time via a generalized inverse transfer function (SphygmoCor). Data were collected in the seated upright position at rest and throughout six-minutes of cycling at 20 Watts during either a placebo or NTG condition (randomized order). Stroke volume (SV) was calculated from cardiac output (Qc, acetylene rebreathe) and heart rate (ECG). NTG lowered stroke volume at rest, but not during exercise (Table); however, SV responses to exercise were similar (Placebo: Δ22 ± 19 vs NTG: Δ25 ± 18 ml, P = 0.1991). Aortic diastolic pressure was not different at rest with NTG compared to placebo (Table). aP1 and aP2 were lower at rest with NTG, with greater reductions in aP2 (Table). NTG lowered aAP during rest (Placebo: 11 ± 11 vs NTG: 0 ± 14 mmHg, P = 0.0002) and exercise (Placebo: 12 ± 11 vs NTG: 4 ± 12 mmHg, P &lt; 0.0001). These results suggest patients with HFpEF do not augment SV responses to exercise despite a lower aAP contribution to (mid-late) systolic afterload with NTG. The inability to take advantage of a lower aAP during exercise may reflect impairments in contractile function and end-systolic reserve.

Abstract 12787: Microrna Administration Attenuates the Contraction Deterioration of Microrna-1 Deficient Heart But Not the Susceptibility of Arrhythmia

Introduction: Aged people are much more likely to suffer arrhythmias or develop heart failure. MicroRNA-1 (miR1), encode by two distinct genes in the mammalian genome, is an evolutionarily conserved, striated muscle-enriched microRNA with a critical role in regulating cardiac physiology. Full knockout of miR1 in mice causes postnatal lethal. Interestingly, miR1 expression was significantly decreased to the level of 12.5±4.0% in the heart of 18-month aged animals, in comparison with that in young mice (age 3-4 months). Aims: We hypothesize that miR1 deficiency is one of the major etiologies in diseased and/or aged hearts and study the therapeutic effect of miR1 administration. Methods and Results: We generated miR1-75% knockdown (75%KD) mice and confirmed that miR1 expression was 4-5 fold lower in 75%KD hearts than in WT hearts. Echocardiogram showed that 75%KD mice demonstrated impaired cardiac contractile function, evidenced by decreased ejection fraction, and fraction shortening. Surface ECG recording showed that 75%KD mice had significantly lower heart rates, shorter PR interval, prolonged QRS interval as well as premature ventricular contractions. Consistently, optical mapping of ex vivo hearts revealed that 75%KD hearts had slower ventricular conduction velocity. Whole transcriptome RNA-sequencing and Proteomic assays demonstrated cardiac electrical and metabolic remodeling in miR1-deficient hearts. Compared to WT cells, 75%KD ventricular cardiomyocytes showed more hyperpolarized RMPs with prolonged APD; the sarcomere shortening was significantly decreased at a 5Hz fast pacing rate. Structurally, 75%KD myocytes had disrupted mitochondria with a shorter length of sarcomeres. We delivered miR into the animals through intravenous tail vein injection and found that 12-week miR1 administration could significantly attenuate the decrease of ejection fraction of 75%KD hearts, however, miR1-treated hearts had significantly higher inducibility of arrhythmia than control-RNA treated hearts. Conclusions: MicroRNA-1 deficiency causes cardiac electrical and metabolic remodeling. Restoring miR1 prevents the contraction deterioration of miR1-deficient hearts but increases the risk of arrhythmia.

Disruption of Sarcoplasmic Reticulum-Mitochondrial Contacts Underlies Contractile Dysfunction in Experimental and Human Atrial Fibrillation: A Key Role of Mitofusin 2.

Background Atrial fibrillation (AF) is the most common and progressive tachyarrhythmia. Diabetes is a common risk factor for AF. Recent research findings revealed that microtubule network disruption underlies AF. The microtubule network mediates the contact between sarcoplasmic reticulum and mitochondria, 2 essential organelles for normal cardiomyocyte function. Therefore, disruption of the microtubule network may impair sarcoplasmic reticulum and mitochondrial contacts (SRMCs) and subsequently cardiomyocyte function. The current study aims to determine whether microtubule-mediated SRMCs disruption underlies diabetes-associated AF. Methods and Results Tachypacing (mimicking AF) and high glucose (mimicking diabetes) significantly impaired contractile function in HL-1 cardiomyocytes (loss of calcium transient) and Drosophila (reduced heart rate and increased arrhythmia), both of which were prevented by microtubule stabilizers. Furthermore, both tachypacing and high glucose significantly reduced SRMCs and the key SRMC tether protein mitofusin 2 (MFN2) and resulted in consequent mitochondrial dysfunction, all of which were prevented by microtubule stabilizers. In line with pharmacological interventions with microtubule stabilizers, cardiac-specific knockdown of MFN2 induced arrhythmia in Drosophila and overexpression of MFN2 prevented tachypacing- and high glucose-induced contractile dysfunction in HL-1 cardiomyocytes and/or Drosophila. Consistently, SRMCs/MFN2 levels were significantly reduced in right atrial appendages of patients with persistent AF compared with control patients, which was aggravated in patients with diabetes. Conclusions SRMCs may play a critical role in clinical AF, especially diabetes-related AF. Furthermore, SRMCs can be regulated by microtubules and MFN2, which represent novel potential therapeutic targets for AF.

Citrullination is linked to reduced Ca2+ sensitivity in hearts of a murine model of rheumatoid arthritis.

Cardiac contractile dysfunction is prevalent in rheumatoid arthritis (RA), with an increased risk for heart failure. A hallmark of RA has increased levels of peptidyl arginine deaminases (PAD) that convert arginine to citrulline leading to ubiquitous citrullination, including in the heart. We aimed to investigate whether PAD-dependent citrullination in the heart was linked to contractile function in a mouse model of RA during the acute inflammatory phase. We used hearts from the collagen-induced arthritis (CIA) mice, with overt arthritis, and control mice to analyze cardiomyocyte Ca2+ handling and fractional shortening, the force-Ca2+ relationship in isolated myofibrils, the levels of PAD, protein post-translational modifications, and Ca2+ handling protein. Then, we used an in vitro model to investigate the role of TNF-α in the PAD-mediated citrullination of proteins in cardiomyocytes. Cardiomyocytes from CIA mice displayed larger Ca2+ transients than controls, whereas cell shortening was similar in the two groups. Myofibrils from CIA hearts required higher [Ca2+ ] to reach 50% of maximum shortening, ie Ca2+ sensitivity was lower. This was associated with increased PAD2 expression and α-actin citrullination. TNF-α increased PAD-mediated citrullination which was blocked by pre-treatment with the PAD inhibitor 2-chloroacetamide. Using a mouse RA model we found evidence of impaired cardiac contractile function linked to reduced Ca2+ sensitivity, increased expression of PAD2, and citrullination of α-actin, which was triggered by TNF-α. This provides molecular and physiological evidence for acquired cardiomyopathy and a potential mechanism for RA-associated heart failure.

THE SOLUBLE ST2 BLOOD LEVEL IN PATIENTS WITH DEPRESSIVE DISORDERS AND ISCHEMIC HEART FAILURE SYNDROME

&#x0D; Mental disorders are among the leading causes of the global health-related burden. This burden was high across the entire lifespan, for both sexes, and across many locations. Heart failure (HF) persists as an increasing global epidemic; indeed, its estimated prevalence is currently more than 37.7 million people worldwide. ST2 (growth stimulation expressed gene 2) is a member of the interleukin (IL)-1 receptor family. Recent studies revealed that elevated levels of sST2 were associated with adverse outcomes in patients with acute dyspnoea, acute coronary syndromes, and HF. The purpose of this study: to evaluate the relationship between ST2 levels in the blood of patients with heart failure and depression. Material and methods: there were examined 129 patients with depression of varying severity together with the syndrome of HF of ischemic genesis, FC II-IV (NYHA). Depressive disorders were screened using the Hospital Anxiety and Depression Scale, which showed 8 or more points to indicate depressive episodes. The severity of depressive disorders was determined using the Hamilton scale. Transthoracic echocardiography was performed; the plasma content of soluble ST2 (sST2) and the titers of the N-terminal fragment of the brain natriuretic peptide (NT-proBNP) in the blood were determined by ELISA. Statistical analysis was performed using the standard software package "Statistica for Windows 12.0" (StatSoft, Tulsa, OK, USA).Results: The average age of the examined patients was (67.21 ± 11.73) years; among them - 41 women (31.8%). It was noted that the levels of soluble ST2 and NT-pro-BNP in the blood of patients with depressive disorders and HF of ischemic origin increased with decreasing LV EF. According to the Hamilton questionnaire, the majority of patients with HF suffered from depressive disorders of moderate intensity - 96 (74.4%) people. Univariate correlation analysis showed a direct relationship between soluble ST2 levels in the blood of the examined patients and the severity of depressive symptoms (rs = 0.33; p = 0.041); blood content of NT-pro-BNP (rs = 0.51; p = 0.015); resting heart rate (rs = 0.31; p = 0.011) and feedback from LV EF (rs = -0.39; p = 0.043).Conclusion. Patients with depressive disorders and concomitant heart failure of ischemic origin more often showed elevated levels of soluble ST2 molecule in the blood, which depend on the intensity of depression and impaired myocardial contractile function.

Abstract P1090: Polycystin-1 Regulates Calcium Handling And Contractility In Human IPSC-derived Ventricular Cardiomyocytes

Mutations in polycystin-1 (PC1) cause 85% of autosomal dominant polycystic kidney disease cases. This disease is characterized by progressive renal disease and multiple cardiovascular alterations. Evidence suggests that cardiovascular disease precedes the onset of renal failure. We recently demonstrated that PC1 ablation shortens cardiomyocyte action potential, decreases Ca 2+ transient amplitude, and reduces contractility in adult cardiomyocytes isolated from cardiomyocyte-specific PC1 knock-out mice compared to control mice. Alterations in Ca 2+ cycling in PC1-deficient cardiomyocytes were demonstrated to be due to action potential duration shortening and impaired sarcoendoplasmic reticulum ATPase activity. However, mouse and human cardiomyocytes have considerable differences in their action potential shapes and the ion channels involved. It remains unknown whether PC1 regulates these events in human cardiomyocytes. Here, we sought to investigate whether PC1 regulates excitation-contraction coupling in human ventricular cardiomyocytes. We differentiated commercially available human-induced pluripotent stem cell lines (hiPSC) into ventricular cardiomyocytes (hiPSC-CM) using published protocols (measurements were performed 60-90 days after differentiation). We developed a protocol to simultaneously measure action potentials (FluoVolt), intracellular Ca 2+ (Calbryte-590) and contractility using the IonOptix algorithm to detect changes in contrast as contractility measurements. PC1 ablation using siRNA decreased action potential duration and impaired Ca 2+ transients and contractility in hiPSC-CMs. Because hiPSC-CMs have several limitations due to their immature state, we employed a well-validated method to improve their maturation that utilizes a Matrigel mattress and hormone supplementation (dexamethasone; Dexa / thyroid hormone; T3; PMID: 28974554; developed by Dr. Bjorn Knollmann’s lab). Using the Matrigel mattress/Dexa/T3 technique, we increased hiPSC-CM maturation measured as follows: cardiomyocytes were oriented and contracted in the longitudinal axis, ryanodine receptor type 2 and alpha-actinin localized with a striated pattern, exhibited robust inward K + rectifier currents (I K1 ) and fired action potentials that resemble human adult cardiomyocytes. In this model, PC1 knockdown also decreased action potential duration and impaired Ca 2+ transients and decreased contractility measured as fractional shortening. Altogether, this evidence shows for the first time that PC1 regulates human cardiomyocyte physiology and suggests that cardiac alterations in patients with autosomal dominant polycystic kidney disease may emerge as a result of impaired cardiomyocyte contractile function.

Abstract P2032: Cardiac Nuclear Receptor Subfamily 4 Group A Member 2 Activates Cell Cycle Progression And Induces Heart Failure

Transcription factors play a fundamental role in cardiovascular adaptation to stress. Nuclear receptor subfamily 4 group A member 2 (NR4A2; NURR1) is an immediate-early gene and transcription factor with a versatile role throughout many organs. In the adult mammalian heart and particularly in cardiac myocytes, NR4A2 is strongly up-regulated in response to beta-adrenergic stimulation. The physiological implications of this increase remain unknown. In this study, we aimed to interrogate the consequences of cardiac NR4A2 up-regulation under normal conditions and in response to pressure overload. In mice, tamoxifen-dependent, cardiomyocyte-restricted overexpression of NR4A2 led to left ventricular hypertrophy and dilation, heart failure, and death within 40 days. Chronic NR4A2 induction also precipitated cardiac decompensation during transverse aortic constriction (TAC)-induced pressure overload. Mechanistically, NR4A2 caused adult cardiac myocytes to return to a fetal-like phenotype, with a switch to glycolytic metabolism and disassembly of sarcomeric structures. NR4A2 also re-activated cell cycle progression and stimulated DNA replication and karyokinesis but failed to induce cytokinesis, thereby promoting polyploidization and multinucleation of cardiac myocytes. Activation of cell cycle checkpoints led to induction of an apoptotic response which ultimately resulted in excessive loss of cardiac myocytes and impaired left ventricular contractile function. In summary, myocyte-specific overexpression of NR4A2 in the postnatal mammalian heart results in increased cell cycle re-entry and DNA replication but does not result in cardiac myocyte division. Our findings expose a novel function for the nuclear receptor as a critical regulator in the self-renewal of the cardiac myocyte and heart regeneration.

Prolonged cardiac NR4A2 activation causes dilated cardiomyopathy in mice

Transcription factors play a fundamental role in cardiovascular adaptation to stress. Nuclear receptor subfamily 4 group A member 2 (NR4A2; NURR1) is an immediate-early gene and transcription factor with a versatile role throughout many organs. In the adult mammalian heart, and particularly in cardiac myocytes, NR4A2 is strongly up-regulated in response to beta-adrenergic stimulation. The physiologic implications of this increase remain unknown. In this study, we aimed to interrogate the consequences of cardiac NR4A2 up-regulation under normal conditions and in response to pressure overload. In mice, tamoxifen-dependent, cardiomyocyte-restricted overexpression of NR4A2 led to cardiomyocyte hypertrophy, left ventricular dilation, heart failure, and death within 40 days. Chronic NR4A2 induction also precipitated cardiac decompensation during transverse aortic constriction (TAC)-induced pressure overload. Mechanistically, NR4A2 caused adult cardiac myocytes to return to a fetal-like phenotype, with a switch to glycolytic metabolism and disassembly of sarcomeric structures. NR4A2 also re-activated cell cycle progression and stimulated DNA replication and karyokinesis but failed to induce cytokinesis, thereby promoting multinucleation of cardiac myocytes. Activation of cell cycle checkpoints led to induction of an apoptotic response which ultimately resulted in excessive loss of cardiac myocytes and impaired left ventricular contractile function. In summary, myocyte-specific overexpression of NR4A2 in the postnatal mammalian heart results in increased cell cycle re-entry and DNA replication but does not result in cardiac myocyte division. Our findings expose a novel function for the nuclear receptor as a critical regulator in the self-renewal of the cardiac myocyte and heart regeneration.

Secretion of miRNA-326-3p by senescent adipose exacerbates myocardial metabolism in diabetic mice

BackgroundAdipose tissue homeostasis is at the heart of many metabolic syndromes such as diabetes. Previously it has been demonstrated that adipose tissues from diabetic patients are senescent but whether this contributes to diabetic cardiomyopathy (DCM) remains to be elucidated.MethodsThe streptozotocin (STZ) type 1 diabetic mice were established as animal model, and adult mouse ventricular myocytes (AMVMs) isolated by langendorff perfusion as well as neonatal mouse ventricular myocytes (NMVMs) were used as cell models. Senescent associated β galactosidase (SA-β-gal) staining and RT-qPCR were used to identify the presence of adipose senescence in diabetic adipose tissue. Senescent adipose were removed either by surgery or by senolytic treatment. Large extracellular vesicles (LEVs) derived from adipose tissue and circulation were separated by ultracentrifugation. Cardiac systolic and diastolic function was evaluated through cardiac ultrasound. Cardiomyocytes contraction function was evaluated by the Ionoptix HTS system and live cell imaging, mitochondrial morphology and functions were evaluated by transmission electron microscope, live cell fluorescent probe and seahorse analysis. RNA-seq for AMVMs and miRNA-seq for LEVs were performed, and bioinformatic analysis combined with RT-qPCR and Western blot were used to elucidate underlying mechanism that senescent adipose derives LEVs exacerbates myocardial metabolism.ResultsSA-β-gal staining and RT-qPCR identified the presence of adipose tissue senescence in STZ mice. Through surgical as well as pharmacological means we show that senescent adipose tissue participates in the pathogenesis of DCM in STZ mice by exacerbates myocardial metabolism through secretion of LEVs. Specifically, expression of miRNA-326-3p was up-regulated in LEVs isolated from senescent adipose tissue, circulation, and cardiomyocytes of STZ mice. Up-regulation of miRNA-326-3p coincided with myocardial transcriptomic changes in metabolism. Functionally, we demonstrate that miRNA-326-3p inhibited the expression of Rictor and resulted in impaired mitochondrial and contractile function in cardiomyocytes.ConclusionWe demonstrate for the first time that senescent adipose derived LEVs exacerbates myocardial metabolism through up-regulated miRNA-326-3p which inhibits Rictor in cardiomyocytes. Furthermore, reducing senescence burden in adipose tissue is capable of relieving myocardial metabolism disorder in diabetes mellitus.