Adult Ventricular Cardiomyocytes Research Articles

Unlocking cardiac motion: assessing software and machine learning for single-cell and cardioid kinematic insights

The heart coordinates its functional parameters for optimal beat-to-beat mechanical activity. Reliable detection and quantification of these parameters still represent a hot topic in cardiovascular research. Nowadays, computer vision allows the development of open-source algorithms to measure cellular kinematics. However, the analysis software can vary based on analyzed specimens. In this study, we compared different software performances in in-silico model, in-vitro mouse adult ventricular cardiomyocytes and cardioids. We acquired in-vitro high-resolution videos during suprathreshold stimulation at 0.5-1-2 Hz, adapting the protocol for the cardioids. Moreover, we exposed the samples to inotropic and depolarizing substances. We analyzed in-silico and in-vitro videos by (i) MUSCLEMOTION, the gold standard among open-source software; (ii) CONTRACTIONWAVE, a recently developed tracking software; and (iii) ViKiE, an in-house customized video kinematic evaluation software. We enriched the study with three machine-learning algorithms to test the robustness of the motion-tracking approaches. Our results revealed that all software produced comparable estimations of cardiac mechanical parameters. For instance, in cardioids, beat duration measurements at 0.5 Hz were 1053.58 ms (MUSCLEMOTION), 1043.59 ms (CONTRACTIONWAVE), and 937.11 ms (ViKiE). ViKiE exhibited higher sensitivity in exposed samples due to its localized kinematic analysis, while MUSCLEMOTION and CONTRACTIONWAVE offered temporal correlation, combining global assessment with time-efficient analysis. Finally, machine learning reveals greater accuracy when trained with MUSCLEMOTION dataset in comparison with the other software (accuracy > 83%). In conclusion, our findings provide valuable insights for the accurate selection and integration of software tools into the kinematic analysis pipeline, tailored to the experimental protocol.

Cardiotoxicity in light chain amyloidosis: Insights from the first murine model with intramyocardial injection of patient-derived amyloidogenic light chains

Abstract Background Light chain amyloidosis (AL) is a plasma cell dyscrasia in which cardiac involvement determines the prognosis. Cardiotoxicity of light chains (LCs) complicates the management of heart failure in AL and has not been addressed in the clinical praxis. We have previously genetically identified and biotechnologically produced full-length LCs from patients with AL and cardiac involvement and isotype LCs (WT) from healthy volunteers. We characterized the LCs’ toxicity on primary adult ventricular murine cardiomyocytes (CMs) and our investigation led to the selection of three cardiotoxic and amyloidogenic patient-derived LCs (one from the kappa and two from the lambda family) for further characterization. Purpose We aimed to 1) generate a murine model that recapitulates LCs’ cardiotoxicity in vivo, 2) characterize the mechanism of cardiotoxicity and 3) identify cardioprotective therapies. Methods C57BL6 mice were randomized into 6 groups as follows: 1) PBS (vehicle), 2) WT_kappa, 3) AL_kappa, 4) WT_lambda, 5) AL_lambda1 and 6) AL_lambda2. The animals received the LCs (1 mg/mouse) or vehicle via intramyocardial injection (IMI). Echocardiography evaluation and blood sampling was performed weekly. The animals were sacrificed on the 4th week post IMI for histology, electron microscopy and investigation of the cardiotoxicity mechanism. Results On the 4th week, the AL-lambda LCs significantly reduced the fractional shortening and ejection fraction compared to the vehicle and isotype control groups. The AL_lambda2 also increased mortality of the mice. The AL-kappa did not alter the systolic function of the mice. The circulating lactate dehydrogenase was significantly increased on the 2nd and 4th week in the AL-LCs treated mice indicative of the LCs’ toxicity. The IMI of AL_kappa led to signs of apoptosis including the shrinkage of cardiomyocytes and increased condensation of nuclear chromatin to the nuclear membranes. The IMI of AL_lambda LCs led to increased cardiomyocyte size indicative of hypertrophy, interstitial fibrosis, and increased number of autophagosomes. None of the animals bared amyloid deposits in the heart. The AL-LCs induced different molecular responses in vivo and confirmed the histology findings. The AL-kappa increased apoptosis via the CHOP-Bax pathway, and the ΑL-lambda increased Beclin-1 mediated autophagy without inducing apoptosis. We identified that the common upstream mediator of AL-LCs cardiotoxicity in vivo is the overexpression of the endoplasmic reticulum stress (ERS) markers, IRE-1a and Bip. In vitro, we confirmed that the AL-LCs induce cell death in CMs through ERS. Treatment with the IRE-1a inhibitor STF-083010 and the molecular chaperone tauroursodeoxycholic acid restored the LCs’ induced cardiotoxicity. Conclusions The IMI of AL-LCs stands as a method for the investigation of the LCs cardiotoxicity in vivo in the absence of amyloid deposits via which we identified ERS as a target to alleviate AL-LCs toxicity.

Abstract 17305: Diet-Induced Obesity Drives Phospholamban Acetylation and Promotes Cardiomyocyte Calcium Reuptake Into the Sarcoplasmic Reticulum

Background: The rising incidence of obesity and metabolic syndrome in the US has profound implications for the pathology of heart disease in coming years. Excessive fat and sugar intake is linked to elevated acetyl-CoA, which in turn is linked to protein acetylation. Although an increasing body of work has determined that non-nuclear lysine acetylation can alter the activity and stability of key proteins involved in cardiac health, the impact of acetylation on calcium handling sarcoplasmic reticulum (SR) proteins, particularly the SERCA2a regulator phospholamban, remains poorly understood. Hypothesis: Metabolic dysregulation and obesity lead to the acetylation of phospholamban, relieving its inhibition on SERCA2a and increasing calcium uptake into the SR. APPROACH: C57BL/6J Mice were subjected to high fat diet-induced obesity (DIO). Left ventricle (LV) tissue was collected and analyzed using immunoblotting and acetylation was quantified by immunoprecipitation with acetyl-lysine antibodies. Human LV tissue from control and diabetic patients was similarly processed and evaluated. Adult ventricular cardiomyocytes were isolated from DIO and control mice, and DIO cells were incubated in media containing excess lipid substrates (1% Lipid Mix 1, Millipore Sigma) and acutely administered HDAC activator ITSA-1 to promote deacetylation. Changes in stimulated calcium dynamics were assessed using Fura-2AM and Fluo-4AM imaging. Results: Phospholamban acetylation significantly increased in LV tissue from both DIO mice (p=0.003) and human diabetic patients (p=0.034) compared to their respective controls. Primary cardiomyocytes isolated from DIO mice and incubated for 24hrs in a high-fat environment exhibited increased calcium transient amplitude and reuptake time (tau) into the SR, while incubation with pan HDAC activators significantly reduced these parameters (p=0.0004, and p=0.003 respectively). Conclusions: Obesity increases the acetylation of phospholamban and enhances the reuptake of calcium into the SR, while deacetylation reverses these effects. These data directly tie nutritive status to a novel phospholamban post-translational modification that may directly impact myocardial calcium handling and contractile function.

Abstract P2184: Isocitrate Dehydrogenase 1 Regulates Cardiac Metabolic Adaptation During Oncometabolic Stress

Cardiovascular disease and cancer are the two leading causes of morbidity and mortality in the world. Metabolic dysregulation of cancer cells extends beyond the tumor microenvironment and increases risk for cardiovascular diseases. One common somatic mutation in cancer cells affects isocitrate dehydrogenase (IDH) 1 and 2, which catalyzes the oxidative decarboxylation of isocitrate to alpha-ketoglutarate in the cytosol and mitochondria, respectively. IDH1 and 2 mutations cause the production of the oncometabolite D-2-hydroxyglutarate (D2-HG) which allosterically inhibits alpha-ketoglutarate dehydrogenase (alpha-KGDH) and is associated with reduced cardiac contractile function. We combined stable isotope tracer studies with computational modeling to study the fundamental role of IDH isoforms for cardiac adaptation during oncometabolic stress. We uncovered an unexpected cardiac phenotype that expands the role of IDH1 in the heart beyond oxidative metabolism. Using mass spectrometry-based metabolomics, we quantified the stable isotopomer distributions from glucose and glutamine in perfused working rat hearts and isolated adult ventricular cardiomyocytes. Our analysis revealed that defective mitochondrial metabolism causes redirection of carbon flux oxidative towards reductive pathways. Reductive carboxylation of alpha-KGDH increases glutamine uptake and glutamine-derived citrate formation in both working rat heart perfusions and cultured adult mouse ventricular cardiomyocytes. To identify which IDH isoform is responsible for redirecting carbon flux, we developed knockout models of IDH1, 2, and 3 in adult mouse ventricular cardiomyocytes. Loss of IDH1 expression impaired reductive formation of citrate and caused functional defects in cardiomyocytes. Lastly, epigenetic analyses of histone marks revealed that IDH1 causes widespread alterations in histone acetylation and tri-methylation. Our results highlight a novel role for IDH1 in cardiac metabolism and transcriptional control of metabolic adaptation in response to tumor-mediated stress and provide evidence of how reductive-citrate formation may induce epigenetic modifications in the heart.

Does Enhanced Structural Maturity of hiPSC-Cardiomyocytes Better for the Detection of Drug-Induced Cardiotoxicity?

Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) are currently used following the Comprehensive in vitro Proarrhythmic Assay (CiPA) initiative and subsequent recommendations in the International Council for Harmonization (ICH) guidelines S7B and E14 Q&A, to detect drug-induced cardiotoxicity. Monocultures of hiPSC-CMs are immature compared to adult ventricular cardiomyocytes and might lack the native heterogeneous nature. We investigated whether hiPSC-CMs, treated to enhance structural maturity, are superior in detecting drug-induced changes in electrophysiology and contraction. This was achieved by comparing hiPSC-CMs cultured in 2D monolayers on the current standard (fibronectin matrix, FM), to monolayers on a coating known to promote structural maturity (CELLvo™ Matrix Plus, MM). Functional assessment of electrophysiology and contractility was made using a high-throughput screening approach involving the use of both voltage-sensitive fluorescent dyes for electrophysiology and video technology for contractility. Using 11 reference drugs, the response of the monolayer of hiPSC-CMs was comparable in the two experimental settings (FM and MM). The data showed no functionally relevant differences in electrophysiology between hiPSC-CMs in standard FM and MM, while contractility read-outs indicated an altered amplitude of contraction but not changes in time course. RNA profiling for cardiac proteins shows similarity of the RNA expression across the two forms of 2D culture, suggesting that cell-to-matrix adhesion differences may explain account for differences in contraction amplitude. The results support the view that hiPSC-CMs in both 2D monolayer FM and MM that promote structural maturity are equally effective in detecting drug-induced electrophysiological effects in functional safety studies.

Nitro-oxidative stress impairs single cardiomyocyte adrenergic reserve in a murine model of HFpEF

Abstract Background Exercise intolerance is the central symptom of patients with heart failure and preserved ejection fraction (HFpEF). Underlying reduced cardiac functional reserve in response to adrenergic stimuli (stress testing) has been suggested but the molecular mechanisms are insufficiently understood. In cardiomyocytes, nitric oxide (NO) modifies contractility and is required to achieve a full adrenergic response. Recently, dysregulation of NO release has been described to contribute to HFpEF. In a murine model of HFpEF, we investigated cardiomyocyte's adrenergic functional reserve and the role of NO in cardiomyocyte contractility, calcium handling and adrenergic reserve. Methods Firstly, the effects of NO on sarcomere shortening and calcium handling (Fura-2) were studied in isolated, adult ventricular cardiomyocytes (AMVMs) from 8–10 weeks old, male C57BL/6J mice. Secondly, male C57BL/6J mice (12w) were fed regular CHOW (Sham) or a high fat diet (D12492, Research Diet) and L-NAME (1g/l, via the drinking water) for 15 weeks to induce HFpEF. At week 27, mice underwent echocardiography and exercise testing (treadmill). In AMVMs isolated from HFpEF and Sham mice, we quantified sarcomere shortening, calcium handling (Fura-2), release of NO (CuFL2) and reactive oxygen species (DCF) before and after the addition of isoproterenol (ISO, 1μM), in the absence and presence (40 min preincubation) of an inhibitor of inducible NO Synthase (1400W) or a denitrosylating agent (glutathione). Results In AMVMs, addition of the NO donor SNAP (10μM) increased calcium transient amplitude and accelerated relaxation time. Inhibition of NO synthesis with L-NAME (100μM) impaired adrenergic response upon exposure to ISO. HFpEF mice (evident by diastolic dysfunction (e/e' ratio) and lung edema (wet lung weight / TL)) exhibited significantly reduced exercise capacity (running distance). In AMVMs isolated from HFpEF mice, NO and ROS release were increased at baseline, associated with an increased sarcomere shortening amplitude and faster relaxation and calcium removal as compared to Sham. Strikingly, after the addition of ISO, the adrenergic functional reserve (cellular inotropy and lusitropy) was significantly lower in HFpEF vs. Sham AMVMs. Preincubation with the iNOS inhibitor 1400W or glutathione restored adrenergic inotropic and lusitropic reserve in HFpEF AMVMs. Conclusion In HFpEF, adrenergic reserve is impaired on a single cardiomyocyte level. This is at least partially related to an increase in NO release. Pharmacologic inhibition of iNOS improved adrenergic reserve in HFpEF. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): DFG

Empagliflozin rescues primary murine cardiac fibroblasts, but not cardiomyocytes, from hypoxia-reoxygenation injury through STAT3-mediated cascades

Abstract Introduction Empagliflozin (EMPA) is a selective sodium glucose co-transporter 2 inhibitor (SGLT2i) and an established drug against type 2 diabetes mellitus. Cardiovascular clinical outcome trials on EMPA have shown a reduced mortality rate related to major cardiovascular events, but the precise mechanism of action remains elusive. We have previously proved that chronic EMPA administration reduces infarct size in an in vivo murine model of ischemia / reperfusion injury, irrespective of the diabetic status. The cardioprotective effect of EMPA was attributed to the increase of Signal Transducer and Activator of Transcription 3 (STAT3) phosphorylation. Purpose In this study we aimed to decipher the cell specific effects of EMPA on primary adult ventricular murine cardiomyocytes (pAVMCs) and primary murine cardiac fibroblasts (pMCFs), subjected to hypoxia/reoxygenation (HYP/REO) and to 1) investigate the effect of EMPA on cell death, and 2) reveal the mechanism of cardioprotection. Methods PAVMCs and pMCFs were isolated from adult C57BL/6 murine hearts (n=4–6) and were incubated with EMPA (500nM–100nM) for 24 hours. Cells were then subjected to 3 hours of HYP (99% N2), followed by 1 hour of REO. STATTIC, a selective STAT3 inhibitor was used (500nM–1μM) to determine the contribution of STAT3 on the observed effect. MTT assay was performed at the end of REO to determine cell viability. To unravel the mechanism of cardioprotection, the experimental protocol was repeated, and the cells were either stained with dihydroethidium (DHE) dye to determine relative oxidative stress changes or were harvested for isolation of total protein content. We focused on STAT3 and Akt kinase as key mediators of the cardioprotective survivor activating factor enhancement (SAFE) and the reperfusion injury salvage kinase (RISK) pathways. We also investigated the expression of Cardiotrophin-1 (CT-1), as a potential upstream mediator of STAT3 phosphorylation. Results The evaluation of cell death revealed that pMCFs viability was significantly increased upon EMPA treatment. Added to this, STATTIC co-treatment with EMPA blunted the protective effect on pMCFs, which indicates that EMPA's protection is STAT3-mediated. Oxidative stress remained unaltered by the treatments. Investigation of the molecular pathways that are responsible for the increased viability of pMCFs revealed that EMPA treatment induces an increased phosphorylation of STAT3 and an increase in CT-1 expression, which are both reversed by STATTIC. Akt phosphorylation and expression remained unchanged by EMPA treatment. Notably, the effect of EMPA on pAVMCs' viability was negligible. Conclusion(s) EMPA does not protect pAVMCs, from HYP/REO injury. EMPA rescues the pMCFs in a STAT-3 dependent manner which indicates that EMPA's cardioprotective effect is cell specific and implicates the SAFE pathway. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): Boehringer Ingelheim International GmbHGrant title: “Investigation of the distinct effects of Empagliflozin on different cell populations of the healthy myocardium”

Understanding the molecular mosaic of cardiotoxicity of light chains in plasma cell dyscrasias and cardiac light chain amyloidosis with the use of patient derived full-length light chains

Abstract Background Cardiac light chain amyloidosis (AL-CA) is a life-threatening disease and the major determinant of prognosis in AL amyloidosis. The management of heart failure (HF) in AL is challenging and gold standard therapies for HF are poorly tolerated or ineffective. Cardiac toxicity of LCs in AL-CA is poorly understood and the comparison of cardiotoxicity of LCs derived from plasma cell dyscrasias (PCDs) such as multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS), will improve our understanding of the mechanisms of cardiac damage. Purpose We aimed to 1) genetically identify and biotechnologically produce full-length LCs from patients with AL-CA, MM, MGUS or non-clonal LCs from healthy volunteers (HV), 2) identify LCs' cardiotoxicity and 3) investigate the underlying mechanisms of cardiotoxicity in vitro. Methods Bone marrow derived CD138+ cells from n=7 patients with AL-CA, n=2 patients with MM and n=2 patients with MGUS and peripheral blood mononuclear cells (PBMCs) from n=2 HV were isolated for RNA extraction and characterization of the LC gene family repertoire. At the protein level, LC expression was confirmed by immunoprecipitation in patients' serum followed by top-down proteomics. The overexpressed LC genes in each patient, encoding the full-length clonal LCs were cloned and produced in Shuffle E. coli cells. Two LCs were produced from HV based on the primary protein structure similarity with the patients' LCs. Primary adult ventricular murine cardiomyocytes (pAVMCs) were isolated and exposed at various LC concentrations for evaluation of cell death and investigation of the cardiotoxicity mechanisms via gene and protein expression. LCs folding, oligomerization and amyloidogenic potential were assessed via circular dichroism (CD), SDS page and electron microscopy respectively. Results We successfully identified the LCs responsible for the disease and isolated the respective proteins in all cases (7 AL-CA, 2 MM, 2 MGUS and 3 HV). Despite the similarity of the LCs in conformation as beta-sheet and oligomerization mainly as dimers, 5 out of 7 AL-CA derived LCs led to a different extent of cardiotoxicity in pAVMCs compared to the HV, MM and MGUS derived LCs which did not alter cell viability. Interestingly, these 5 LCs bared the highest amyloidogenic potency. LCs induced different molecular responses leading to cardiomyocyte death. AL-CA proteins κ-type induced apoptosis and overexpression of endoplasmic reticulum stress (ERS) markers while LCs λ-type increased unfolded protein response (UPR) markers and autophagy without inducing apoptosis. All LCs of κ-type including from MM and MGUS patients led to inteleukin-6 mediated inflammation indicating that this mechanism is independent of the observed toxicity. Conclusions AL-CA derived LCs induce cardiotoxicity, which correlates to their amyloidogenic potential via ERS, UPR, autophagy and apoptosis which can be considered targets for cardioprotection. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Hellenic Foundation for Research and Innovation

Klotho relieves inflammation and exerts a cardioprotective effect during renal ischemia/reperfusion-induced cardiorenal syndrome

BackgroundRenal ischemia and reperfusion injury (IRI) is the main cause of acute kidney injury (AKI). AKI induces the development of cardiac hypertrophy (CH) during cardiorenal syndrome (CRS), and cardiomyocyte calcium mishandling though systemic inflammation after 8 days of renal IRI. Klotho has recently been described as an anti-inflammatory component. Given this, Klotho treatment could prevent or attenuate the inflammation, thereby also preventing electrical cardiac outcomes incurred by CRS. The aim of this study was to investigate the therapeutic role of Klotho in CRS after unilateral renal IRI through its anti-inflammatory action. MethodsWe examined renal tissue structure and function, intracellular Ca2+ dynamics in adult ventricular cardiomyocytes and serum cytokine levels from C57BL/6 mice that suffered unilateral renal IRI by occluding the left pedicle for 60 min and reperfusion for 8 days. The animals were treated with recombinant Klotho protein starting from the day of the surgery, then daily for 8 days. ResultsAfter Klotho treatment for 8 days, the left renal tissue remained damaged, however the renal function was restored due to the right kidney tissue preservation. In parallel, Klotho also prevented an increase in serum interleukin (IL-) 6, IL-1β, and tumor necrosis factor alpha (TNF-α) levels. CH and low cell contraction were also prevented, as well as a decrease in systolic Ca2+ transients and sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) activity measured as Ca2+ transient decay, an increase in spontaneous Ca2+ release and the incidence of pro-arrhythmic events. ConclusionsThe Klotho treatment showed promise, playing an important role in the pathophysiology of CRS. We were unable to observe a total renoprotective role of the compound in the model; in turn, a cardioprotective role of Klotho was demonstrated through the prevention of hypertrophy and normalization of the Ca2+ cycle dysfunction of cardiomyocytes. We propose that Klotho acts in the cardiorenal syndrome by systematically preventing inflammation and increased FGF23, alleviating cardiac outcomes.

Abstract P1050: Activation Of Bk Ca Channel Increases Myocardial Infarction After Ischemia-Reperfusion Injury In Neonatal Hearts

Large-conductance calcium and voltage-activated potassium channels (BK Ca ) are heterogeneously expressed in a wide array of cells. In adult ventricular cardiomyocytes, activation of mitochondrial BK Ca channels is implicated in cardioprotection against ischemia-reperfusion (IR) injury. However, plasma membrane BK Ca channel activity has never been detected in adult ventricular cardiomyocytes. In this study, we report the presence of the BK Ca channel in the plasma membrane and mitochondria of neonatal murine and rodent cardiomyocytes. In neonatal cardiomyocyte (NCM), whole-cell potassium currents measured were sensitive to iberiotoxin (IbTx), suggesting the presence of BK Ca channels in the plasma membrane. Moreover, the open probability of single-channel recording decreased in presence of IbTx by 46+5% (n=10) and 32+5% (n=10) for rats and mice, respectively. Ex vivo studies with isolated neonatal hearts (p6) subjected to ischemia and post-conditioned with NS1619 during reoxygenation increased (58+3% as opposed to 48+5% for control, n>5 pups) the myocardial infarction whereas IbTx reduced (32+2%) the infarct size. In agreement, isolated NCM also presented increased apoptosis on treatment with NS1619 during hypoxia-reoxygenation (HR), whereas IbTx reduced TUNEL positive cells. In NCMs, activation of BK Ca channels increased the intracellular reactive oxygen species post HR injury. Electrophysiological characterization of NCMs indicated that NS1619 increased the beat period, field, and action potential duration, and decreased the conduction velocity and spike amplitude. In contrast, IbTx had no impact on the electrophysiological properties of NCMs. Taken together, our data established that inhibition of plasma membrane BK Ca channels in the neonates protects the heart/cardiomyocytes from IR injury. Furthermore, the functional disparity observed towards the cardioprotective activity of BK Ca channels in adults compared to neonatal heart could be attributed to their differential localization.

MiR-8485 alleviates the injury of cardiomyocytes through TP53INP1.

MicroRNAs (miRNAs) feature prominently in regulating the progression of chronic heart failure (CHF). This study was performed to investigate the role of miR-8485 in the injury of cardiomyocytes and CHF. It was found that miR-8485 level was markedly reduced in the plasma of CHF patients, compared with the healthy controls. H2 O2 treatment increased tumor necrosis factor-α, interleukin (IL)-6, and IL-1β levels, inhibited the viability of human adult ventricular cardiomyocyte cell line AC16, and increased the apoptosis, while miR-8485 overexpression reversed these effects. Tumor protein p53 inducible nuclear protein 1 (TP53INP1) was identified as a downstream target of miR-8485, and TP53INP1 overexpression weakened the effects of miR-8485 on cell viability, apoptosis, as well as inflammatory responses. Our data suggest that miR-8485 attenuates the injury of cardiomyocytes by targeting TP53INP1, suggesting it is a protective factor against CHF.

Inhibition of BKCa channels protects neonatal hearts against myocardial ischemia and reperfusion injury

BKCa channels are large-conductance calcium and voltage-activated potassium channels that are heterogeneously expressed in a wide array of cells. Activation of BKCa channels present in mitochondria of adult ventricular cardiomyocytes is implicated in cardioprotection against ischemia-reperfusion (IR) injury. However, the BKCa channel’s activity has never been detected in the plasma membrane of adult ventricular cardiomyocytes. In this study, we report the presence of the BKCa channel in the plasma membrane and mitochondria of neonatal murine and rodent cardiomyocytes, which protects the heart on inhibition but not activation. Furthermore, K+ currents measured in neonatal cardiomyocyte (NCM) was sensitive to iberiotoxin (IbTx), suggesting the presence of BKCa channels in the plasma membrane. Neonatal hearts subjected to IR when post-conditioned with NS1619 during reoxygenation increased the myocardial infarction whereas IbTx reduced the infarct size. In agreement, isolated NCM also presented increased apoptosis on treatment with NS1619 during hypoxia and reoxygenation, whereas IbTx reduced TUNEL-positive cells. In NCMs, activation of BKCa channels increased the intracellular reactive oxygen species post HR injury. Electrophysiological characterization of NCMs indicated that NS1619 increased the beat period, field, and action potential duration, and decreased the conduction velocity and spike amplitude. In contrast, IbTx had no impact on the electrophysiological properties of NCMs. Taken together, our data established that inhibition of plasma membrane BKCa channels in the NCM protects neonatal heart/cardiomyocytes from IR injury. Furthermore, the functional disparity observed towards the cardioprotective activity of BKCa channels in adults compared to neonatal heart could be attributed to their differential localization.

Unilateral Acute Renal Ischemia-Reperfusion Injury Induces Cardiac Dysfunction through Intracellular Calcium Mishandling.

Background: Acute renal failure (ARF) following renal ischemia-reperfusion (I/R) injury is considered a relevant risk factor for cardiac damage, but the underlying mechanisms, particularly those triggered at cardiomyocyte level, are unknown. Methods: We examined intracellular Ca2+ dynamics in adult ventricular cardiomyocytes isolated from C57BL/6 mice 7 or 15 days following unilateral renal I/R. Results: After 7 days of I/R, the cell contraction was significantly lower in cardiomyocytes compared to sham-treated mice. It was accompanied by a significant decrease in both systolic Ca2+ transients and sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) activity measured as Ca2+ transients decay. Moreover, the incidence of pro-arrhythmic events, measured as the number of Ca2+ sparks, waves or automatic Ca2+ transients, was greater in cardiomyocytes from mice 7 days after I/R than from sham-treated mice. Ca2+ mishandling related to systolic Ca2+ transients and contraction were recovered to sham values 15 days after I/R, but Ca2+ sparks frequency and arrhythmic events remained elevated. Conclusions: Renal I/R injury causes a cardiomyocyte Ca2+ cycle dysfunction at medium (contraction-relaxation dysfunction) and long term (Ca2+ leak), after 7 and 15 days of renal reperfusion, respectively.

Investigation of the Effects of the Short QT Syndrome D172N Kir2.1 Mutation on Ventricular Action Potential Profile Using Dynamic Clamp.

The congenital short QT syndrome (SQTS) is a cardiac condition that leads to abbreviated ventricular repolarization and an increased susceptibility to arrhythmia and sudden death. The SQT3 form of the syndrome is due to mutations to the KCNJ2 gene that encodes Kir2.1, a critical component of channels underlying cardiac inwardly rectifying K+ current, IK1. The first reported SQT3 KCNJ2 mutation gives rise to the D172N Kir2.1 mutation, the consequences of which have been studied on recombinant channels in vitro and in ventricular cell and tissue simulations. The aim of this study was to establish the effects of the D172N mutation on ventricular repolarization through real-time replacement of IK1 using the dynamic clamp technique. Whole-cell patch-clamp recordings were made from adult guinea-pig left ventricular myocytes at physiological temperature. Action potentials (APs) were elicited at 1 Hz. Intrinsic IK1 was inhibited with a low concentration (50 µM) of Ba2+ ions, which led to AP prolongation and triangulation, accompanied by a ∼6 mV depolarization of resting membrane potential. Application of synthetic IK1 through dynamic clamp restored AP duration, shape and resting potential. Replacement of wild-type (WT) IK1 with heterozygotic (WT-D172N) or homozygotic (D172N) mutant formulations under dynamic clamp significantly abbreviated AP duration (APD90) and accelerated maximal AP repolarization velocity, with no significant hyperpolarization of resting potential. Across stimulation frequencies from 0.5 to 3 Hz, the relationship between APD90 and cycle length was downward shifted, reflecting AP abbreviation at all stimulation frequencies tested. In further AP measurements at 1 Hz from hiPSC cardiomyocytes, the D172N mutation produced similar effects on APD and repolarization velocity; however, resting potential was moderately hyperpolarized by application of mutant IK1 to these cells. Overall, the results of this study support the major changes in ventricular cell AP repolarization with the D172N predicted from prior AP modelling and highlight the potential utility of using adult ventricular cardiomyocytes for dynamic clamp exploration of functional consequences of Kir2.1 mutations.

Abstract P386: Supplementation Of Tyrode With Fatty Acids And 3-hydroxybutyrate Improves Adult Cardiomyocytes Contractility Of Failing Human Hearts.

Due to its high energy consumption and limited ability to store ATP, the heart is highly dependent of exogenous metabolic substrates. Prior in vivo studies have reported that the development of heart failure is accompanied by a transition from the normal preferential metabolism of free fatty acids (FFA) to increases in glucose utilization and even ketone bodies, which normally provide a modest contribution to energy balance. However, the functional significance of the upregulated ketone metabolism in the failing heart is poorly understood. Recognizing that nearly all prior studies examining isolated cardiomyocyte physiology have used glucose as the sole metabolic substrate, we initiated studies to examine the impact of alternative metabolic substrates on contractility in isolated human cardiomyocytes. To understand the role of substrate alteration cardiomyocyte functionalities, we employed freshly isolated adult human left ventricular cardiomyocytes from 11 non-failing hearts (NF) obtained from organ donors and 13 failing hearts (HF) obtained from transplant recipients. Cardiomyocytes were resuspended in a conventional 5mM Glucose Tyrode solution with alternative substrates (Glucose, FFA, R-3-OHB or Mix (Glucose + FFA + 3-OHB)). Myocytes were field stimulated at 1 Hz and sarcomere length, fractional shortening, contraction velocity and relaxation velocity were measured using a video-based sarcomere length detection system (IonOptix Corp). Studies using isolated cardiac myocyte contractility as readout confirm that myocytes from NF human hearts are omnivorous: high levels of myocyte fractional shortening (FS) can be achieved under unstressed conditions (1 Hz, unloaded) with any substrate (FS Glucose : 0.1315±0.012; FS FFA : 0.1428±0.0127; FS 3OHB : 0.1343±0.014; FS MIX : 0.15467±0.02). In the failing heart, glucose alone is insufficient to produce normal unstressed myocyte fractional shortening (FS Glucose : 0.088±0.009***, p<0.001 compare to NF). However, in failing myocytes, supplementation of physiological levels of glucose with FFA or ketones each enhances myocyte contractility and rates of shortening and re-lengthening (FS FFA : 0.109±0.0127; FS 3OHB : 0.107±0.012; FS MIX : 0.112±0.016). These results suggest that future comparisons of NF vs. HF human myocyte contractility should include conditions with a physiological mix of metabolic substrates.

Analysis of the response of human iPSC-derived cardiomyocyte tissue to ICaL block. A combined in vitro and in silico approach

The high incidence of cardiac arrythmias underlines the need for the assessment of pharmacological therapies. In this field of drug efficacy, as in the field of drug safety highlighted by the Comprehensive in Vitro Proarrhythmia Assay initiative, new pillars for research have become crucial: firstly, the integration of in-silico experiments, and secondly the evaluation of fully integrated biological systems, such as human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). In this study, we therefore aimed to combine in-vitro experiments and in-silico simulations to evaluate the antiarrhythmic effect of L-type calcium current (ICaL) block in hiPSC-CMs. For this, hiPSC-CM preparations were cultured and an equivalent virtual tissue was modeled. Re-entry patterns of electrical activation were induced and several biomarkers were obtained before and after ICaL block. The virtual hiPSC-CM simulations were also reproduced using a tissue composed of adult ventricular cardiomyocytes (hAdultV-CMs). The analysis of phases, currents and safety factor for propagation showed an increased size of the re-entry core when ICaL was blocked as a result of depressed cellular excitability. The bigger wavefront curvature yielded reductions of 12.2%, 6.9%, and 4.2% in the frequency of the re-entry for hiPSC-CM cultures, virtual hiPSC-CM, and hAdultV-CM tissues, respectively. Furthermore, ICaL block led to a 47.8% shortening of the vulnerable window for re-entry in the virtual hiPSC-CM tissue and to re-entry vanishment in hAdultV-CM tissue. The consistent behavior between in-vitro and in-silico hiPSC-CMs and between in-silico hiPSC-CMs and hAdultV-CMs evidences that virtual hiPSC-CM tissues are suitable for assessing cardiac efficacy, as done in the present study through the analysis of ICaL block.

The Non‐Neuronal Cholinergic System is Causal for Cardioprotection by Hypoxic Preconditioning in Isolated Adult Rat Cardiomyocytes

Introduction Brief episodes of non-lethal myocardial ischemia/reperfusion preceding more sustained ischemia reduce lethal ischemia/reperfusion injury in all species tested so far, including humans. Although this phenomenon is known for more than 3 decades, the underlying signaling mechanisms are unclear in their details. Among various cardioprotective mediators, neuronal acetylcholine (ACh) and its signaling through cholinergic receptors is certainly involved. However, a non-neuronal cardiac cholinergic system (NNCCS) has been recently identified, suggesting a nervous system-independent, cardiomyocyte-derived ACh signaling with signal amplification by autocrine cholinergic receptor activation. Aim We aimed to examine the role of NNCCS-derived ACh as a mediator of cardioprotection. We therefore in a reductionist approach used isolated adult cardiomyocytes with hypoxic preconditioning (HPC). Methods Hearts from male Lewis rats were harvested, and adult ventricular cardiomyocytes were isolated. In suspended cardiomyocytes HPC was induced by one cycle of 10 min brief hypoxia and 20 min reoxygenation. Thirty min normoxia served as control. Afterwards, cardiomyocytes were exposed to 30 min sustained hypoxia and 5 min reoxygenation (H/R) or to 35 min normoxia as time control (TC). Cardiomyocyte viability was quantified before and after H/R or TC, respectively, as the percent fraction of rod-shaped, unstained (0.5% trypan blue) cells over all cells. In parallel experiments, physostigmine (0.2 mmol/L) was added to cardiomyocyte suspensions to block the ubiquitous ACh esterase. Intracellular ACh was quantified with and without HPC via liquid chromatography-coupled tandem mass spectrometry. In subsets, the nicotinic cholinergic receptor antagonists hexamethonium (1.0 µmol/L) and α-bungarotoxine (0.1 µmol/L) or the muscarinic cholinergic receptor antagonist atropine (0.1 µmol/L) were added to cardiomyocyte suspensions with and without HPC, respectively, and exposure to H/R or TC, respectively, afterwards. Again, cardiomyocyte viability was quantified. Results With HPC, the viability of cardiomyocytes subjected to H/R was better preserved than without (24±1% vs. 7±1%, figure A). Also, intracellular ACh was slightly (n.s.) increased after sustained hypoxia (6±2 pmol/mg protein), but significantly with HPC (13±3 pmol/mg protein) in comparison to baseline (3±1 pmol/mg protein) or TC (3±1 pmol/mg protein, figure B). However, this increase was only detectable in the presence of physostigmine (figure B). Addition of nicotinic and muscarinic cholinergic receptor antagonists attenuated the protection by HPC to the same extent (hexamethonium: 12±2%, α-bungarotoxine: 11±1%, atropine: 10±1%, figure A). Conclusion The NNCCS is activated by HPC and causal for HPC´s protection. HPC increases intracellular ACh, and cardiomyocyte protection is mediated via both nicotinic and muscarinic cholinergic receptors, suggesting an autocrine signal amplification on the cardiomyocyte level.

Cardioprotection by post-conditioning with exogenous triiodothyronine in isolated perfused rat hearts and isolated adult rat cardiomyocytes

Ischemic post-conditioning (iPoCo) by coronary re-occlusion/reperfusion during immediate reperfusion after prolonged myocardial ischemia reduces infarct size. Mechanical manipulation of culprit lesions, however, carries the risk of coronary microembolization which may obscure iPoCo’s cardioprotection. Pharmacological post-conditioning with exogenous triiodothyronine (T3) could serve as an alternative conditioning strategy. Similar to iPoCo, T3 may activate cardioprotective prosurvival pathways. We aimed to study T3’s impact on infarct size and its underlying signal transduction. Hearts were isolated from male Lewis rats (200–380 g), buffer-perfused and subjected to 30 min/120 min global zero-flow ischemia/reperfusion (I/R). In additional hearts, either iPoCo (2 × 30 s/30 s I/R) was performed or T3 (100–500 µg/L) infused at reperfusion. Infarct size was demarcated with triphenyl tetrazolium chloride staining and calculated as percent of ventricular mass. Infarct size was reduced with iPoCo to 16 ± 7% vs. 36 ± 4% with I/R only. The maximum infarct size reduction was observed with 300 µg/L T3 (14 ± 2%). T3 increased the phosphorylation of protein kinase B and mitogen extracellular-regulated-kinase 1/2, both key enzymes of the reperfusion injury salvage kinase (RISK) pathway. Pharmacological RISK blockade (RISK-BL) during reperfusion abrogated T3’s cardioprotection (35 ± 10%). Adult ventricular cardiomyocytes were isolated from buffer-perfused rat hearts and exposed to 30 min/5 min hypoxia/reoxygenation (H/R); reoxygenation was initiated without or with T3, respectively, and without or with RISK-BL, respectively. Maximal preservation of viability was observed with 500 µg/L T3 after H/R (27 ± 4% of all cells vs. 5 ± 3% in time-matched controls). Again, RISK-BL abrogated protection (11 ± 3%). Mitochondria were isolated at early reperfusion from buffer-perfused rat hearts without or with iPoCo or 300 µg/L T3, respectively, at reperfusion. T3 improved mitochondrial function (i.e.: increased respiration, adenosine triphosphate production, calcium retention capacity, and decreased reactive oxygen species formation) to a similar extent as iPoCo. T3 at reperfusion reduces infarct size by activation of the RISK pathway. T3’s protection is a cardiomyocyte phenomenon and targets mitochondria.

Modeling the complete kinetics of coxsackievirus B3 reveals human determinants of host-cell feedback.

Complete kinetic models are pervasive in chemistry but lacking in biological systems. We encoded the complete kinetics of infection for coxsackievirus B3 (CVB3), a compact and fast-acting RNA virus. The model consists of separable, detailed modules describing viral binding-delivery, translation-replication, and encapsidation. Specific module activities are dampened by the type I interferon response to viral double-stranded RNAs (dsRNAs), which is itself disrupted by viral proteinases. The experimentally validated kinetics uncovered that cleavability of the dsRNA transducer mitochondrial antiviral signaling protein (MAVS) becomes a stronger determinant of viral outcomes when cells receive supplemental interferon after infection. Cleavability is naturally altered in humans by a common MAVS polymorphism, which removes a proteinase-targeted site but paradoxically elevates CVB3 infectivity. These observations are reconciled with a simple nonlinear model of MAVS regulation. Modeling complete kinetics is an attainable goal for small, rapidly infecting viruses and perhaps viral pathogens more broadly. A record of this paper's transparent peer review process is included in the Supplemental information.

Oxidized Low-Density Lipoprotein Associates with Ventricular Stress in Young Adults and Triggers Intracellular Ca2+ Alterations in Adult Ventricular Cardiomyocytes.

Oxidized low-density lipoprotein (oxLDL) is associated with cardiac damage and causes injury to multiple cell types. We aimed to investigate the role of oxLDL in ventricular stress. We first examined the association between circulating oxLDL and N-terminal pro-brain natriuretic peptide (NT-proBNP), a marker of myocardial stress, in young subjects (30–50 years) with or without stable coronary artery disease (SCAD). oxLDL and NT-proBNP were significantly higher in subjects at high cardiovascular risk (CVR) than in subjects at low CVR and were associated independently of traditional CVR factors and C-reactive protein. Furthermore, the levels of oxLDL and NT-proBNP were significantly lower in subjects with SCAD than in peers at high CVR. To determine the intracellular mechanisms involved in the cardiac effects of oxLDL, we analyzed the in vitro effect of oxLDL on intracellular Ca2+ handling in adult rat ventricular cardiomyocytes using confocal microscopy. Acute challenge of adult ventricular cardiomyocytes to oxLDL reduced systolic Ca2+ transients and sarcoplasmic reticulum Ca2+ load. Moreover, diastolic spontaneous Ca2+ leak increased significantly after acute exposure to oxLDL. Thus, we demonstrate that oxLDL associates with NT-proBNP in young subjects, and can directly induce Ca2+ mishandling in adult ventricular cardiomyoyctes, predisposing cardiomyocytes to cardiac dysfunction and arrhythmogenicity.