Murine Ventricular Cardiomyocytes Research Articles

Establishment of an in vivo murine model of immune checkPoint inhibitors cardiotoxicity: emerging role of vascular permeability in pembrolizumab-induced cardiotoxicity

Abstract Introduction Advances in oncology have led to the discovery of immune checkpoint inhibitors (ICIs) that exhibit remarkable results in cancer patients’ survival and tumour regression. Despite their antitumor potential, some life-threatening immune-related adverse effects (AEs) including cardiotoxicity have been reported, while the pathomechanism of the cardiac AEs is obscure. Purpose We herein investigated the cardiotoxic effects of ipilimumab (IPI, anti-CTLA-4), pembrolizumab (PEM, anti-PD-1) and avelumab (AVE, anti-PD-L1) in primary murine ventricular cardiomyocytes (mAVCs) and spleenocytes in vitro and established the mechanism and progression of Pem’s cardiotoxicity in an in vivo murine model. Methods Primary mAVCs and spleenocytes were isolated and incubated with IPI, PEM and AVE (0-100 μg/ml) for 24h. Cell viability was assessed by MTT assay. Subsequently conditioned media from spleenocytes treated with the aforementioned ICIs was transferred onto the mAVCs for 24h. The experiments were repeated for molecular analyses. For the confirmation of PEM binding on the murine PD-1, human and murine PD-1 extracellular domains (ED) were biotechnologically produced and PEM binding was confirmed by circular dichroism (CD). C57BL6/J male mice were randomized into i. IgG4 and ii. Pem groups and treated for 2 (n = 5/group) and 5 weeks (n = 9/group). IgG4 and Pem were administered intraperitoneally once weekly at 2mg/ml. Pem dose was directly translated from humans. Mice underwent weekly echocardiography analysis, while at the endpoint, mice were sacrificed for myocardial sampling and histology analyses. Results IPI, PEM and AVE did not induce direct cytotoxicity on primary mAVCs, whereas IPI was excluded from the study due to unexpected spleenocyte cytotoxicity. Incubation of mAVCs with PEM and AVE conditioned media, revealed that only PEM could induce Immune cell (IC)-dependent cytotoxicity at 50 and 100 μg/ml and establishment of inflammation and autophagy in vitro. CD experiments showed that PEM binds on the murine PD-1 ED, which supports for the first time the use of the murine model for the extraction of translational results. In vivo Pem led to %Fractional shortening (FS%) decline, which was significantly reduced versus baseline at 2 weeks and further depressed at 5 weeks. At two weeks, Pem increased ICAM-1 expression as well as iNOS and beclin-1 expression in the myocardium, which was accompanied by intra-cardiac IC infiltration. At five weeks, Pem further increased vascular permeability, as shown by e-selectin and ICAM-1 upregulation and led to increase of cardiac autophagy and inflammation markers. Conclusions Herein we established for the first time a murine in vivo model of PEM cardiotoxicity. PEM induces an IC-mediated mAVCs cytotoxicity, via induction of inflammation, autophagy and ER-stress. Increased vascular permeability and dysregulated endothelial homeostasis seem to orchestrate early-on Pem’s cardiotoxicity manifestation in vivo.

Abstract 17334: Association of BIN1 Deletion With Electrical and Structural Remodeling in a Transgenic BIN1 KO Mouse Model of Heart Failure-Novel Option for Effective Therapy Using VK-II-86

Introduction: Cardiac deletion of Bridging Integrator 1 (BIN1) membrane protein produces an age-related cardiomyopathy in mice. Our hypothesis was to determine whether electrical accompanies this structural remodeling and examine the utility of VK-II-86, a structural analog of carvedilol with minimum β-blocking activity, as a potential antiarrhythmic therapeutic strategy. Methods: A cardiac-specific transgenic BIN1 KO mouse was employed. Structural and electrical cardiac changes were assessed using echocardiography and EKG (Visual Sonics-Vevo Heart Image). Murine ventricular cardiomyocytes were studied using patch-clamp techniques to examine changes in ion channel function and to study the effects of VK-II-86 on the action potential (AP). Results: Loss of BIN1 (KO) significantly reduced Left Ventricular Ejection Fraction (LVEF) and Cardiac Output (CO) when compared to WT mice, with a relative reduction of nearly 20% and 35% in EF and CO, respectively. Heterozygotic mice (HET) also exhibited significant reduction in EF and CO compared to WT mice. Loss of BIN1 (KO and HET) significantly prolonged the QT interval on surface ECG by approximately 15%. This finding correlates with the increase in AP duration (APD) demonstrated in ventricular cardiomyocytes using current clamp. BIN1 deficient hearts displayed significant reduction in expression of I to , I K1 and peak I Na (80%, 40% and 20% respectively) as well as augmented expression of late I Na (50%). This electrical remodeling is likely responsible for the increased expression of early (EAD) and late (DAD) afterdepolarization observed in APs. In BIN1 deficient ventricular cardiomyocytes, the addition of 1 μM VK-II-86 prevented the development of APD prolongation and suppressed EAD and DAD development. Conclusions: Our study adds to the understanding the role of BIN1. We demonstrate the first-time electrical remodeling in the form of QT and APD prolongation associated with BIN1 deficiency, due to reduced I to and I K1 and increase in late I Na , an increase in EAD and DAD events. Additionally, VK-II-86 is shown to normalize APD in isolated cardiomyocytes and suppress afterdepolarization events, making it a potentially novel and effective therapeutic strategy in HF associated with BIN1 deficiency.

33P Immune checkpoint inhibitor-induced cardiotoxicity is driven through inflammation, autophagy and stress

Immune checkpoint inhibitors (ICIs) are novel drugs with profound anti-cancer activity but also with adverse events. We investigated the cardiotoxic effects of ipilimumab (IPI, anti-CTLA-4), pembrolizumab (PEM, anti-PD-1) and avelumab (AVE, anti-PD-L1), in primary murine ventricular cardiomyocytes (mAVCs) and spleenocytes, which mainly consist of T- and B- lymphocytes. C57BL6/J mice were sacrificed and primary mAVCs and spleenocytes were isolated. Primary cells were incubated with IPI, PEM and AVE at a concentration range of 0-100 μg/ml for 24h and cell viability was assessed by MTT assay. Furthermore, spleenocytes were treated with IPI, PEM and AVE for 24h and their conditioned medium was added onto the mAVCs for 24h. For the confirmation of PEM binding on the murine PD-1, human and murine PD-1 extracellular domains (ED) were produced and PEM binding was confirmed by circular dichroism (CD). Human IgG4 was used as a negative control. Only IPI led to cytotoxicity in primary spleenocytes and was therefore excluded from the study, as ICIs should not induce cytotoxicity in immune cells (IC). Incubation of mAVCs with PEM and AVE conditioned media, revealed that only PEM could induce IC-dependent cytotoxicity at 50 and 100 μg/ml. The latter was accompanied by an increase in Tnfa, Inos, Rela, Ifng mRNA expression in primary spleenocytes after PEM treatment and Tnfa, Tgfb, Inos, Rela, Ifng as well as autophagy markers Lc3b, Lc3a, Becn2, Atg5 and Endoplasmic Reticulum (ER) stress markers Canx and Ddit3 increase in the primary mAVCs after treatment with PEM conditioned medium. The latter were confirmed with Western Blot analysis. We then investigated whether PEM binds on the murine PD-1 or whether the observed effects are due to an off-target effect. CD experiments showed that PEM binds on the murine PD-1 ED in a similar manner as with the human PD-1 ED used as the positive control, which supports the use of the murine model for the extraction of translational results. The binding of PEM on the murine PD-1 ED was also confirmed by in silico protein-protein docking experiments. PEM induces IC-dependent cytotoxicity on primary mAVCs via induction of inflammation, autophagy and ER-stress.

Targeting the Microtubule EB1-CLASP2 Complex Modulates NaV1.5 at Intercalated Discs.

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MiRNA-26a inhibits myocardial infarction-induced apoptosis by targeting PTEN via JAK/STAT pathways.

Acute myocardial infarction (MI) is a common cause of the morbidity and mortality of cardiovascular diseases in the world. Acute MI lead to cardiovascular output after formation of myocardial ischemia and circulatory arrest in coronary heart diseases. However, the mechanisms underlying MI injury are poorly understood. We explored the part played by miR-26a in myocardial infarction (MI). Decreased miR-26a expression in H2O2-treated newborn murine ventricular cardiomyocytes (NMVCs) was observed, as well as in the infarcted heart of MI mouse model, compared to untreated NMVCs and healthy mouse heart tissue, respectively. Conversely, the upregulation of phosphatase and tensin homolog (PTEN) was observed in H2O2-treated NMVCs, and in infarcted hearts. An MTT assay and BrdU staining showed that H2O2 treatment attenuated cell viability in NMVCs, whereas miR-26a overexpression increased cell viability. Both TUNEL assay and flow cytometry (FC) displayed that miR-26a expression suppressed H2O2-induced cell apoptosis. Besides, miR-26a overexpression suppressed the upregulation of PTEN expression in H2O2-treated NMVCs by directly binding to PTEN 3'-UTR. PI3K/Akt and JAK/STAT signal transduction pathways were found to be regulated through cross-talk between miR-26a and PTEN. Furthermore, agomiR-26a treatment in MI mouse model considerably suppressed the size of the infarcted regions, and improved cardiac activity. MiR-26a expression in MI cardiac tissues was downregulated in response to H2O2 stress, whereas it could still protect against cell death by modulation of the PI3K/Akt and JAK/STAT signal transduction pathways by directly targeting PTEN.

MicroRNA-1278 ameliorates the inflammation of cardiomyocytes during myocardial ischemia by targeting both IL-22 and CXCL14

AimsThis study aimed to elucidate the role of microRNAs (miRNAs) during myocardial infarction (MI) development in vivo and in vitro. Main methodsDifferentially expressed miRNAs between heart tissue from the MI mouse model and the control mouse were identified via microarray. Quantitative PCR (qPCR) and western blotting (WB) were performed to examine the expression levels of miRNAs and proteins, respectively. EdU-staining and colony formation assay were performed to assess cell viability and growth. Annexin V- and PI-staining-based flow cytometry was used to assess cell apoptosis. An MI mouse model was also established to study the function of miR-1278 in vivo. Key findingsThe levels of miR-1278 were reduced in the infarct regions of heart tissues of the MI mouse model and in H2O2-treated newborn murine ventricular cardiomyocytes (NMVCs) compared to those in the heart tissues of healthy mice and non-treated NMVCs. H2O2 treatment suppressed the proliferation of NMVCs, while miR-1278 upregulation improved it. Moreover, we found that miR-1278 inhibited the upregulation of IL-22 and CXCL14 expression in H2O2-treated NMVCs by directly binding with the 3′-UTRs of both IL-22 and CXCL14. Furthermore, restoration of IL-22 and CXCL14 in H2O2-treated NMVCs promoted miR-1278-induced inflammation and apoptosis. Administration of agomiR-1278 to the MI mouse model significantly improved cardiac activity. SignificanceCollectively, our findings illustrate that the expression of miR-1278 is low in H2O2-treated NMVCs and post-MI cardiac tissues, and the overexpression of miR-1278 in these protects against cell death by modulating IL-22 and CXCL14 expression.

MicroRNA-200a represses myocardial infarction-related cell death and inflammation by targeting the Keap1/Nrf2 and β-catenin pathways

BackgroundAcute myocardial infarction (MI) is a main cause of emergency death in the world. MicroRNAs (miRs/miRNAs) are a series of small non-coding RNA molecules, which regulate cardiovascular disorders that involve MI. In this study, we explored the function of miR-200a in MI treatment. MethodsWe observed down-regulation of miR-200a levels and up-regulation of Keap1 and β-catenin levels in H2O2-treated newborn murine ventricular cardiomyocytes (NMVCs) and the infarcted heart tissues of MI mouse models, compared to the non-treated NMVCs and normal heart tissues of healthy mice. ResultsCCK-8 and colony formation assays indicated the reduction in NMVC vitality due to H2O2 treatment and the recovery of cell vitality due to miR-200a overexpression, respectively. Flow cytometry with Annexin and PI staining indicated the inhibition of H2O2-triggered cell apoptosis through ectopically expressed miR-200a. Western blotting and ELISA analyses that detected pro-inflammatory cell factors [interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α] confirmed that miR-200a prevented H2O2-induced NMVC inflammation. Moreover, miR-200a inhibited up-regulation of Keap1 and β-catenin expression in H2O2-treated NMVCs by directly binding with the 3′-UTR regions of both Keap1 and β-catenin. Furthermore, overexpression of Keap1 and β-cateninin in H2O2-treated NMVCs with recovered miR-200a elevated inflammation and apoptosis, respectively. ConclusionThe results showed that miR-200a expression was inhibited in murine cardiomyocytes due to H2O2 stress in MI cardiac tissues and overexpressed miR-200a could protect the cells from death by regulating the Keap1/Nrf2 and β-catenin signal transduction pathways.

Inhibition of cardiac potassium currents by oxidation-activated protein kinase A contributes to early afterdepolarizations in the heart.

Reactive oxygen species (ROS) have been shown to prolong cardiac action potential duration resulting in afterdepolarizations, the cellular basis of triggered arrhythmias. As previously shown, protein kinase A type I (PKA I) is readily activated by oxidation of its regulatory subunits. However, the relevance of this mechanism of activation for cardiac pathophysiology is still elusive. In this study, we investigated the effects of oxidation-activated PKA I on cardiac electrophysiology. Ventricular cardiomyocytes were isolated from redox-dead PKA-RI Cys17Ser knock-in (KI) and wild-type (WT) mice and exposed to H2O2 (200 µmol/L) or vehicle (Veh) solution. In WT myocytes, exposure to H2O2 significantly increased oxidation of the regulatory subunit I (RI) and thus its dimerization (threefold increase in PKA RI dimer). Whole cell current clamp and voltage clamp were used to measure cardiac action potentials (APs), transient outward potassium current (Ito) and inward rectifying potassium current (IK1), respectively. In WT myocytes, H2O2 exposure significantly prolonged AP duration due to significantly decreased Ito and IK1 resulting in frequent early afterdepolarizations (EADs). Preincubation with the PKA-specific inhibitor Rp-8-Br-cAMPS (10 µmol/L) completely abolished the H2O2-dependent decrease in Ito and IK1 in WT myocytes. Intriguingly, H2O2 exposure did not prolong AP duration, nor did it decrease Ito, and only slightly enhanced EAD frequency in KI myocytes. Treatment of WT and KI cardiomyocytes with the late INa inhibitor TTX (1 µmol/L) completely abolished EAD formation. Our results suggest that redox-activated PKA may be important for H2O2-dependent arrhythmias and could be important for the development of specific antiarrhythmic drugs.NEW & NOTEWORTHY Oxidation-activated PKA type I inhibits transient outward potassium current (Ito) and inward rectifying potassium current (IK1) and contributes to ROS-induced APD prolongation as well as generation of early afterdepolarizations in murine ventricular cardiomyocytes.

Empagliflozin inhibits Na+ /H+ exchanger activity in human atrial cardiomyocytes.

AimsRecent clinical trials have proven gliflozins to be cardioprotective in diabetic and non‐diabetic patients. However, the underlying mechanisms are incompletely understood. A potential inhibition of cardiac Na+/H+ exchanger 1 (NHE1) has been suggested in animal models. We investigated the effect of empagliflozin on NHE1 activity in human atrial cardiomyocytes.Methods and resultsExpression of NHE1 was assessed in human atrial and ventricular tissue via western blotting. NHE activity was measured as the maximal slope of pH recovery after NH4 + pulse in isolated carboxy‐seminaphtarhodafluor 1 (SNARF1)‐acetoxymethylester‐loaded murine ventricular and human atrial cardiomyocytes. NHE1 is abundantly expressed in human atrial and ventricular tissue. Interestingly, compared with patients without heart failure (HF), atrial NHE1 expression was significantly increased in patients with HF with preserved ejection fraction and atrial fibrillation. The largest increase in atrial and ventricular NHE1 expression, however, was observed in patients with end‐stage HF undergoing heart transplantation. Importantly, acute exposure to empagliflozin (1 μmol/L, 10 min) significantly inhibited NHE activity to a similar extent in human atrial myocytes and mouse ventricular myocytes. This inhibition was also achieved by incubation with the well‐described selective NHE inhibitor cariporide (10 μmol/L, 10 min).ConclusionsThis is the first study systematically analysing NHE1 expression in human atrial and ventricular myocardium of HF patients. We show that empagliflozin inhibits NHE in human cardiomyocytes. The extent of NHE inhibition was comparable with cariporide and may potentially contribute to the improved outcome of patients in clinical trials.

Sacubitrilat reduces pro-arrhythmogenic sarcoplasmic reticulum Ca2+ leak in human ventricular cardiomyocytes of patients with end-stage heart failure.

AimsInhibition of neprilysin and angiotensin II receptor by sacubitril/valsartan (Val) (LCZ696) reduces mortality in heart failure (HF) patients compared with sole inhibition of renin–angiotensin system. Beneficial effects of increased natriuretic peptide levels upon neprilysin inhibition have been proposed, whereas direct effects of sacubitrilat (Sac) (LBQ657) on myocardial Ca2+ cycling remain elusive.Methods and resultsConfocal microscopy (Fluo‐4 AM) was used to investigate pro‐arrhythmogenic sarcoplasmic reticulum (SR) Ca2+ leak in freshly isolated murine and human ventricular cardiomyocytes (CMs) upon Sac (40 μmol/L)/Val (13 μmol/L) treatment. The concentrations of Sac and Val equalled plasma concentrations of LCZ696 treatment used in PARADIGM‐HF trial. Epifluorescence microscopy measurements (Fura‐2 AM) were performed to investigate effects on systolic Ca2+ release, SR Ca2+ load, and Ca2+‐transient kinetics in freshly isolated murine ventricular CMs. The impact of Sac on myocardial contractility was evaluated using in toto‐isolated, isometrically twitching ventricular trabeculae from human hearts with end‐stage HF. Under basal conditions, the combination of Sac/Val did not influence diastolic Ca2+‐spark frequency (CaSpF) nor pro‐arrhythmogenic SR Ca2 leak in isolated murine ventricular CMs (n CMs/hearts = 80/7 vs. 100/7, P = 0.91/0.99). In contrast, Sac/Val treatment reduced CaSpF by 35 ± 9% and SR Ca2+ leak by 45 ± 9% in CMs put under catecholaminergic stress (isoproterenol 30 nmol/L, n = 81/7 vs. 62/7, P < 0.001 each). This could be attributed to Sac, as sole Sac treatment also reduced both parameters by similar degrees (reduction of CaSpF by 57 ± 7% and SR Ca2+ leak by 76 ± 5%; n = 101/4 vs. 108/4, P < 0.01 each), whereas sole Val treatment did not. Systolic Ca2+ release, SR Ca2+ load, and Ca2+‐transient kinetics including SERCA activity (k SERCA) were not compromised by Sac in isolated murine CMs (n = 41/6 vs. 39/6). Importantly, the combination of Sac/Val and Sac alone also reduced diastolic CaSpF and SR Ca2+ leak (reduction by 74 ± 7%) in human left ventricular CMs from patients with end‐stage HF (n = 71/8 vs. 78/8, P < 0.05 each). Myocardial contractility of human ventricular trabeculae was not acutely affected by Sac treatment as the developed force remained unchanged over a time course of 30 min (n trabeculae/hearts = 3/3 vs. 4/3).ConclusionThis study demonstrates that neprilysin inhibitor Sac directly improves Ca2+ homeostasis in human end‐stage HF by reducing pro‐arrhythmogenic SR Ca2+ leak without acutely affecting systolic Ca2+ release and inotropy. These effects might contribute to the mortality benefits observed in the PARADIGM‐HF trial.

Activation of endogenous protein phosphatase 1 enhances the calcium sensitivity of the ryanodine receptor type 2 in murine ventricular cardiomyocytes.

Increased protein phosphatase 1 (PP-1) activity has been found in end stage human heart failure. Although PP-1 has been extensively studied, a detailed understanding of its role in the excitation-contraction coupling mechanism, in normal and diseased hearts, remains elusive. The present study investigates the functional effect of the PP-1 activity on local Ca2+ release events in ventricular cardiomyocytes, by using an activating peptide (PDP3) for the stimulation of the endogenous PP-1 protein. We report that acute de-phosphorylation may increase the sensitivity of RyR2 channels to Ca2+ in situ, and that the RyR2-serine2808 phosphorylation site may mediate such a process. Our approach unmasks the functional importance of PP-1 in the regulation of RyR2 activity, suggesting a potential role in the generation of a pathophysiological sarcoplasmic reticulum Ca2+ leak in the diseased heart. Changes in cardiac ryanodine receptor (RyR2) phosphorylation are considered to be important regulatory and disease related post-translational protein modifications. The extent of RyR2 phosphorylation is mainly determined by the balance of the activities of protein kinases and phosphatases, respectively. Increased protein phosphatase-1 (PP-1) activity has been observed in heart failure, although the regulatory role of this enzyme on intracellular Ca2+ handling remains poorly understood. To determine the physiological and pathophysiological significance of increased PP-1 activity, we investigated how the PP-1 catalytic subunit (PP-1c) alters Ca2+ sparks in permeabilized cardiomyocytes and we also applied a PP-1-disrupting peptide (PDP3) to specifically activate endogenous PP-1, including the one anchored on the RyR2 macromolecular complex. We compared wild-type and transgenic mice in which the usually highly phosphorylated site RyR2-S2808 has been ablated to investigate its involvement in RyR2 modulation (S2808A+/+ ). In wild-type myocytes, PP-1 increased Ca2+ spark frequency by two-fold, followed by depletion of the sarcoplasmic reticulum Ca2+ store. Similarly, PDP3 transiently increased spark frequency and decreased sarcoplasmic reticulum Ca2+ load. RyR2 Ca2+ sensitivity, which was assessed by Ca2+ spark recovery analysis, was increased in the presence of PDP3 compared to a negative control peptide. S2808A+/+ cardiomyocytes did not respond to both PP-1c and PDP3 treatment. Our results suggest an increased Ca2+ sensitivity of RyR2 upon de-phosphorylation by PP-1. Furthermore, we have confirmed the S2808 site as a target for PP-1 and as a potential link between RyR2s modulation and the cellular response.

Mepivacaine reduces calcium transients in isolated murine ventricular cardiomyocytes

BackgroundThe potential mechanism of mepivacaine’s myocardial depressant effect observed in papillary muscle has not yet been investigated at cellular level. Therefore, we evaluated mepivacaine’s effects on Ca2+ transient in isolated adult mouse cardiomyocytes.MethodsSingle ventricular myocytes were enzymatically isolated from wild-type C57Bl/6 mice and loaded with 10 μM fluorescent Ca2+ indicator Fluo-4-AM to record intracellular Ca2+ transients upon electrical stimulation. The mepivacaine effects at half-maximal inhibitory concentration (IC50) was determined on calibrated cardiomyocytes’ Ca2+ transients by non-parametric statistical analyses on biophysical parameters. Combination of mepivacaine with NCX blockers ORM-10103 or NiCl2 were used to test a possible mechanism to explain mepivacaine-induced Ca2+ transients’ reduction.ResultsA significant inhibition at mepivacaine’s IC50 (50 μM) on Ca2+ transients was measured in biophysical parameters such as peak (control: 528.6 ± 73.61 nM vs mepivacaine: 130.9 ± 15.63 nM; p < 0.05), peak area (control: 401.7 ± 63.09 nM*s vs mepivacaine: 72.14 ± 10.46 nM*s; p < 0.05), slope (control: 7699 ± 1110 nM/s vs mepivacaine: 1686 ± 226.6 nM/s; p < 0.05), time to peak (control: 107.9 ± 8.967 ms vs mepivacaine: 83.61 ± 7.650 ms; p < 0.05) and D50 (control: 457.1 ± 47.16 ms vs mepivacaine: 284.5 ± 22.71 ms; p < 0.05). Combination of mepivacaine with NCX blockers ORM-10103 or NiCl2 showed a significant increase in the baseline of [Ca2+] and arrhythmic activity upon electrical stimulation.ConclusionAt cellular level, mepivacaine blocks Na+ channels, enhancing the reverse mode activity of NCX, leading to a significant reduction of Ca2+ transients. These results suggest a new mechanism for the mepivacaine-reduction contractility effect.

RyR2 regulates Cx43 hemichannel intracellular Ca2+-dependent activation in cardiomyocytes.

Connexin-based gap junctions are crucial for electrical communication in the heart; they are each composed of two docked hemichannels (HCs), supplied as unpaired channels via the sarcolemma. When open, an unpaired HC forms a large pore, high-conductance and Ca2+-permeable membrane shunt pathway that may disturb cardiomyocyte function. HCs composed of connexin 43 (Cx43), a major cardiac connexin, can be opened by electrical stimulation but only by very positive membrane potentials. Here, we investigated the activation of Cx43 HCs in murine ventricular cardiomyocytes voltage-clamped at -70 mV. Using whole-cell patch-clamp, co-immunoprecipitation, western blot analysis, immunocytochemistry, proximity ligation assays, and protein docking studies, we found that stimulation of ryanodine receptors (RyRs) triggered unitary currents with a single-channel conductance of ∼220 pS, which were strongly reduced by Cx43 knockdown. Recordings under Ca2+-clamp conditions showed that both RyR activation and intracellular Ca2+ elevation were necessary for HC opening. Proximity ligation studies indicated close Cx43-RyR2 apposition (<40 nm), and both proteins co-immunoprecipitated indicating physical interaction. Molecular modelling suggested a strongly conserved RyR-mimicking peptide sequence (RyRHCIp), which inhibited RyR/Ca2+ HC activation but not voltage-triggered activation. The peptide also slowed down action potential repolarization. Interestingly, alterations in the concerned RyR sequence are known to be associated with primary familial hypertrophic cardiomyopathy. Our results demonstrate that Cx43 HCs are intimately linked to RyRs, allowing them to open at negative diastolic membrane potential in response to RyR activation.

P1596Interaction of CaMKII and NaV1.8 modulates cardiac electrophysiology in human heart failure

Abstract Introduction In human heart failure, electrical remodeling contributes to the risk of arrhythmia generation. Increased expression of Ca/Calmodulin-dependent protein kinase IIδ (CaMKIIδ) and an enhanced persistent Na current (INaL) have been linked to arrhythmogenesis. CaMKIIδ increases INaL via regulation of sodium channels thereby contributing to arrhythmias through early- and delayed-afterdepolarizations (EADs and DADs). Genome-wide association studies (GWAS) have described the implication of the neuronal sodium channel isoform NaV1.8 (SCN10A) in cardiac electrophysiology showing modulation in cardiac conduction. We showed that the expression of the isoform Nav1.8 is significantly increased in human failing cardiomyocytes and contributes substantially to the enhanced INaL. Purpose We investigated a potential interaction of CaMKIIδ and NaV1.8 and thereby its role in arrhythmia generation and electrophysiology in human and murine failing hearts. Methods Cardiomyocytes were isolated from explanted failing hearts and CaMKIIδ transgenic (TG) mice. We performed immunostainings and co-immunoprecipitation (Co-IP) to show interactions of CaMKIIδ and Nav1.8 in isolated cardiomyocytes and homogenates. Whole-cell patch clamp experiments were conducted in isolated human and murine ventricular cardiomyocytes. Additionally, Ca2+ transients were measured using epifluorescence microscopy with the Ca2+ dye fura-2 (10μmol/L) whereas Ca2+ sparks measurements were performed by using confocal microscopy with the Ca2+ dye fluo-4 (10μmol/L). PF-01247324 is a novel specific NaV1.8 inhibitor (orally bioavailable; 1 μmol/L) and autocamtide inhibitory peptide (AIP, 1 μmol/L) was used to inhibit CaMKIIδ. Results Co-immunoprecipitation experiments revealed an association of CaMKIIδ and Nav1.8 in human homogenates compared to healthy controls. Furthermore, immunohistochemistry stainings in isolated human cardiomyocytes showed a co-localization of CaMKIIδ and NaV1.8 at the intercalated disc and t-tubules. We observed a significant reduction of INaL integral and proarrhythmic SR-Ca2+ spark frequency (CaSpF) after addition of either PF-01247324 or the CaMKIIδ inhibitor AIP in failing human and murine ventricular cardiomyocytes. When PF-01247324 and AIP were added together, the decrease in INaL integral and CaSpF was comparable to PF-01247324 alone in human failing cardiomyocytes. Inhibition of NaV1.8 did not show an effect on Ca2+ transient amplitude or Ca2+ transient decay at different stimulation frequencies in CaMKIIδ TG cardiomyocytes. Conclusion Our results demonstrate the significance of both CaMKIIδ and NaV1.8 in INaL generation and their detrimental interaction. This data suggest that increased CaMKIIδ activity plays a substantial role for the activation of NaV1.8-mediated late sodium current and SR-Ca2+ leak.

P3829Antiarrhythmic effects of Sacubitrilat (LBQ657) on Ca2+ homeostasis in ventricular cardiomyocytes

Abstract Background and objectives Simultaneous inhibition of neprilysin and angiotensin II receptors by sacubitril/valsartan was shown to significantly reduce morbidity and mortality in heart failure patients compared to sole interference with the renin angiotensin system. Beneficial effects of increased levels of natriuretic peptides following neprilysin inhibition have been suggested, whereas direct effects of sacubitrilat on myocardial Ca2+ cycling properties remain elusive. Methods and results Under basal conditions the combination of active neprilysin-inhibitior sacubitrilat (LBQ657) and angiotensin II receptor inhibitor valsartan did not influence diastolic Ca2+ spark frequency (CaSpF) nor arrhythmogenic SR Ca2+ leak in murine ventricular cardiomyocytes (confocal microscopy, n CMs/hearts=80/7 vs. 100/7, P=0.91/0.99). In contrast, sacubitrilat/valsartan treatment significanty reduced CaSpF by 35±9% and SR Ca2+ leak by 45±9% in CMs that had been put under catecholaminergic stress (isoproterenol 10nM, n=81/7 vs. 62/7, P&lt;0.001 both). This effect could be clearly be attributed to the neprilysin inhibitor sacubitrilat as sole sacubitrilat treatment also reduced both parameters by similar degrees (reduction of CaSpF by 57±7% and SR Ca2+ leak by 76±5%; n=101/4 vs. 108/4, P&lt;0.01 both) whereas sole valsartan treatment did not affect diastolic SR Ca2+ leak. Of note, systolic Ca2+ release, SR Ca2+ load and Ca2+ transient kinetics of murine CMs were not compromised upon treatment with sacubitrilat (epifluorescence microscopy, n=41/6 vs. 39/6). Importantly, sacubitrilat/valsartan in combination as well as sacubitrilat alone also reduced diastolic CaSpF and SR Ca2+ leak by 40–74% in human left-ventricular CMs from patients with end-stage heart failure (n=71/8 vs. 78/8, P&lt;0.05). Conclusion This study demonstrates that neprilysin-inhibition directly exerts beneficial effects on Ca2+ homeostasis in human heart failure. We can show for the first time that neprilysin-inhibition by sacubitrilat yields a strong reduction of arrhythmogenic SR Ca2+ leak without affecting systolic Ca2+ release. These effects might contribute to the mortality benefit of sacubitril/valsartan treatment in the PARADIGM Study. Acknowledgement/Funding THF was funded by the Deutsche Forschungsgemeinschaft (DFG) through the SFB 1002 (A11). SS is supported by the Marga und Walter Boll-Stiftung.

A Distinct Pool of Nav1.5 Channels at the Lateral Membrane of Murine Ventricular Cardiomyocytes.

Background: In cardiac ventricular muscle cells, the presence of voltage-gated sodium channels Nav1.5 at the lateral membrane depends in part on the interaction between the dystrophin–syntrophin complex and the Nav1.5 C-terminal PDZ-domain-binding sequence Ser-Ile-Val (SIV motif). α1-Syntrophin, a PDZ-domain adaptor protein, mediates the interaction between Nav1.5 and dystrophin at the lateral membrane of cardiac cells. Using the cell-attached patch-clamp approach on cardiomyocytes expressing Nav1.5 in which the SIV motif is deleted (ΔSIV), sodium current (INa) recordings from the lateral membrane revealed a SIV-motif-independent INa. Since immunostaining has suggested that Nav1.5 is expressed in transverse (T-) tubules, this remaining INa might be carried by channels in the T-tubules. Of note, a recent study using heterologous expression systems showed that α1-syntrophin also interacts with the Nav1.5 N-terminus, which may explain the SIV-motif independent INa at the lateral membrane of cardiomyocytes.Aim: To address the role of α1-syntrophin in regulating the INa at the lateral membrane of cardiac cells.Methods and Results: Patch-clamp experiments in cell-attached configuration were performed on the lateral membranes of wild-type, α1-syntrophin knockdown, and ΔSIV ventricular mouse cardiomyocytes. Compared to wild-type, a reduction of the lateral INa was observed in myocytes from α1-syntrophin knockdown hearts. Similar to ΔSIV myocytes, a remaining INa was still recorded. In addition, cell-attached INa recordings from lateral membrane did not differ significantly between non-detubulated and detubulated ΔSIV cardiomyocytes. Lastly, we obtained evidence suggesting that cell-attached patch-clamp experiments on the lateral membrane cannot record currents carried by channels in T-tubules such as calcium channels.Conclusion: Altogether, these results suggest the presence of a sub-pool of sodium channels at the lateral membrane of cardiomyocytes that is independent of α1-syntrophin and the PDZ-binding motif of Nav1.5, located in membrane domains outside of T-tubules. The question of a T-tubular pool of Nav1.5 channels, however, remains open.

Cardiac sodium-dependent glucose cotransporter 1 is a novel mediator of ischaemia/reperfusion injury

We previously reported that sodium-dependent glucose cotransporter 1 (SGLT1) is highly expressed in cardiomyocytes and is further up-regulated in ischaemia. This study aimed to determine the mechanisms by which SGLT1 contributes to ischaemia/reperfusion (I/R) injury. Mice with cardiomyocyte-specific knockdown of SGLT1 (TGSGLT1-DOWN) and wild-type controls were studied. In vivo, the left anterior descending coronary artery was ligated for 30 min and reperfused for 48 h. Ex vivo, isolated perfused hearts were exposed to 20 min no-flow and up to 2 h reperfusion. In vitro, HL-1 cells and isolated adult murine ventricular cardiomyocytes were exposed to 1 h hypoxia and 24 h reoxygenation (H/R). We found that TGSGLT1-DOWN hearts were protected from I/R injury in vivo and ex vivo, with decreased infarct size, necrosis, dysfunction, and oxidative stress. 5'-AMP-activated protein kinase (AMPK) activation increased SGLT1 expression, which was abolished by extracellular signal-related kinase (ERK) inhibition. Co-immunoprecipitation studies showed that ERK, but not AMPK, interacts directly with SGLT1. AMPK activation increased binding of the hepatocyte nuclear factor 1 and specificity protein 1 transcription factors to the SGLT1 gene, and HuR to SGLT1 mRNA. In cells, up-regulation of SGLT1 during H/R was abrogated by AMPK inhibition. Co-immunoprecipitation studies showed that SGLT1 interacts with epidermal growth factor receptor (EGFR), and EGFR interacts with protein kinase C (PKC). SGLT1 overexpression activated PKC and NADPH oxidase 2 (Nox2), which was attenuated by PKC inhibition, EGFR inhibition, and/or disruption of the interaction between EGFR and SGLT1. During ischaemia, AMPK up-regulates SGLT1 through ERK, and SGLT1 interacts with EGFR, which in turn increases PKC and Nox2 activity and oxidative stress. SGLT1 may represent a novel therapeutic target for mitigating I/R injury.

Excitation of murine cardiac myocytes by nanosecond pulsed electric field.

Opening of voltage-gated sodium channels takes tens to hundreds of microseconds, and mechanisms of their opening by nanosecond pulsed electric field (nsPEF) stimuli remain elusive. This study was aimed at uncovering the mechanisms of how nsPEF elicits action potentials (APs) in cardiomyocytes. Fluorescent imaging of optical APs (FluoVolt) and Ca2+ -transients (Fluo-4) was performed in enzymatically isolated murine ventricular cardiomyocytes stimulated by 200-nanosecond trapezoidal pulses. nsPEF stimulation evoked tetrodotoxin-sensitive APs accompanied or preceded by slow sustained depolarization (SSD) and, in most cells, by transient afterdepolarization waves. SSD threshold was lower than the AP threshold (1.26 ± 0.03 vs 1.34 ± 0.03 kV/cm, respectively, P < 0.001). Inhibition of l-type calcium and sodium-calcium exchanger currents reduced the SSD amplitude and increased the AP threshold ( P < 0.05). The threshold for Ca 2+ -transients (1.40 ± 0.04 kV/cm) was not significantly affected by a tetrodotoxin-verapamil cocktail, suggesting the activation of a Ca 2+ entry pathway independent from the opening of Na + or Ca 2+ voltage-gated channels. Removal of external Ca 2+ decreased the SSD amplitude ( P = 0.004) and blocked Ca 2+ -transients but not APs. The incidence of transient afterdepolarization waves was decreased by verapamil and by removal of external Ca 2+ ( P = 0.002). The study established that nsPEF stimulation caused calcium entry into cardiac myocytes (including routes other than voltage-gated calcium channels) and SSD. Tetrodotoxin-sensitive APs were mediated by SSD, whose amplitude depended on the calcium entry. Plasma membrane electroporation was the most likely primary mechanism of SSD with additional contribution from l-type calcium and sodium-calcium exchanger currents.

Transcriptomic analyses of murine ventricular cardiomyocytes

Mice are used universally as model organisms for studying heart physiology, and a plethora of genetically modified mouse models exist to study cardiac disease. Transcriptomic data for whole-heart tissue are available, but not yet for isolated ventricular cardiomyocytes. Our lab therefore collected comprehensive RNA-seq data from wildtype murine ventricular cardiomyocytes as well as from knockout models of the ion channel regulators CASK, dystrophin, and SAP97. We also elucidate ion channel expression from wild-type cells to help forward the debate about which ion channels are expressed in cardiomyocytes. Researchers studying the heart, and especially cardiac arrhythmias, may benefit from these cardiomyocyte-specific transcriptomic data to assess expression of genes of interest.

SR Ca2+-leak and disordered excitation-contraction coupling as the basis for arrhythmogenic and negative inotropic effects of acute ethanol exposure

AimsEthanol has acute negative inotropic and arrhythmogenic effects. The underlying mechanisms, however, are largely unknown. Sarcoplasmic reticulum Ca2+-leak is an important mechanism for reduced contractility and arrhythmias. Ca2+-leak can be induced by oxidative stress and Ca2+/Calmodulin-dependent protein kinase II (CaMKII). Therefore, we investigated the influence of acute ethanol exposure on excitation-contraction coupling in atrial and ventricular cardiomyocytes. Methods and resultsIsolated human atrial and murine atrial or ventricular cardiomyocytes were preincubated for 30 min and then superfused with control solution or solution containing ethanol. Ethanol had acute negative inotropic and positive lusitropic effects in human atrial muscle strips and murine ventricular cardiomyocytes. Accordingly, Ca2+-imaging indicated lower Ca2+-transient amplitudes and increased SERCA2a activity, while myofilament Ca2+-sensitivity was reduced. SR Ca2+-leak was assessed by measuring Ca2+-sparks. Ethanol induced severe SR Ca2+-leak in human atrial cardiomyocytes (calculated leak: 4.60 ± 0.45 mF/F0 vs 1.86 ± 0.26 in control, n ≥ 80). This effect was dose-dependent, while spontaneous arrhythmogenic Ca2+-waves increased ~5-fold, as investigated in murine cardiomyocytes. Delayed afterdepolarizations, which can result from increased SR Ca2+-leak, were significantly increased by ethanol. Measurements using the reactive oxygen species (ROS) sensor CM-H2DCFDA showed increased ROS-stress in ethanol treated cells. ROS-scavenging with N-acetylcysteine prevented negative inotropic and positive lusitropic effects in human muscle strips. Ethanol-induced Ca2+-leak was abolished in mice with knockout of NOX2 (the main source for ROS in cardiomyocytes). Importantly, mice with oxidation-resistant CaMKII (Met281/282Val mutation) were protected from ethanol-induced Ca2+-leak. ConclusionWe show for the first time that ethanol acutely induces strong SR Ca2+-leak, also altering excitation-contraction coupling. Acute negative inotropic effects of ethanol can be explained by reduced systolic Ca2+-release. Mechanistically, ROS-production via NOX2 and oxidative activation of CaMKII appear to play central roles. This provides a mechanism for the arrhythmogenic and negative inotropic effects of ethanol and suggests a druggable target (CaMKII).