Membrane Potential In Cardiomyocytes Research Articles

Epidural administration of large dose of opioid μ receptor agonist may impair cardiac functions and myocardial viability via desensitizing transient receptor potential vanilloid 1

The incidence of postoperative myocardial injury remains high as the underlying pathogenesis is still unknown. The dorsal root ganglion (DRG) neurons express transient receptor potential vanilloid 1 (TRPV1) and its downstream effector, calcitonin gene-related peptide (CGRP) participating in transmitting pain signals and cardiac protection. Opioids remain a mainstay therapeutic option for moderate-to-severe pain relief clinically, as a critical component of multimodal postoperative analgesia via intravenous and epidural delivery. Evidence indicates the interaction of opioids and TRPV1 activities in DRG neurons. Here, we verify the potential impairment of myocardial viability by epidural usage of opioids in postoperative analgesia. We found that large dose of epidural morphine (50 μg) significantly worsened the cardiac performance (+dP/dtmax reduction by 11% and -dP/dtmax elevation by 24%, all P < 0.001), the myocardial infarct size (morphine vs Control, 0.54 ± 0.09 IS/AAR vs. 0.23 ± 0.06 IS/AAR, P < 0.001) and reduced CGRP in the myocardium (morphine vs. Control, 9.34 ± 2.24 pg/mg vs. 21.23 ± 4.32 pg/mg, P < 0.001), while induced definite suppression of nociception in the postoperative animals. It was demonstrated that activation of μ-opioid receptor (μ-OPR) induced desensitization of TRPV1 by attenuating phosphorylation of the channel in the dorsal root ganglion neurons, via inhibiting the accumulation of cAMP. CGRP may attenuated the buildup of ROS and the reduction of mitochondrial membrane potential in cardiomyocytes induced by hypoxia/reoxygenation. The findings of this study indicate that epidurally giving large dose of μ-OPR agonist may aggravate myocardial injury by inhibiting the activity of TRPV1/CGRP pathway.

P18-07: A pesticide, fludioxonil, induced mitochondrial toxicity by increasing reactive oxygen species production and decreasing mitochondrial membrane potential in cardiomyocytes

P18-07: A pesticide, fludioxonil, induced mitochondrial toxicity by increasing reactive oxygen species production and decreasing mitochondrial membrane potential in cardiomyocytes

Abstract 10415: Dysregulation of FOXO-FOXM1 Signaling Inhibits Maturation of IPSC-Derived Cardiomyocytes in 3D Suspension Culture

Directed differentiation of human pluripotent stem cells (PSC) into cardiomyocytes via manipulation of Wnt signaling leads to generation of immature cardiomyocytes, more closely resembling a fetal state. It has become increasingly apparent that metabolic parameters regulate of cardiomyocyte maturation. The forkhead box (FOX) family of transcription factors has previously been shown to regulate metabolic phenotype in neonatal cardiomyocytes through a balance between FOXO and FOXM proteins. Therefore, we hypothesized that dysregulation of FOXO-FOXM1 signaling inhibits maturation of iPSC-derived cardiomyocytes (iPSC-CMs). We cultured iPSC-CMs in 3D suspension culture using an orbital shaker. iPSC-CMs were treated with RCM-1 (FOXM1 inhibitor), LOM612 (FOXO nuclear translocator), or AS1842856 (FOXO inhibitor) starting 2 days after onset of beating. We found that inhibition of FOXO with AS1842856 resulted in loss of expression of cardiac-specific markers such as cardiac troponin T as well as loss of spontaneous beating. In contrast, inhibition of FOXM1 with RCM-1 or activation of FOXO with LOM612 resulted in retention of a cardiomyocyte phenotype with continued expression of cardiac troponin T but with significantly increased expression membrane protein expression of Kir2.1 (Figure), the protein largely responsible for maintaining the resting membrane potential in cardiomyocytes. These results suggest that inhibition of FOXM1 and/or activation of FOXO signaling may facilitate maturation of iPSC-derived cardiomyocytes. Figure. (A) % of TNNT2+ iPSC-derived cardiomyocytes that express Kir2.1 and (B) Mean fluorescence intensity of Kir2.1 in iPSC-derived cardiomyocytes after treatment with DMSO (vehicle control), RCM-1, LOM612, or AS1842856. *p&lt;0.05, ***p&lt;0.001, ****p&lt;0.0001 by one-way ANOVA with Dunnett’s multiple comparisons test.

Impairment of Multiple Mitochondrial Energy Metabolism Pathways in the Heart of Chagas Disease Cardiomyopathy Patients.

Chagas disease cardiomyopathy (CCC) is an inflammatory dilated cardiomyopathy occurring in 30% of the 6 million infected with the protozoan Trypanosoma cruzi in Latin America. Survival is significantly lower in CCC than ischemic (IC) and idiopathic dilated cardiomyopathy (DCM). Previous studies disclosed a selective decrease in mitochondrial ATP synthase alpha expression and creatine kinase activity in CCC myocardium as compared to IDC and IC, as well as decreased in vivo myocardial ATP production. Aiming to identify additional constraints in energy metabolism specific to CCC, we performed a proteomic study in myocardial tissue samples from CCC, IC and DCM obtained at transplantation, in comparison with control myocardial tissue samples from organ donors. Left ventricle free wall myocardial samples were subject to two-dimensional electrophoresis with fluorescent labeling (2D-DIGE) and protein identification by mass spectrometry. We found altered expression of proteins related to mitochondrial energy metabolism, cardiac remodeling, and oxidative stress in the 3 patient groups. Pathways analysis of proteins differentially expressed in CCC disclosed mitochondrial dysfunction, fatty acid metabolism and transmembrane potential of mitochondria. CCC patients’ myocardium displayed reduced expression of 22 mitochondrial proteins belonging to energy metabolism pathways, as compared to 17 in DCM and 3 in IC. Significantly, 6 beta-oxidation enzymes were reduced in CCC, while only 2 of them were down-regulated in DCM and 1 in IC. We also observed that the cytokine IFN-gamma, previously described with increased levels in CCC, reduces mitochondrial membrane potential in cardiomyocytes. Results suggest a major reduction of mitochondrial energy metabolism and mitochondrial dysfunction in CCC myocardium which may be in part linked to IFN-gamma. This may partially explain the worse prognosis of CCC as compared to DCM or IC.

Insulin accelerates recovery from QRS complex widening in a frog heart model of hyperkalemia.

Hyperkalemia is one of the most common electrolyte disorders. By injecting various concentrations of potassium chloride (KCl) solutions intravenously into bullfrogs, we demonstrated characteristic electrocardiogram (ECG) abnormalities of hyperkalemia in frog hearts. The widened QRS complexes induced by 100 mM KCl injection were accompanied by an increase in the resting membrane potential in cardiomyocytes and a decreased slope of phase 0 in the action potential. Recording both ECG waveforms and the cardiac action potential enabled us to reveal the mechanisms of hyperkalemia-induced ECG abnormalities. Additionally, pre-treatment with insulin, a powerful stimulator of Na+/K+-ATPase activity, significantly accelerated the recovery from the widened QRS complexes in the ECG, demonstrating a pronounced shift of extracellular potassium ions into the intracellular space.

Electromechanical Assessment of Optogenetically Modulated Cardiomyocyte Activity.

Over the past two decades, optogenetic tools have been established as potent means to modulate cell-type specific activity in excitable tissues, including the heart. While Channelrhodopsin-2 (ChR2) is a common tool to depolarize the membrane potential in cardiomyocytes (CM), potentially eliciting action potentials (AP), an effective tool for reliable silencing of CM activity has been missing. It has been suggested to use anion channelrhodopsins (ACR) for optogenetic inhibition. Here, we describe a protocol to assess the effects of activating the natural ACR GtACR1 from Guillardia theta in cultured rabbit CM. Primary readouts are electrophysiological patch-clamp recordings and optical tracking of CM contractions, both performed while applying different patterns of light stimulation. The protocol includes CM isolation from rabbit heart, seeding and culturing of the cells for up to 4 days, transduction via adenovirus coding for the light-gated chloride channel, preparation of patch-clamp and carbon fiber setups, data collection and analysis. Using the patch-clamp technique in whole-cell configuration allows one to record light-activated currents (in voltage-clamp mode, V-clamp) and AP (current-clamp mode, I-clamp) in real time. In addition to patch-clamp experiments, we conduct contractility measurements for functional assessment of CM activity without disturbing the intracellular milieu. To do so, cells are mechanically preloaded using carbon fibers and contractions are recorded by tracking changes in sarcomere length and carbon fiber distance. Data analysis includes assessment of AP duration from I-clamp recordings, peak currents from V-clamp recordings and force calculation from carbon fiber measurements. The described protocol can be applied to the testing of biophysical effects of different optogenetic actuators on CM activity, a prerequisite for the development of a mechanistic understanding of optogenetic experiments in cardiac tissue and whole hearts.

Kir2.1 and K2P1 channels reconstitute two levels of resting membrane potential in cardiomyocytes.

Outward and inward background currents across the cell membrane balance, determining resting membrane potential. Inward rectifier K+ channel subfamily 2 (Kir2) channels primarily maintain the resting membrane potential of cardiomyocytes. Human cardiomyocytes exhibit two levels of resting membrane potential at subphysiological extracellular K+ concentrations or pathological hypokalaemia, however, the underlying mechanism is unclear. In the present study, we show that human cardiomyocytes derived from induced pluripotent stem cells with enhanced expression of isoform 1 of Kir2 (Kir2.1) channels and mouse HL-1 cardiomyocytes with ectopic expression of two pore-domain K+ channel isoform 1 (K2P1) recapitulate two levels of resting membrane potential, indicating the contributions of Kir2.1 and K2P1 channels to the phenomenon. In Chinese hamster ovary cells that express the channels, Kir2.1 currents non-linearly counterbalance hypokalaemia-induced K2P1 leak cation currents, reconstituting two levels of resting membrane potential. These findings support the hypothesis that Kir2 currents non-linearly counterbalance inward background cation currents, such as K2P1 currents, accounting for two levels of resting membrane potential in human cardiomyocytes and demonstrating a novel mechanism that regulates excitability. Inward rectifier K+ channel subfamily 2 (Kir2) channels primarily maintain the normal resting membrane potential of cardiomyocytes. At subphysiological extracellular K+ concentrations or pathological hypokalaemia, human cardiomyocytes show both hyperpolarized and depolarized resting membrane potentials; these depolarized potentials cause cardiac arrhythmia; however, the underlying mechanism is unknown. In the present study, we show that inward rectifier K+ channel subfamily 2 isoform 1 (Kir2.1) currents non-linearly counterbalance hypokalaemia-induced two pore-domain K+ channel isoform 1 (K2P1) leak cation currents, reconstituting two levels of resting membrane potential in cardiomyocytes. Under hypokalaemic conditions, both human cardiomyocytes derived from induced pluripotent stem cells with enhanced Kir2.1 expression and mouse HL-1 cardiomyocytes with ectopic expression of K2P1 channels recapitulate two levels of resting membrane potential. These cardiomyocytes display N-shaped current-voltage relationships that cross the voltage axis three times and the first and third zero-current potentials match the two levels of resting membrane potential. Inhibition of K2P1 expression eliminates the phenomenon, indicating contributions of Kir2.1 and K2P1 channels to two levels of resting membrane potential. Second, in Chinese hamster ovary cells that heterologously express the channels, Kir2.1 currents non-linearly counterbalance hypokalaemia-induced K2P1 leak cation currents, yielding the N-shaped current-voltage relationships, causing the resting membrane potential to spontaneously jump from hyperpolarization at the first zero-current potential to depolarization at the third zero-current potential, again recapitulating two levels of resting membrane potential. These findings reveal ionic mechanisms of the two levels of resting membrane potential, demonstrating a previously unknown mechanism for the regulation of excitability, and support the hypothesis that Kir2 currents non-linearly balance inward background cation currents, accounting for two levels of resting membrane potential of human cardiomyocytes.

NPY Impairs Cell Viability and Mitochondrial Membrane Potential Through Ca2+ and p38 Signaling Pathways in Neonatal Rat Cardiomyocytes.

NPY is involved in stress cardiomyopathy. However, the associated mechanism for NPY-induced stress cardiomyopathy remains unclear. In this study, we aimed to explore potential cell signaling pathways that are related to NPY-mediated cell viability in neonatal rat cardiomyocytes. We found that NPY induced cell viability suppression in cultured cardiomyocytes in a dose-dependent manner. After NPY treatment, expression of CaN and p-CAMKII increased significantly, and phosphorylation of p38 but not ERK and JNK was changed. Moreover, NPY treatment significantly increased PGC-1α (the key factor of mitochondrial biogenesis and energy metabolism) expression but decreased mitochondrial membrane potential in cultured cardiomyocytes. More importantly, the blockage of CaN, CAMKII, and p38 signaling pathways by their inhibitors could rescue the reduced cell viability and mitochondrial membrane potential in NPY-treated cardiomyocytes. Collectively, our data demonstrated that NPY mediated cell viability and mitochondrial membrane potential in cardiomyocytes through CaN, CAMKII, and p38 signaling pathways.

The combined effects of ranolazine and dronedarone on human atrial and ventricular electrophysiology

IntroductionPharmacological rhythm control of atrial fibrillation (AF) in patients with structural heart disease is limited. Ranolazine in combination with low dose dronedarone remarkably reduced AF-burden in the phase II HARMONY trial. We thus aimed to investigate the possible mechanisms underlying these results. Methods and resultsPatch clamp experiments revealed that ranolazine (5μM), low-dose dronedarone (0.3μM), and the combination significantly prolonged action potential duration (APD90) in atrial myocytes from patients in sinus rhythm (prolongation by 23.5±0.1%, 31.7±0.1% and 25.6±0.1% respectively). Most importantly, in atrial myocytes from patients with AF ranolazine alone, but more the combination with dronedarone, also prolonged the typically abbreviated APD90 (prolongation by 21.6±0.1% and 31.9±0.1% respectively). It was clearly observed that neither ranolazine, dronedarone nor the combination significantly changed the APD or contractility and twitch force in ventricular myocytes or trabeculae from patients with heart failure (HF). Interestingly ranolazine, and more so the combination, but not dronedarone alone, caused hyperpolarization of the resting membrane potential in cardiomyocytes from AF. As measured by confocal microscopy (Fluo-3), ranolazine, dronedarone and the combination significantly suppressed diastolic sarcoplasmic reticulum (SR) Ca2+ leak in myocytes from sinus rhythm (reduction by ranolazine: 89.0±30.7%, dronedarone: 75.6±27.4% and combination: 78.0±27.2%), in myocytes from AF (reduction by ranolazine: 67.6±33.7%, dronedarone: 86.5±31.7% and combination: 81.0±33.3%), as well as in myocytes from HF (reduction by ranolazine: 64.8±26.5% and dronedarone: 65.9±29.3%). ConclusionsElectrophysiological measurements during exposure to ranolazine alone or in combination with low-dose dronedarone showed APD prolongation, cellular hyperpolarization and reduced SR Ca2+ leak in human atrial myocytes. The combined inhibitory effects on various currents, in particular Na+ and K+ currents, may explain the anti-AF effects observed in the HARMONY trial. Therefore, the combination of ranolazine and dronedarone, but also ranolazine alone, may be promising new treatment options for AF, especially in patients with HF, and merit further clinical investigation.

Pharmacological inhibition of IK1 by PA-6 in isolated rat hearts affects ventricular repolarization and refractoriness.

The inwardly rectifying potassium current (IK 1) conducted through Kir2.X channels contribute to repolarization of the cardiac action potential and to stabilization of the resting membrane potential in cardiomyocytes. Our aim was to investigate the effect of the recently discovered IK 1 inhibitor PA‐6 on action potential repolarization and refractoriness in isolated rat hearts. Transiently transfected HEK‐293 cells expressing IK 1 were voltage‐clamped with ramp protocols. Langendorff‐perfused heart experiments were performed on male Sprague–Dawley rats, effective refractory period, Wenckebach cycle length, and ventricular effective refractory period were determined following 200 nmol/L PA‐6 perfusion. 200 nmol/L PA‐6 resulted in a significant time‐latency in drug effect on the IK 1 current expressed in HEK‐293 cells, giving rise to a maximal effect at 20 min. In the Langendorff‐perfused heart experiments, PA‐6 prolonged the ventricular action potential duration at 90% repolarization (from 41.8 ± 6.5 msec to 72.6 ± 21.1 msec, 74% compared to baseline, P < 0.01, n = 6). In parallel, PA‐6 significantly prolonged the ventricular effective refractory period compared to baseline (from 34.8 ± 4.6 msec to 58.1 ± 14.7 msec, 67%, P < 0.01, n = 6). PA‐6 increased the short‐term beat‐to‐beat variability and ventricular fibrillation was observed in two of six hearts. Neither atrial ERP nor duration of atrial fibrillation was altered following PA‐6 application. The results show that pharmacological inhibition of cardiac IK 1 affects ventricular action potential repolarization and refractoriness and increases the risk of ventricular arrhythmia in isolated rat hearts.

Exosomes secreted from GATA-4 overexpressing mesenchymal stem cells serve as a reservoir of anti-apoptotic microRNAs for cardioprotection

BackgroundExosomes play an important role in intercellular signaling and exert regulatory function by carrying bioactive molecules. This study investigated (1) the cardioprotective capabilities of exosomes derived from mesenchymal stem cells (MSCs) overexpressing GATA-4 (MSCGATA-4) and (2) its underlying regulatory mechanisms for expression of target proteins in recipient cells. Methods and resultsExosomes were isolated and purified from MSCGATA-4 (ExoGATA-4) and control MSCs (ExoNull). Cell injury was investigated in primary cultured rat neonatal cardiomyocytes (CM) and in the rat heart. Exosomes contributed to increased CM survival, reduced CM apoptosis, and preserved mitochondrial membrane potential in CM cultured under a hypoxic environment. Direct intramyocardial transplantation of exosomes at the border of an ischemic region following ligation of the left anterior descending coronary artery significantly restored cardiac contractile function and reduced infarct size. Real-time PCR revealed that several anti-apoptotic miRs were highly expressed in ExoGATA-4. Rapid internalization of ExoGATA-4 by CM was documented using time-lapse imaging. Subsequent expression of these miRs, particularly miR-19a was higher in CM and in the myocardium treated with ExoGATA-4 compared to those treated with ExoNull. The enhanced protective effects observed in CM were diminished by the inhibition of miR-19a. The expression level of PTEN, a predicted target of miR-19a, was reduced in CM treated with ExoGATA-4, which resulted in the activation of the Akt and ERK signaling pathways. ConclusionsExoGATA-4 upon transplantation in the damaged tissue mediate protection by releasing multiple miRs responsible for activation of the cell survival signaling pathway.

Heme Oxygenase-1 Induction Improves Cardiac Function following Myocardial Ischemia by Reducing Oxidative Stress

BackgroundOxidative stress plays a key role in exacerbating diabetes and cardiovascular disease. Heme oxygenase-1 (HO-1), a stress response protein, is cytoprotective, but its role in post myocardial infarction (MI) and diabetes is not fully characterized. We aimed to investigate the protection and the mechanisms of HO-1 induction in cardiomyocytes subjected to hypoxia and in diabetic mice subjected to LAD ligation.MethodsIn vitro: cultured cardiomyocytes were treated with cobalt-protoporphyrin (CoPP) and tin protoporphyrin (SnPP) prior to hypoxic stress. In vivo: CoPP treated streptozotocin-induced diabetic mice were subjected to LAD ligation for 2/24 h. Cardiac function, histology, biochemical damage markers and signaling pathways were measured.ResultsHO-1 induction lowered release of lactate dehydrogenase (LDH) and creatine phospho kinase (CK), decreased propidium iodide staining, improved cell morphology and preserved mitochondrial membrane potential in cardiomyocytes. In diabetic mice, Fractional Shortening (FS) was lower than non-diabetic mice (35±1%vs.41±2, respectively p<0.05). CoPP-treated diabetic animals improved cardiac function (43±2% p<0.01), reduced CK, Troponin T levels and infarct size compared to non-treated diabetic mice (P<0.01, P<0.001, P<0.01 respectively). CoPP-enhanced HO-1 protein levels and reduced oxidative stress in diabetic animals, as indicated by the decrease in superoxide levels in cardiac tissues and plasma TNFα levels (p<0.05). The increased levels of HO-1 by CoPP treatment after LAD ligation led to a shift of the Bcl-2/bax ratio towards the antiapoptotic process (p<0.05). CoPP significantly increased the expression levels of pAKT and pGSK3β (p<0.05) in cardiomyocytes and in diabetic mice with MI. SnPP abolished CoPP's cardioprotective effects.ConclusionsHO-1 induction plays a role in cardioprotection against hypoxic damage in cardiomyocytes and in reducing post ischemic cardiac damage in the diabetic heart as proved by the increased levels of pAKT with a concomitant inhibition of pGSK3β leading to preserved mitochondrial membrane potential.

Mitochondrial chaperone tumour necrosis factor receptor‐associated protein 1 protects cardiomyocytes from hypoxic injury by regulating mitochondrial permeability transition pore opening

Tumour necrosis factor receptor-associated protein 1 (TRAP1) is a mitochondrial chaperone that plays a role in maintaining mitochondrial function and regulating cell apoptosis. The opening of the mitochondrial permeability transition pore (MPTP) is a key step in cell death after hypoxia. However, it is still unclear whether TRAP1 protects cardiomyocytes from hypoxic damage by regulating the opening of the pore. In the present study, primary cultured cardiomyocytes from neonatal rats were used to investigate changes in TRAP1 expression after hypoxia treatment as well as the mechanism and effect of TRAP1 on hypoxic damage. The results obtained showed that TRAP1 expression increased after 1 h of hypoxia and continued to increase for up to 12 h of treatment. Hypoxia caused an increase in cell death and decreased cell viability and mitochondrial membrane potential; overexpressing TRAP1 prevented hypoxia-induced damage to cardiomyocytes. The silencing of TRAP1 induced an increase in cell death and decreased both cell viability and mitochondrial membrane potential in cardiomyocytes under normoxic and hypoxic conditions. Furthermore, cell damage induced by the silencing of TRAP1 was prevented by the mitochondrial permeability transition pore inhibitor, cyclosporin A. These data demonstrate that hypoxia induces an increase in TRAP1 expression in cardiomyocytes, and that TRAP1 plays a protective role by regulating the opening of the mitochondrial permeability transition pore.

016 Evidence for inward rectifier potassium channels in HL-1 cells

Cardiac inward rectifier potassium current (IK1) plays a paramount role in repolarisation and stabilisation of the membrane potential in cardiomyocytes. Its downregulation contributes to arrhythmogenesis in heart failure and post-myocardial...

Postconditioning Prevents Reperfusion Injury by Activating δ-Opioid Receptors

While postconditioning has been proposed to protect the heart by targeting the mitochondrial permeability transition pore (mPTP), the detailed mechanism underlying this action is unknown. The authors hypothesized that postconditioning stimulates opioid receptors, which in turn protect the heart from reperfusion injury by targeting the mPTP. Rat hearts (both in vivo and in vitro) were subjected to 30 min of ischemia and 2 h of reperfusion. Postconditioning was elicited by six cycles of 10-s reperfusion and 10-s ischemia. To measure nitric oxide concentration, cardiomyocytes loaded with 4-amino-5-methylamino-2',7'-difluorofluorescein were imaged using confocal microscopy. Mitochondrial membrane potential was determined by loading cardiomyocytes with tetramethylrhodamine ethyl ester. In open chest rats, postconditioning reduced infarct size, an effect that was reversed by both naloxone and naltrindole. The antiinfarct effect of postconditioning was also blocked by the mPTP opener atractyloside. In isolated hearts, postconditioning reduced infarct size. Morphine mimicked postconditioning to reduce infarct size, which was abolished by both naltrindole and atractyloside. N-nitro-l-arginine methyl ester and guanylyl cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one blocked the action of morphine. Further experiments showed that morphine produces nitric oxide in cardiomyocytes by activating delta-opioid receptors. Moreover, morphine could prevent hydrogen peroxide-induced collapse of mitochondrial membrane potential in cardiomyocytes, which was reversed by naltrindole, N-nitro-l-arginine methyl ester, and the protein kinase G inhibitor KT5823. Postconditioning protects the heart by targeting the mPTP through activation of delta-opioid receptors. The nitric oxide-cyclic guanosine monophosphate-protein kinase G pathway may account for the effect of postconditioning on the mPTP opening.