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Alterations in ion channel expression and function known as "electrical remodeling" contribute to the development of hypertrophy and to the emergence of arrhythmias and sudden cardiac death. However, comparing current density values - an electrophysiological parameter commonly utilized to assess ion channel function - between normal and hypertrophied cells may be flawed when current amplitude does not scale with cell size. Even more, common routines to study equally sized cells or to discard measurements when large currents do not allow proper voltage-clamp control may introduce a selection bias and thereby confound direct comparison. To test a possible dependence of current density on cell size and shape, we employed whole-cell patch-clamp recording of voltage-gated sodium and calcium currents in Langendorff-isolated ventricular cardiomyocytes and Purkinje myocytes, as well as in cardiomyocytes derived from trans-aortic constriction operated mice. Here, we describe a distinct inverse relationship between voltage-gated sodium and calcium current densities and cell capacitance both in normal and hypertrophied cells. This inverse relationship was well fit by an exponential function and may be due to physiological adaptations that do not scale proportionally with cell size or may be explained by a selection bias. Our study emphasizes the need to consider cell size bias when comparing current densities in cardiomyocytes of different sizes, particularly in hypertrophic cells. Conventional comparisons based solely on mean current density may be inadequate for groups with unequal cell size or non-proportional current amplitude and cell size scaling.
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Cardiomegalia , Tamaño de la Célula , Miocitos Cardíacos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Animales , Cardiomegalia/metabolismo , Cardiomegalia/patología , Ratones , Masculino , Técnicas de Placa-ClampRESUMEN
Cardiac arrhythmias significantly contribute to mortality in Duchenne muscular dystrophy (DMD), a severe muscle illness caused by mutations in the gene encoding for the intracellular protein dystrophin. A major source for arrhythmia vulnerability in patients with DMD is impaired ventricular impulse conduction, which predisposes for ventricular asynchrony, decreased cardiac output, and the development of reentrant circuits. Using the dystrophin-deficient mdx mouse model for human DMD, we previously reported that the lack of dystrophin causes a significant loss of peak Na+ current (INa) in ventricular cardiomyocytes. This finding provided a mechanistic explanation for ventricular conduction defects and concomitant arrhythmias in the dystrophic heart. In the present study, we explored the hypothesis that empagliflozin (EMPA), an inhibitor of sodium/glucose cotransporter 2 in clinical use to treat type II diabetes and nondiabetic heart failure, rescues peak INa loss in dystrophin-deficient ventricular cardiomyocytes. We found that INa of cardiomyocytes derived from mdx mice, which had received clinically relevant doses of EMPA for 4 wk, was restored to wild-type level. Moreover, incubation of isolated mdx ventricular cardiomyocytes with 1 µM EMPA for 24 h significantly increased their peak INa. This effect was independent of Na+-H+ exchanger 1 inhibition by the drug. Our findings imply that EMPA treatment can rescue abnormally reduced peak INa of dystrophin-deficient ventricular cardiomyocytes. Long-term EMPA administration may diminish arrhythmia vulnerability in patients with DMD.NEW & NOTEWORTHY Dystrophin deficiency in cardiomyocytes leads to abnormally reduced Na+ currents. These can be rescued by long-term empagliflozin treatment.
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Compuestos de Bencidrilo , Diabetes Mellitus Tipo 2 , Glucósidos , Distrofia Muscular de Duchenne , Animales , Ratones , Humanos , Distrofina/genética , Ratones Endogámicos mdx , Miocitos Cardíacos/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Distrofia Muscular de Duchenne/genética , Arritmias Cardíacas/metabolismo , Sodio/metabolismo , Modelos Animales de EnfermedadRESUMEN
Introduction: Transverse-aortic constriction (TAC) operation is a widely used animal model to induce hypertrophy and heart failure through left-ventricular pressure overload. In mice, the cardiac response to TAC exhibits considerable variability influenced by factors such as strain, sub-strain, age, sex and vendor. Methods: To investigate the impact of suture material (silk versus prolene) and size (6-0 versus 7-0) on the TAC-induced phenotype, we performed surgeries on male C57BL6/N mice at 9 weeks of age defining the aortic constriction by a 27G needle, thereby employing most frequently used methodological settings. The mice were randomly assigned into four separate groups, 6-0 silk, 7-0 silk, 6-0 prolene and 7-0 prolene (10 mice per group). Echocardiography was conducted before TAC and every 4 weeks thereafter to monitor the development of heart failure. Repeated measures correlation analysis was employed to compare disease progression among the different groups. Results: Our findings reveal a significant influence of the chosen suture material on TAC outcomes. Mice operated with prolene showed increased mortality, slower body weight gain, faster left-ventricular mass increase, and a faster decline in left-ventricular ejection fraction, fractional shortening and aortic pressure gradient compared to silk-operated mice. Moreover, despite non significant, using thinner suture threads (7-0) tended to result in a more severe phenotype compared to thicker threads (6-0) across all tested parameters. Discussion: Collectively, our results highlight the importance of suture material selection in determining the cardiac phenotype induced by TAC and emphasize the need to consider this factor when comparing data across different research laboratories.
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Cortisol is a potent human steroid hormone that plays key roles in the central nervous system, influencing processes such as brain neuronal synaptic plasticity and regulating the expression of emotional and behavioral responses. The relevance of cortisol stands out in the disease, as its dysregulation is associated with debilitating conditions such as Alzheimer's Disease, chronic stress, anxiety and depression. Among other brain regions, cortisol importantly influences the function of the hippocampus, a structure central for memory and emotional information processing. The mechanisms fine-tuning the different synaptic responses of the hippocampus to steroid hormone signaling remain, however, poorly understood. Using ex vivo electrophysiology and wild type (WT) and miR-132/miR-212 microRNAs knockout (miRNA-132/212-/-) mice, we examined the effects of corticosterone (the rodent's equivalent to cortisol in humans) on the synaptic properties of the dorsal and ventral hippocampus. In WT mice, corticosterone predominantly inhibited metaplasticity in the dorsal WT hippocampi, whereas it significantly dysregulated both synaptic transmission and metaplasticity at dorsal and ventral regions of miR-132/212-/- hippocampi. Western blotting further revealed significantly augmented levels of endogenous CREB and a significant CREB reduction in response to corticosterone only in miR-132/212-/- hippocampi. Sirt1 levels were also endogenously enhanced in the miR-132/212-/- hippocampi but unaltered by corticosterone, whereas the levels of phospo-MSK1 were only reduced by corticosterone in WT, not in miR-132/212-/- hippocampi. In behavioral studies using the elevated plus maze, miRNA-132/212-/- mice further showed reduced anxiety-like behavior. These observations propose miRNA-132/212 as potential region-selective regulators of the effects of steroid hormones on hippocampal functions, thus likely fine-tuning hippocampus-dependent memory and emotional processing.
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Corticosterona , MicroARNs , Ratones , Humanos , Animales , Corticosterona/farmacología , Corticosterona/metabolismo , Hidrocortisona/metabolismo , Hipocampo/metabolismo , MicroARNs/metabolismo , Plasticidad NeuronalRESUMEN
The muscular dystrophies caused by dystrophin deficiency, the so-called dystrophinopathies, are associated with impaired cardiac contractility and arrhythmias, which considerably contribute to disease morbidity and mortality. Impaired Ca handling in ventricular cardiomyocytes has been identified as a causative factor for complications in the dystrophic heart, and restoration of normal Ca handling in myocytes has emerged as a promising new therapeutic strategy. In the present study, we explored the hypothesis that ivabradine, a drug clinically approved for the treatment of heart failure and stable angina pectoris, improves Ca handling in dystrophic cardiomyocytes and thereby enhances contractile performance in the dystrophic heart. Therefore, ventricular cardiomyocytes were isolated from the hearts of adult dystrophin-deficient DMDmdx rats, and the effects of acutely applied ivabradine on intracellular Ca transients were tested. In addition, the drug's acute impact on cardiac function in DMDmdx rats was assessed by transthoracic echocardiography. We found that administration of ivabradine to DMDmdx rats significantly improved cardiac function. Moreover, the amplitude of electrically induced intracellular Ca transients in ventricular cardiomyocytes isolated from DMDmdx rats was increased by the drug. We conclude that ivabradine enhances Ca release from the sarcoplasmic reticulum in dystrophic cardiomyocytes and thereby improves contractile performance in the dystrophic heart.
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Distrofina , Distrofia Muscular de Duchenne , Ratones , Ratas , Animales , Distrofia Muscular de Duchenne/complicaciones , Distrofia Muscular de Duchenne/tratamiento farmacológico , Ivabradina/farmacología , Ivabradina/uso terapéutico , Ratones Endogámicos mdx , Miocitos Cardíacos , Modelos Animales de EnfermedadRESUMEN
Alterations in cardiac impulse conduction may exert both beneficial and detrimental effects. The assessment of ventricular conduction properties is of paramount importance both in clinical and in experimental settings. Currently the duration of the QRS complex is regarded as hallmark of in-vivo assessment of global ventricular conduction time. In addition, the amplitude of the QRS complex has been suggested to reflect ventricular conduction time in man and in rats. Here, for the first time, we systematically investigated the relationship between QRS duration ("QRS") and QRS amplitude ("RS-height"; RSh) in the murine ECG obtained during anesthesia. In mice harbouring a homozygous knockout of the transmembrane protein podoplanin (PDPN-/-; n = 10) we found both a shorter QRS and a greater RSh than in wild-type animals (n = 13). In both genotypes cumulative i.p. administration of 5 mg/kg and 10 mg/kg of the Na channel blocker flecainide resulted in dose-dependent QRS increase and RSh decrease, whereby the drug-induced changes in RSh were greater than in QRS. In both genotypes the flecainide-induced changes in QRS and in RSh were significantly correlated with each other (R = -0.56, P = 0.004). Whereas dispersion of QRS and RSh was similar between genotypes, dispersion of the ratio QRS/RSh was significantly smaller in PDPN-/- than in wild-types. We conclude that in the murine ECG QRS is inversely related to RSh. We suggest that both parameters should be considered in the analysis of ventricular conduction time in the murine ECG.
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Flecainida , Sistema de Conducción Cardíaco , Ratas , Animales , Ratones , Flecainida/farmacología , Electrocardiografía , Ventrículos Cardíacos , Frecuencia CardíacaRESUMEN
T-type Ca channels are strongly expressed and important in the developing heart. In the adult heart, these channels play a significant role in pacemaker tissues, but there is uncertainty about their presence and physiological relevance in the working myocardium. Here, we show that the T-type Ca channel isoforms Cav3.1 and Cav3.2 are expressed at a protein level in ventricular cardiomyocytes from healthy adult C57/BL6 mice. Myocytes isolated from adult wild-type and Cav3.2 KO mice showed considerable whole cell T-type Ca currents under beta-adrenergic stimulation with isoprenaline. We further show that the detectability of basal T-type Ca currents in murine wild-type cardiomyocytes depends on the applied experimental conditions. Together, these findings reveal the presence of functional T-type Ca channels in the membrane of ventricular myocytes. In addition, electrically evoked Ca release from the sarcoplasmic reticulum was significantly impaired in Cav3.2 KO compared to wild-type cardiomyocytes. Our work implies a physiological role of T-type Ca channels in the healthy adult murine ventricular working myocardium.
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Background and purpose: Ivabradine is clinically administered to lower the heart rate, proposedly by inhibiting hyperpolarization-activated cyclic nucleotide-gated cation channels in the sinoatrial node. Recent evidence suggests that voltage-gated sodium channels (VGSC) are inhibited within the same concentration range. VGSCs are expressed within the sinoatrial node and throughout the conduction system of the heart. A block of these channels thus likely contributes to the established and newly raised clinical indications of ivabradine. We, therefore, investigated the pharmacological action of ivabradine on VGSCs in sufficient detail in order to gain a better understanding of the pro- and anti-arrhythmic effects associated with the administration of this drug. Experimental Approach: Ivabradine was tested on VGSCs in native cardiomyocytes isolated from mouse ventricles and the His-Purkinje system and on human Nav1.5 in a heterologous expression system. We investigated the mechanism of channel inhibition by determining its voltage-, frequency-, state-, and temperature-dependence, complemented by a molecular drug docking to the recent Nav1.5 cryoEM structure. Automated patch-clamp experiments were used to investigate ivabradine-mediated changes in Nav1.5 inactivation parameters and inhibition of different VGSC isoforms. Key results: Ivabradine inhibited VGSCs in a voltage- and frequency-dependent manner, but did not alter voltage-dependence of activation and fast inactivation, nor recovery from fast inactivation. Cardiac (Nav1.5), neuronal (Nav1.2), and skeletal muscle (Nav1.4) VGSC isoforms were inhibited by ivabradine within the same concentration range, as were sodium currents in native cardiomyocytes isolated from the ventricles and the His-Purkinje system. Molecular drug docking suggested an interaction of ivabradine with the classical local anesthetic binding site. Conclusion and Implications: Ivabradine acts as an atypical inhibitor of VGSCs. Inhibition of VGSCs likely contributes to the heart rate lowering effect of ivabradine, in particular at higher stimulation frequencies and depolarized membrane potentials, and to the observed slowing of intra-cardiac conduction. Inhibition of VGSCs in native cardiomyocytes and across channel isoforms may provide a potential basis for the anti-arrhythmic potential as observed upon administration of ivabradine.
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Psilocybin, a hallucinogen contained in "magic" mushrooms, holds great promise for the treatment of various psychiatric disorders, and early clinical trials are encouraging. Adverse cardiac events after intake of high doses of psilocybin and a trial reporting QT interval prolongation in the electrocardiogram attributed to the drug's main metabolite, psilocin, gave rise to safety concerns. Here we show that clinical concentrations of psilocin do not cause significant human ether-a-go-go-related gene (hERG) potassium channel inhibition, a major risk factor for adverse cardiac events. We conclude that hERG channel blockage by psilocin is not liable for psilocybin- associated cardiotoxic effects.
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Alucinógenos , Trastornos Mentales , Cardiotoxicidad , Alucinógenos/efectos adversos , Humanos , Trastornos Mentales/inducido químicamente , Trastornos Mentales/tratamiento farmacológico , Canales de Potasio , Psilocibina/efectos adversosRESUMEN
Current knowledge on store-operated Ca2+ entry (SOCE) regarding its localization, kinetics, and regulation is mostly derived from studies performed in non-excitable cells. After a long time of relative disinterest in skeletal muscle SOCE, this mechanism is now recognized as an essential contributor to muscle physiology, as highlighted by the muscle pathologies that are associated with mutations in the SOCE molecules STIM1 and Orai1. This review mainly focuses on the peculiar aspects of skeletal muscle SOCE that differentiate it from its counterpart found in non-excitable cells. This includes questions about SOCE localization and the movement of respective proteins in the highly organized skeletal muscle fibers, as well as the diversity of expressed STIM isoforms and their differential expression between muscle fiber types. The emerging evidence of a phasic SOCE, which is activated during EC coupling, and its physiological implication is described as well. The specific issues related to the use of SOCE modulators in skeletal muscles are discussed. This review highlights the complexity of SOCE activation and its regulation in skeletal muscle, with an emphasis on the most recent findings and the aim to reach a current picture of this mesmerizing phenomenon.
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Señalización del Calcio/fisiología , Calcio/metabolismo , Músculo Esquelético/metabolismo , Animales , Humanos , Proteína ORAI1/metabolismo , Molécula de Interacción Estromal 1/metabolismoRESUMEN
Since their discovery in the 1960s, the term paroxysmal depolarization shift (PDS) has been applied to a wide variety of reinforced neuronal discharge patterns. Occurrence of PDS as cellular correlates of electrographic spikes during latent phases of insult-induced rodent epilepsy models and their resemblance to giant depolarizing potentials (GDPs) nourished the idea that PDS may be involved in epileptogenesis. Both GDPs and - in analogy - PDS may lead to progressive changes of neuronal properties by generation of pulsatile intracellular Ca2+ elevations. Herein, a key element is the gating of L-type voltage gated Ca2+ channels (LTCCs, Cav1.x family), which may convey Ca2+ signals to the nucleus. Accordingly, the present study investigates various insult-associated neuronal challenges for their propensities to trigger PDS in a LTCC-dependent manner. Our data demonstrate that diverse disturbances of neuronal function are variably suited to induce PDS-like events, and the contribution of LTCCs is essential to evoke PDS in rat hippocampal neurons that closely resemble GDPs. These PDS appear to be initiated in the dendritic sub-compartment. Their morphology critically depends on the position of recording electrodes and on their rate of occurrence. These results provide novel insight into induction mechanisms, origin, variability, and co-existence of PDS with other discharge patterns and thereby pave the way for future investigations regarding the role of PDS in epileptogenesis.
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Epilepsia , Alta del Paciente , Animales , Hipocampo , Humanos , Neuronas , RatasRESUMEN
Besides skeletal muscle abnormalities, Duchenne muscular dystrophy (DMD) patients present with dilated cardiomyopathy development, which considerably contributes to morbidity and mortality. Because the mechanisms responsible for the cardiac complications in the context of DMD are largely unknown, evidence-based therapy approaches are still lacking. This has increased the need for basic research efforts into animal models for DMD. Here, we characterized in detail the cardiovascular abnormalities of Dmdmdx rats, with the aim of determining the suitability of this recently established dystrophin-deficient small animal as a model for DMD.Various methods were applied to compare cardiovascular properties between wild-type and Dmdmdx rats, and to characterize the Dmdmdx cardiomyopathy. These methods comprised echocardiography, invasive assessment of left ventricular hemodynamics, examination of adverse remodeling and endothelial cell inflammation, and evaluation of vascular function, employing wire myography. Finally, intracellular Ca2+ transient measurements, and recordings of currents through L-type Ca2+ channels were performed in isolated single ventricular cardiomyocytes. We found that, similar to respective observations in DMD patients, the hearts of Dmdmdx rats show significantly impaired cardiac function, fibrosis and inflammation, consistent with the development of a dilated cardiomyopathy. Moreover, in Dmdmdx rats, vascular endothelial function is impaired, which may relate to inflammation and oxidative stress, and Ca2+ handling in Dmdmdx cardiomyocytes is abnormal.These findings indicate that Dmdmdx rats represent a promising small-animal model to elucidate mechanisms of cardiomyopathy development in the dystrophic heart, and to test mechanism-based therapies aiming to combat cardiovascular complications in DMD.
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Cardiomiopatías/patología , Sistema Cardiovascular , Modelos Animales de Enfermedad , Distrofina/genética , Distrofia Muscular de Duchenne/genética , Miocardio/patología , Miocitos Cardíacos/patología , Animales , Calcio/metabolismo , Canales de Calcio Tipo L/metabolismo , Cardiomiopatía Dilatada/complicaciones , Distrofina/metabolismo , Endotelio Vascular/patología , Fibrosis/patología , Ventrículos Cardíacos/fisiopatología , Hemodinámica , Homeostasis , Humanos , Inflamación , Riñón/metabolismo , Pulmón/metabolismo , Músculo Esquelético/patología , Miocitos Cardíacos/metabolismo , Estrés Oxidativo , Peptidil-Dipeptidasa A , Fenotipo , Ratas , Estrés MecánicoRESUMEN
Ivabradine blocks hyperpolarisation-activated cyclic nucleotide-gated (HCN) channels, thereby lowering the heart rate, an action that is used clinically for the treatment of heart failure and angina pectoris. We and others have shown previously that ivabradine, in addition to its HCN channel blocking activity, also inhibits voltage-gated Na channels in vitro at concentrations that may be clinically relevant. Such action may reduce conduction velocity in cardiac atria and ventricles. Here, we explore the effect of administration of ivabradine on parameters of ventricular conduction and repolarization in the surface ECG of anesthetized mice. We found that 5 min after i.p. administration of 10 mg/kg ivabradine spontaneous heart rate had declined by ~13%, which is within the range observed in human clinical studies. At the same time a significant increase in QRS duration by ~18% was observed, suggesting a reduction in ventricular conduction velocity. During transesophageal pacing at heart rates between 100 and 220 beats/min there was no obvious rate-dependence of ivabradine-induced QRS prolongation. On the other hand, ivabradine produced substantial rate-dependent slowing of AV nodal conduction. We conclude that ivabradine prolongs conduction in the AV-node and in the ventricles in vivo.
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Antiarrítmicos/farmacología , Arritmias Cardíacas/tratamiento farmacológico , Nodo Atrioventricular/efectos de los fármacos , Frecuencia Cardíaca/efectos de los fármacos , Ivabradina/farmacología , Potenciales de Acción/efectos de los fármacos , Animales , Arritmias Cardíacas/etiología , Arritmias Cardíacas/fisiopatología , Nodo Atrioventricular/fisiopatología , Estimulación Cardíaca Artificial , Modelos Animales de Enfermedad , Electrocardiografía , Femenino , Ratones Endogámicos C57BL , Factores de TiempoRESUMEN
Store-operated calcium entry (SOCE) plays a pivotal role in skeletal muscle physiology as, when impaired, the muscle is prone to early fatigue and the development of different myopathies. A chronic mode of slow SOCE activation is carried by stromal interaction molecule 1 (STIM1) and calcium-release activated channel 1 (ORAI1) proteins. A phasic mode of fast SOCE (pSOCE) occurs upon single muscle twitches in synchrony with excitation-contraction coupling, presumably activated by a local and transient depletion at the terminal cisternae of the sarcoplasmic reticulum Ca2+-stores. Both SOCE mechanisms are poorly understood. In particular, pSOCE has not been described in detail because the conditions required for its detection in mouse skeletal muscle have not been established to date. Here we report the first measurements of pSOCE in mouse extensor digitorum longus muscle fibers using electrical field stimulation (EFS) in a skinned fiber preparation. We show moderate voluntary wheel running to be a prerequisite to render muscle fibers reasonably susceptible for EFS, and thereby define an experimental paradigm to measure pSOCE in mouse muscle. Continuous monitoring of the physical activity of mice housed in cages equipped with running wheels revealed an optimal training period of 5-6 days, whereby best responsiveness to EFS negatively correlated with running distance and speed. A comparison of pSOCE kinetic data in mouse with those previously derived from rat muscle demonstrated very similar properties and suggests the existence and similar function of pSOCE across mammalian species. The new technique presented herein enables future experiments with genetically modified mouse models to define the molecular entities, presumably STIM1 and ORAI1, and the physiological role of pSOCE in health and under conditions of disease.
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Plasmalogens (Pls) are a class of membrane phospholipids which serve a number of essential biological functions. Deficiency of Pls is associated with common disorders such as Alzheimer's disease or ischemic heart disease. A complete lack of Pls due to genetically determined defective biosynthesis gives rise to rhizomelic chondrodysplasia punctata (RCDP), characterized by a number of severe disabling pathologic features and death in early childhood. Frequent cardiac manifestations of RCDP include septal defects, mitral valve prolapse, and patent ductus arteriosus. In a mouse model of RCDP, reduced nerve conduction velocity was partially rescued by dietary oral supplementation of the Pls precursor batyl alcohol (BA). Here, we examine the impact of Pls deficiency on cardiac impulse conduction in a similar mouse model (Gnpat KO). In-vivo electrocardiographic recordings showed that the duration of the QRS complex was significantly longer in Gnpat KO mice than in age- and sex-matched wild-type animals, indicative of reduced cardiac conduction velocity. Oral supplementation of BA for 2 months resulted in normalization of cardiac Pls levels and of the QRS duration in Gnpat KO mice but not in untreated animals. BA treatment had no effect on the QRS duration in age-matched wild-type mice. These data suggest that Pls deficiency is associated with increased ventricular conduction time which can be rescued by oral BA supplementation.
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Arritmias Cardíacas/tratamiento farmacológico , Condrodisplasia Punctata Rizomélica/tratamiento farmacológico , Éteres de Glicerilo/farmacología , Plasmalógenos/biosíntesis , Administración Oral , Animales , Arritmias Cardíacas/etiología , Condrodisplasia Punctata Rizomélica/fisiopatología , Suplementos Dietéticos , Modelos Animales de Enfermedad , Electrocardiografía , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Éteres Fosfolípidos/farmacologíaRESUMEN
Cardiac arrhythmias significantly contribute to mortality in Duchenne muscular dystrophy (DMD), a degenerative muscle disease triggered by mutations in the gene encoding for the intracellular protein dystrophin. A major source for the arrhythmias in patients with DMD is impaired ventricular impulse conduction, which predisposes for ventricular asynchrony, decreased cardiac output, and the development of reentrant mechanisms. The reason for ventricular conduction impairments and the associated arrhythmias in the dystrophic heart has remained unidentified. In the present study, we explored the hypothesis that dystrophin-deficient cardiac Purkinje fibers have reduced Na+ currents (INa), which would represent a potential mechanism underlying slowed ventricular conduction in the dystrophic heart. Therefore, by using a Langendorff perfusion system, we isolated Purkinje fibers from the hearts of adult wild-type control and dystrophin-deficient mdx mice. Enhanced green fluorescent protein (eGFP) expression under control of the connexin 40 gene allowed us to discriminate Purkinje fibers from eGFP-negative ventricular working cardiomyocytes after cell isolation. Finally, we recorded INa from wild-type and dystrophic mdx Purkinje fibers for comparison by means of the whole cell patch clamp technique. We found substantially reduced INa densities in mdx compared with wild-type Purkinje fibers, suggesting that dystrophin deficiency diminishes INa. Because Na+ channels in the Purkinje fiber membrane represent key determinants of ventricular conduction velocity, we propose that reduced INa in Purkinje fibers at least partly explains impaired ventricular conduction and the associated arrhythmias in the dystrophic heart.NEW & NOTEWORTHY Dystrophic cardiac Purkinje fibers have abnormally reduced Na+ current densities. This explains impaired ventricular conduction in the dystrophic heart.
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Arritmias Cardíacas/metabolismo , Trastorno del Sistema de Conducción Cardíaco/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Ramos Subendocárdicos/metabolismo , Canales de Sodio/metabolismo , Potenciales de Acción/fisiología , Animales , Arritmias Cardíacas/complicaciones , Arritmias Cardíacas/fisiopatología , Trastorno del Sistema de Conducción Cardíaco/complicaciones , Trastorno del Sistema de Conducción Cardíaco/fisiopatología , Masculino , Ratones , Ratones Endogámicos mdx , Distrofia Muscular de Duchenne/complicaciones , Distrofia Muscular de Duchenne/fisiopatología , Sodio/metabolismoRESUMEN
Neuronal nitric oxide synthase (nNOS) is considered a regulator of Cav1.2 L-type Ca2+ channels and downstream Ca2+ cycling in the heart. The commonest view is that nitric oxide (NO), generated by nNOS activity in cardiomyocytes, reduces the currents through Cav1.2 channels. This gives rise to a diminished Ca2+ release from the sarcoplasmic reticulum, and finally reduced contractility. Here, we report that nNOS inhibitor substances significantly increase intracellular Ca2+ transients in ventricular cardiomyocytes derived from adult mouse and rat hearts. This is consistent with an inhibitory effect of nNOS/NO activity on Ca2+ cycling and contractility. Whole cell currents through L-type Ca2+ channels in rodent myocytes, on the other hand, were not substantially affected by the application of various NOS inhibitors, or application of a NO donor substance. Moreover, the presence of NO donors had no effect on the single-channel open probability of purified human Cav1.2 channel protein reconstituted in artificial liposomes. These results indicate that nNOS/NO activity does not directly modify Cav1.2 channel function. We conclude that-against the currently prevailing view-basal Cav1.2 channel activity in ventricular cardiomyocytes is not substantially regulated by nNOS activity and NO. Hence, nNOS/NO inhibition of Ca2+ cycling and contractility occurs independently of direct regulation of Cav1.2 channels by NO.
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Potenciales de Acción , Canales de Calcio Tipo L/metabolismo , Señalización del Calcio , Miocitos Cardíacos/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Animales , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Femenino , Ventrículos Cardíacos/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/fisiología , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico Sintasa de Tipo III/antagonistas & inhibidores , Ornitina/análogos & derivados , Ornitina/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND/AIMS: Ivabradine lowers the heart rate by inhibition of hyperpolarisation-activated cyclic nucleotide-gated (HCN) channels mediating the 'funny' pacemaker current If in the sinoatrial node. It is clinically approved for the treatment of heart failure and angina pectoris. Due to its proposed high selectivity for If administration of ivabradine is not associated with the side effects commonly observed following the application of other heart rate lowering agents. Recent evidence, however, has shown significant affinity of ivabradine towards Kv11.1 (ether-a-go-go related gene, ERG) potassium channels. Despite the inhibition of Kv11.1 channels by ivabradine, cardiac action potential (AP) duration and heart rate corrected QT interval (QTc) of the human electrocardiogram (ECG) were not prolonged. We thus surmised that compensatory mechanisms might counteract the drug's inhibitory action on Kv11.1. METHODS: The effects of ivabradine on human Kv11.1 and Kv7.1 potassium, Cav1.2 calcium, and Nav1.5 sodium channels, heterologously expressed in tsA-201 cells, were studied in the voltage-clamp mode of the whole cell patch clamp technique. In addition, changes in action potential parameters of human induced pluripotent stem cell (iPSC) derived cardiomyocytes upon application of ivabradine were studied with current-clamp experiments. RESULTS: Here we show that ivabradine exhibits significant affinity towards cardiac ion channels other than HCN. We demonstrate for the first time inhibition of human voltage-gated Nav1.5 sodium channels at therapeutically relevant concentrations. Within this study we also confirm recent findings of human Kv11.1 inhibition by low µM concentrations of ivabradine and observed no prolongation of ventricular-like APs in cardiomyocytes derived from iPSCs. CONCLUSION: Our results provide an explanation why ivabradine, despite its affinity for Kv11.1 channels, does not prolong the cardiac AP and QTc interval. Furthermore, our results suggest the inhibition of voltage-gated Nav1.5 sodium channels to underlie the recent observations of slowed atrioventricular conduction by increased atrial-His bundle intervals upon administration of ivabradine.
Asunto(s)
Potenciales de Acción/efectos de los fármacos , Fármacos Cardiovasculares/farmacología , Canales Iónicos/metabolismo , Ivabradina/farmacología , Canales de Calcio Tipo L/química , Canales de Calcio Tipo L/metabolismo , Línea Celular , Canal de Potasio ERG1/antagonistas & inhibidores , Canal de Potasio ERG1/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Canales Iónicos/antagonistas & inhibidores , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Canal de Sodio Activado por Voltaje NAV1.5/química , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Técnicas de Placa-ClampRESUMEN
Skeletal muscle fibres support store-operated Ca2+-entry (SOCE) across the t-tubular membrane upon exhaustive depletion of Ca2+ from the sarcoplasmic reticulum (SR). Recently we demonstrated the presence of a novel mode of SOCE activated under conditions of maintained [Ca2+]SR. This phasic SOCE manifested in a fast and transient manner in synchrony with excitation contraction (EC)-coupling mediated SR Ca2+-release (Communications Biology 1:31, doi: https://doi.org/10.1038/s42003-018-0033-7). Stromal interaction molecule 1 (STIM1) and calcium release-activated calcium channel 1 (ORAI1), positioned at the SR and t-system membranes, respectively, are the considered molecular correlate of SOCE. The evidence suggests that at the triads, where the terminal cisternae of the SR sandwich a t-tubule, STIM1 and ORAI1 proteins pre-position to allow for enhanced SOCE transduction. Here we show that phasic SOCE is not only shaped by global [Ca2+]SR but provide evidence for a local activation within nanodomains at the terminal cisternae of the SR. This feature may allow SOCE to modulate [Ca2+]SR during EC coupling. We define SOCE to occur on the same timescale as EC coupling and determine the temporal coherence of SOCE activation to SR Ca2+ release. We derive a delay of 0.3â¯ms reflecting diffusive Ca2+-equilibration at the luminal ryanodine receptor 1 (RyR1) channel mouth upon SR Ca2+-release. Numerical simulations of Ca2+-calsequestrin binding estimates a characteristic diffusion length and confines an upper limit for the spatial distance between STIM1 and RyR1. Experimental evidence for a 4- fold change in t-system Ca2+-permeability upon prolonged electrical stimulation in conjunction with numerical simulations of Ca2+-STIM1 binding suggests a Ca2+ dissociation constant of STIM1 below 0.35â¯mM. Our results show that phasic SOCE is intimately linked with RyR opening and closing, with only µs delays, because [Ca2+] in the terminal cisternae is just above the threshold for Ca2+ dissociation from STIM1 under physiological resting conditions. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.