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Cardiomyocytes derived from human embryonic stem cells constitute a promising cell source for the regeneration of damaged hearts. The assessment of their in vitro functional properties is mandatory to envisage appropriate cardiac cell-based therapies. In this study, we characterized human embryonic stem cell-derived cardiomyocytes over a 3-month period, using patch-clamp or intracellular recordings to assess their functional maturation and reverse transcriptase-polymerase chain reaction to evaluate the expression of ion channel-encoding subunits. I(to1) and I(K1), the transient outward and inward rectifier potassium currents, were present in cardiomyocytes only, whereas the rapid delayed rectifier potassium current (I(Kr)), pacemaker current (I(f)), and L-type calcium current (I(Ca,L)) could be recorded both in undifferentiated human embryonic stem cells and in cardiomyocytes. Most of the currents underwent developmental maturation in cardiomyocytes, as assessed by modifications in current density (I(to1), I(K1), and I(Ca,L)) and properties (I(f)). Ion-channel mRNAs were always present when the current was recorded. Intracellular recordings in spontaneously beating clusters of cardiomyocytes revealed changes in action potential parameters and in response to pharmacological tools according to time of differentiation. In summary, human embryonic stem cell-derived cardiomyocytes mature over time during in vitro differentiation, approaching an adult phenotype. Disclosure of potential conflicts of interest is found at the end of this article.

Optogenetics, utilising light-reactive proteins to manipulate tissue activity, are a relatively novel approach in the field of cardiac electrophysiology. We here provide an overview of light-activated transmembrane channels (optogenetic actuators) currently applied in strategies to modulate cardiac activity, as well as newly developed variants yet to be implemented in the heart. In addition, we touch upon genetically encoded indicators (optogenetic sensors) and fluorescent dyes to monitor tissue activity, including cardiac transmembrane potential and ion homeostasis. The combination of the two allows for all-optical approaches to monitor and manipulate the heart without any physical contact. However, spectral congestion poses a major obstacle, arising due to the overlap of excitation/activation and emission spectra of various optogenetic proteins and/or fluorescent dyes, resulting in optical crosstalk. Therefore, optogenetic proteins and fluorescent dyes should be carefully selected to avoid optical crosstalk and consequent disruptions in readouts and/or cellular activity. We here present a novel approach to simultaneously monitor transmembrane potential and cytosolic calcium, while also performing optogenetic manipulation. For this, we used the novel voltage-sensitive dye ElectroFluor 730p and the cytosolic calcium indicator X-Rhod-1 in mouse hearts expressing channelrhodopsin-2 (ChR2). By exploiting the isosbestic point of ElectroFluor 730p and avoiding the ChR2 activation spectrum, we here introduce a novel optical imaging and manipulation approach with minimal crosstalk. Future developments in both optogenetic proteins and fluorescent dyes will allow for additional and more optimised strategies, promising a bright future for all-optical approaches in the field of cardiac electrophysiology. [ABSTRACT FROM AUTHOR]

Current techniques for fast characterization of cardiac electrophysiology employ optical technologies to control and monitor action potential features of single cells or cellular monolayers placed in multiwell plates. High-speed investigation capacities are commonly achieved by serially analyzing well after well employing fully automated fluorescence microscopes. Here, we describe an alternative cost-effective optical approach (MULTIPLE) that exploits high-power LED arrays to globally illuminate a culture plate and an sCMOS sensor for parallel detection of the fluorescence coming from multiple wells. MULTIPLE combines optical detection of action potentials using a red-shifted voltage-sensitive fluorescent dye (di-4-ANBDQPQ) with optical stimulation, employing optogenetic actuators, to ensure excitation of cardiomyocytes at constant rates. MULTIPLE was first characterized in terms of interwell uniformity of the illumination intensity and optical detection performance. Then, it was applied for probing action potential features in HL-1 cells (i.e., mouse atrial myocyte-like cells) stably expressing the blue light-activatable cation channel CheRiff. Under proper stimulation conditions, we were able to accurately measure action potential dynamics across a 24-well plate with variability across the whole plate of the order of 10%. The capability of MULTIPLE to detect action potential changes across a 24-well plate was demonstrated employing the selective K v 11.1 channel blocker (E-4031), in a dose titration experiment. Finally, action potential recordings were performed in spontaneous beating human induced pluripotent stem cell derived cardiomyocytes following pharmacological manipulation of their beating frequency. We believe that the simplicity of the presented optical scheme represents a valid complement to sophisticated and expensive state-of-the-art optical systems for high-throughput cardiac electrophysiological investigations. [ABSTRACT FROM AUTHOR]

Human Chronic Atrial Fibrillation and If Introduction Despite the evidence that the hyperpolarization-activated current (If) is highly modulated in human cardiomyopathies, no definite data exist in chronic atrial fibrillation (cAF). We investigated the expression, function, and modulation of If in human cAF. Methods and Results Right atrial samples were obtained from sinus rhythm (SR, n = 49) or cAF (duration >1 year, n = 31) patients undergoing corrective cardiac surgery. Among f-channel isoforms expressed in the human atrium (HCN1, 2 and 4), HCN4 mRNA levels measured by RT-PCR were significantly reduced. However, protein expression was preserved in cAF compared to SR (+85% for HCN4); concurrently, miR-1 expression was significantly reduced. In patch-clamped atrial myocytes, current-specific conductance (gf) was significantly increased in cAF at voltages around the threshold for If activation (−60 to −80 mV); accordingly, a 10-mV rightward shift of the activation curve occurred (P < 0.01). β-Adrenergic and 5-HT4 receptor stimulation exerted similar effects on If in cAF and SR cells, while the ANP-mediated effect was significantly reduced (P < 0.02), suggesting downregulation of natriuretic peptide signaling. Conclusions In human cAF modifications in transcriptional and posttranscriptional mechanisms of HCN channels occur, associated with a slight yet significant gain-of-function of If, which may contribute to enhanced atrial ectopy. [ABSTRACT FROM AUTHOR]

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BACKGROUND AND PURPOSE Selective hyperpolarization activated, cyclic nucleotide-gated channel (HCN) blockers represent an important therapeutic goal due to the wide distribution and multiple functions of these proteins, representing the molecular correlate of f- and h-current ( If or Ih). Recently, new compounds able to block differentially the homomeric HCN isoforms expressed in HEK293 have been synthesized. In the present work, the electrophysiological and pharmacological properties of these new HCN blockers were characterized and their activities evaluated on native channels. EXPERIMENTAL APPROACH HEK293 cells expressing mHCN1, mHCN2 and hHCN4 isoforms were used to verify channel blockade. Selected compounds were tested on native guinea pig sinoatrial node cells and neurons from mouse dorsal root ganglion (DRG) by patch-clamp recordings and on dog Purkinje fibres by intracellular recordings. KEY RESULTS In HEK293 cells, EC18 was found to be significantly selective for HCN4 and MEL57A for HCN1 at physiological membrane potential. When tested on guinea pig sinoatrial node cells, EC18 (10 µM) maintained its activity, reducing If by 67% at −120 mV, while MEL57A (3 µM) reduced If by 18%. In contrast, in mouse DRG neurons, only MEL57A (30 and 100 µM) significantly reduced Ih by 60% at −80 mV. In dog cardiac Purkinje fibres, EC18, but not MEL57A, reduced the amplitude and slowed the slope of the spontaneous diastolic depolarization. CONCLUSIONS Our results have identified novel and highly selective HCN isoform blockers, EC18 and MEL57A; the selectivity found in recombinant system was maintained in various tissues expressing different HCN isoforms. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the effect of chronic heart rate (HR) reduction with the hyperpolarization-activated current inhibitor ivabradine on the global phenotype of left ventricular (LV) remodeling in a ligated rat model. Seven days after coronary artery ligation, Wistar rats received ivabradine (10 mg∙kg-1∙day-1 administered in drinking water) [myocardial infarction + ivabradine (MI+IVA), n = 22] or vehicle only (drinking water) (MI, n = 20) for 90 days. A sham group (n = 20) was included for model validation. MI+IVA rats had 12% lower HR (P < 0.01), improved LV volumes, 15% higher LV ejection fraction (LVEF, P < 0.01) than MI rats, and 33% reductions in both plasma atrial natriuretic peptide (ANP, P = 0.052) and cardiac hydroxyproline. Using patch-clamp, action potential duration was reduced and transient outward current density increased (P < 0.05). Cardiac energy metabolism was also improved (+33% creatine phosphate, P < 0.001; +15% ATP; and +9% energy charge, P < 0.05). Significant correlations were found between HR and parameters of cardiac metabolism, ANP, and LVEF (all P < 0.05). The HR-reducing properties of ivabradine prevent changes in the global phenotype of LV remodeling in the rat, optimize energy consumption, and avoid electrophysiological and structural remodeling. [ABSTRACT FROM AUTHOR]

Tegaserod (Teg), a 5-hydroxytryptamine type-4 (5-HT4) receptor partial agonist, represents a novel treatment for irritable bowel syndrome with constipation and chronic constipation. Cardiovascular safety data from pooled clinical studies showed a signal suggestive of increased occurrence of ischaemic cardiovascular events in patients exposed to Teg versus placebo. Thereafter, marketing of Teg was suspended in the USA and other countries. The clinical data did not demonstrate a causative effect but raised questions of whether a non-recognized effect on the heart was present. Our aim was to evaluate for arrhythmogenic potential of Teg on human cardiomyocytes. Cells isolated from human atrial specimens during cardiac surgery were used to assess the effects of Teg (1, 10, 30 and 100 nM) on action potential and If (funny current) by patch-clamp technique. Results showed that Teg (at all concentrations tested) did not significantly affect action potential characteristics of atrial myocytes when driven at different rates (0.2, 0.5 and 1 Hz). In contrast, 5HT significantly prolonged action potential duration (1 and 10 nM) and caused cell un-excitability (100 nM). Teg, at the highest concentration tested (100 nM, corresponding to 10 times Cmax, produced by the recommended dose of 6 mg b.i.d.) increased the If amplitude and caused a shift of its activation curve. This effect of a high concentration of Teg is not considered clinically relevant. When evaluated on single human atrial cells, Teg does not appear to exhibit arrhythmogenic properties, as it did not affect the action potential profile. [ABSTRACT FROM AUTHOR]

Heart failure is a common clinical syndrome occurring as a result of cardiac overload, injury, and a complex interplay among genetic, neurohormonal, inflammatory, and biochemical factors. Occurrence of arrhythmias in heart failure is largely a consequence of disease-induced electrical remodeling of cardiac myocytes, a phenomenon consisting of alterations of ion channels and the ion-transport function that predispose patients to develop lethal arrhythmias. In most cases, the mechanism is the rapid onset of a ventricular tachyarrhythmia progressing to ventricular fibrillation and hemodynamic compromise. This paper highlights some of the important changes in ion channel expression and function that underlie electrical remodeling of the failing heart. Particular attention will be focused on the presence, features, and pharmacologic modulation of f channels expressed in ventricular cardiac myocytes. L’insuffisance cardiaque est un syndrome clinique courant se produisant après une surcharge cardiaque, une lésion et une interaction complexe entre des facteurs génétiques, neurohormonaux, inflammatoires et biochimiques. L’apparition d’arythmies dans l’insuffisance cardiaque découle en grande partie du remodelage électrique des myocytes cardiaques induit par la maladie, phénomène consistant en des modifications des canaux ioniques et de la fonction de transport des ions qui prédisposent les patients au développement d’arythmies mortelles. Dans la plupart des cas, le mécanisme est l’apparition rapide de tachyarythmies ventriculaires progressant vers une fibrillation ventriculaire et un compromis hémodynamique. Le présent article met en lumière quelques modifications importantes dans l’expression et la fonction des canaux ioniques qui sous-tendent le remodelage électrique du cœur défaillant. La présence, les caractéristiques et la modulation pharmacologique des canaux f exprimés dans les myocytes cardiaques ventriculaires feront l’objet d’une attention particulière. [ABSTRACT FROM AUTHOR]

Objectives: The relationship between atrial stretching and changes in cell excitability is well documented. Once stretched, human atrial myocytes (HuAM) release atrial natriuretic peptide (hANP). Receptors for hANP (NPR) are coupled to a guanylyl cyclase (GC) activity, and are present on HuAM, but the electrophysiological effects of hANP are largely unknown. We investigated the effect of hANP on If, the hyperpolarization-activated current present in HuAM, and the underlying intracellular pathway. Methods: HuAM were isolated from atrial appendages and utilized for patch-clamp recording. Results: hANP caused a significant and concentration dependent shift of the midpoint activation potential (ΔVh) toward less negative potentials of 6.9±1.0 mV at 0.1 nM; 13.0±2.6 mV at 1 nM and 15.3±2.2 mV at 10 nM (p<0.001 for all); a parallel increase of If rate of activation occurred. The effect of hANP was completely blocked by isatin, a potent antagonist of NPR (p<0.01 vs. hANP). In the presence of the inhibitors of guanylyl cyclase (ODQ and LY83583), hANP caused a significantly smaller ΔVh (p<0.01 vs. hANP for both). 8Br-cGMP mimicked the effect of hANP, both in the presence and absence of KT5823, a selective inhibitor of Protein kinase G. Pretreatment with pertussis toxin (PTX) did not change the effect of hANP, thus excluding a major role for the coupling of NPR with the Gi-Proteins system. Pretreating cells with cyclopentyladenosine (CPA), an A1-adenosine receptor agonist, completely blocked hANP effect. Adding hANP to maximal serotonin concentration produced an additive response. Conclusions: Our data demonstrate for the first time that ANP is able to increase If, likely through a modulation of intracellular cGMP and cAMP levels. This effect could have implications in the relationship between stretch and arrhythmogenesis in the human atrium. [Copyright &y& Elsevier]

Recent evidence suggests that blockade of the renin-angiotensin system ameliorates diabetes-induced cardiac dysfunction, but the mechanisms involved in this process remain elusive. We investigated the effect of treatment with an angiotensin II receptor blocker, losartan, on the metabolic and electrophysiological properties of cardiomyocytes isolated from streptozotocin-induced diabetic (STZ) rats. Glucose uptake and electrophysiological properties were measured in ventricular cardiomyocytes from normoglycemic and STZ-induced diabetic rats given vehicle or 20 mg x kg(-1) x day(-1) losartan for 8 weeks. Insulin and beta-adrenergic stimulation failed to increase the glucose uptake rate in STZ cardiomyocytes, whereas the alpha-adrenergic effect persisted. Concurrently, a typical prolongation of action potential duration (APD) and a decrease of transient outward current (I(to)) were recorded in patch-clamped STZ myocytes. Treatment with losartan did not affect body weight or glycemia of diabetic or control animals. However, in losartan-treated STZ-induced diabetic rats, beta-adrenergic-mediated enhancement of glucose uptake was completely recovered. APD and I(to) were similar to those measured in losartan-treated control rats. A significant (P < 0.0001) correlation between metabolic and electrophysiological parameters was found in control, diabetic, and losartan-treated diabetic rats. Thus, angiotensin receptor blockade protects the heart from the development of cellular alterations typically associated with diabetes. These data suggest that angiotensin receptor blockers may represent a new therapeutic strategy for diabetic cardiomyopathy. [ABSTRACT FROM AUTHOR]

Both L- and T-type calcium channels are present in the heart. In cardiac myocytes L-type calcium channels are blocked by the classical calcium channel blockers, while T-type calcium channels are thought to be insensitive to these drugs and to be selectively blocked by mibefradil. We aimed to compare the T/L calcium channel blocking selectivity of several calcium channel blockers by evaluating their effects on both components evoked in the same cell from a holding potential corresponding to the normal physiological value (−90 mV). Currents were recorded in single patch-clamped guinea-pig ventricular myocytes, superfused with a Na+- and K+-free solution to abolish overlapping currents. Two dihydropyridines (amlodipine and lacidipine), verapamil diltiazem and mibefradil were tested; for each compound concentrations equieffective on L-type Ca2+ current were used. All calcium channel blockers, at concentrations blocking less than 30% of L-type Ca2+ current, inhibited a significant amount of T-type Ca2+ current, varying from 0.8% (diltiazem) to 28% (mibefradil). We calculated for each compound the T/L ratio. As expected, mibefradil showed the highest T selectivity; lacidipine and diltiazem resulted to be L selective. Verapamil and amlodipine were not selective. Thus, the calcium channel blockers can be differentiated on the basis of their T/L selectivity. [Copyright &y& Elsevier]

Objective: If is a hyperpolarization-activated current, which plays a key role in determining the spontaneous rate of cardiac pacemaker cells. We have previously shown that If is also expressed in left ventricular myocytes isolated from spontaneously hypertensive rats; in these cells, its occurrence and density is linearly related with the severity of myocardial hypertrophy. Since hypertrophy induces a re-expression of genes encoding fetal proteins, we investigated changes in If properties during post-natal development. Methods: Fresh ventricular myocytes were enzymatically isolated from the heart of 1–2- to 28-day-old Wistar rats. The whole-cell configuration of the patch-clamp technique was employed to record the action potential and If. Results: Membrane capacitance, an index of cell size, progressively increased from 13±1 pF at 1–2 days to 66±4 pF at 28 days of age (p<0.01). At 1–2 days, a cesium-sensitive hyperpolarization-activated inward current (If) was recorded in the majority of tested cells (n=51). The midpoint of the activation curve (V1/2) was −78±2 mV (n=32), and specific current conductance of fully activated If (gf,max) was 60±11 pS/pF. Reversal potential (Vrev) measured by tail-current analysis was −24±3 mV (n=8). Reduction of extracellular Na+ from 140 to 35 mM or extracellular K+ from 25 to 5.4 mM caused a shift of −12±1 mV (n=3) or −11±2 mV (n=5) of Vrev, respectively. Occurrence of If decreased with aging, being present in 64%, 48% and 32% of cells at 10, 15 and 28 days, respectively. When present, If density was significantly smaller than at 1–2 days (p<0.05), reaching a value of 8±2 pS/pF at 28 days. However, V1/2 did not change in the older rats, being −80±2, −83±4 and −85±3 mV at 10, 15 and 28 days, respectively. Vrev at 10 and 15 days was −27 and −28 mV, respectively, thus suggesting that channel selectivity did not change. Conclusions: The pacemaker current, If, is expressed in ventricular myocytes from neonatal rats and progressively disappears; when present, it shows electrophysiological properties similar to If re-expressed in hypertrophied adult rat ventricular myocytes. Thus, it is likely that the occurrence of If in ventricular myocytes of hypertrophied and failing hearts is due to the re-expression of a fetal gene. [ABSTRACT FROM PUBLISHER]

Abstract: Ivabradine is a novel antianginal agent which inhibits the pacemaker current. The effects of ivabradine on maximum rate of depolarization (Vmax), repolarization and spontaneous depolarization have not yet been reported in human isolated cardiac preparations. The same applies to large animals close to human in heart size and spontaneous frequency. Using microelectrode technique action potential characteristics and by applying patch-clamp technique ionic currents were studied. Ivabradine exerted concentration-dependent (0.1–10μM) decrease in the amplitude of spontaneous diastolic depolarization and reduction in spontaneous rate of firing of action potentials and produced a concentration- and frequency-dependent Vmax block in dog Purkinje fibers while action potential duration measured at 50% of repolarization was shortened. In the presence of ivabradine, at 400ms cycle length, Vmax block developed with an onset kinetic rate constant of 13.9±3.2beat−1 in dog ventricular muscle. In addition to a fast recovery of Vmax from inactivation (τ=41–46ms) observed in control, a second slow component for recovery of Vmax was expressed (offset kinetics of Vmax block) having a time constant of 8.76±1.34s. In dog after attenuation of the repolarization reserve ivabradine moderately but significantly lengthened the repolarization. In human, significant prolongation of repolarization was only observed at 10μM ivabradine. Ivabradine in addition to the Class V antiarrhythmic effect also has Class I/C and Class III antiarrhythmic properties, which can be advantageous in the treatment of patients with ischemic heart disease liable to disturbances of cardiac rhythm. [Copyright &y& Elsevier]

Abstract: If overexpression has been functionally demonstrated in ventricular myocytes from failing human hearts. Altered expression of If-channels as a consequence of electrophysiological remodeling may represent an arrhythmogenic mechanism in heart failure; however, the molecular basis of If overexpression in human cardiac disease is unknown. HCN1, 2 and 4 subtypes, which encode If-channels, have been identified in the heart. The present study was designed to characterize HCN isoform expression in failing and non-failing hearts. Ventricular and atrial samples were obtained from normal or failing hearts explanted from patients with end-stage ischemic cardiomyopathy. If was recorded in patch-clamped left ventricular myocytes. mRNA and protein expression of HCN subunits were measured in both atria and ventricles of control and diseased hearts. HCN2 and HCN4 were detected in human myocardium. Both mRNA and protein levels of HCN2/4 were significantly augmented in failing ventricles (p <0.01 for mRNA, p <0.05 for protein). These results are consistent with the electrophysiological data showing that, in failing ventricular myocytes, If is of larger amplitude and activates at less negative potential. Changes in mRNA and protein expression of both HCN2/4 isoforms in atrial specimens from patients with heart failure mirrored those observed in ventricles (p <0.001 for mRNA, p <0.05 for protein). No disease-dependent alteration was detected for MiRP1, the putative β-subunit of the If-channel. In conclusion, HCN4 is the predominant channel subtype in normal human heart, and its expression is further amplified by disease. HCN upregulation likely contributes to increased If and may play a role in ventricular and atrial arrhythmogenesis in heart failure. [Copyright &y& Elsevier]