Elisabetta Cerbai

Developmental Changes in Cardiomyocytes Differentiated from Human Embryonic Stem Cells: A Molecular and Electrophysiological Approach

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. Ito1 and IK1, the transient outward and inward rectifier potassium currents, were present in cardiomyocytes only, whereas the rapid delayed rectifier potassium current (IKr), pacemaker current (If), and L‐type calcium current (ICa,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 (Ito1, IK1, and ICa,L) and properties (If). 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.

Characterization of the Hyperpolarization-Activated Current, If, in Ventricular Myocytes From Human Failing Heart

BACKGROUND Disease-associated electrophysiological alterations may contribute to the increased predisposition to arrhythmias of the hypertrophied or failing myocardium. An I(f)-like current is expressed in rat left ventricular myocytes (LVMs), its amplitude being linearly related to the severity of cardiac hypertrophy. Here, we report the occurrence and electrophysiological properties of I(f) in human LVMs. METHODS AND RESULTS LVMs were isolated from hearts of three male patients undergoing cardiac transplantation for terminal heart failure due to ischemic dilated cardiomyopathy. The patch-clamp technique was used to record I(f), ie, a barium-insensitive, cesium-sensitive, time-dependent increasing inward current elicited on hyperpolarization. Membrane capacitance was 244 +/- 27 pF (n = 25). I(f) occurred in all cells tested; its density measured at -120 mV was 2.1 +/- 0.3 pA/pF. Activation curves of I(f) (n = 24) were fitted by a Boltzmann function; the threshold was -55 mV; midpoint, -70.9 +/- 2.1 mV; slope, -5.4 +/- 0.3 mV; and maximal specific conductance, 19.6 +/- 2.5 pS/pF. I(f) blockade by extracellular cesium was voltage dependent. Reducing extracellular potassium concentration from 25 to 5.4 mmol/L caused a shift of the reversal potential from -12.7 +/- 0.5 to -24.8 +/- 2.1 mV and a 64% decrease of current conductance. CONCLUSIONS I(f) is present in human LVMs. Its electrophysiological characteristics resemble those previously described in hypertrophied rat LVMs and suggest that I(f) could be an arrhythmogenic mechanism in patients with severe heart failure.

Late Sodium Current Inhibition Reverses Electromechanical Dysfunction in Human Hypertrophic Cardiomyopathy

Background— Hypertrophic cardiomyopathy (HCM), the most common mendelian heart disorder, remains an orphan of disease-specific pharmacological treatment because of the limited understanding of cellular mechanisms underlying arrhythmogenicity and diastolic dysfunction. Methods and Results— We assessed the electromechanical profile of cardiomyocytes from 26 HCM patients undergoing myectomy compared with those from nonfailing nonhypertrophic surgical patients by performing patch-clamp and intracellular Ca2+ (Ca2+i) studies. Compared with controls, HCM cardiomyocytes showed prolonged action potential related to increased late Na+ (INaL) and Ca2+ (ICaL) currents and decreased repolarizing K+ currents, increased occurrence of cellular arrhythmias, prolonged Ca2+i transients, and higher diastolic Ca2+i. Such changes were related to enhanced Ca2+/calmodulin kinase II (CaMKII) activity and increased phosphorylation of its targets. Ranolazine at therapeutic concentrations partially reversed the HCM-related cellular abnormalities via INaL inhibition, with negligible effects in controls. By shortening the action potential duration in HCM cardiomyocytes, ranolazine reduced the occurrence of early and delayed afterdepolarizations. Finally, as a result of the faster kinetics of Ca2+i transients and the lower diastolic Ca2+i, ranolazine accelerated the contraction-relaxation cycle of HCM trabeculae, ameliorating diastolic function. Conclusions— We highlighted a specific set of functional changes in human HCM myocardium that stem from a complex remodeling process involving alterations of CaMKII-dependent signaling, rather than being a direct consequence of the causal sarcomeric mutations. Among the several ion channel and Ca2+i handling proteins changes identified, an enhanced INaL seems to be a major contributor to the electrophysiological and Ca2+i dynamic abnormalities of ventricular myocytes and trabeculae from patients with HCM, suggesting potential therapeutic implications of INaL inhibition.

Occurrence and Properties of the Hyperpolarization-Activated Current If in Ventricular Myocytes From Normotensive and Hypertensive Rats During Aging

BACKGROUND Cellular electrophysiological alterations may contribute to arrhythmogenesis in cardiac hypertrophy. An L-like current occurs in left ventricular myocytes (LVMs) isolated from the hypertrophied heart of old spontaneously hypertensive rats (SHR). Factors that influence L occurrence during development of cardiac hypertrophy were studied by determining its presence, amplitude, characteristics, and beta-adrenoceptor modulation. METHODS AND RESULTS Patch-clamped LVMs from young (2 to 3 months old) or old (18 to 24 months old) normotensive Wistar-Kyoto rats (WKY) and SHR were used. A diastolic depolarization phase was present in old SHR. An If-like current occurred in > 90% of LVMs from old SHR and WKY and in approximately = 15% of LVMs from young rats (P < .05). Activation curves of If were similar in old rats, with the midpoint at -92.9 +/- 2.9 mV in WKY (n = 42) and -88.1 +/- 1.5 mV in SHR (n = 25); maximal specific conductance was 54.4 +/- 1.7 in SHR and 20.1 +/- 0.5 picosiemens/picofarad in WKY (P < .05). In WKY, If amplitude was linearly related to membrane capacitance, an index of cell size (r = .53, P < .001). This relation was absent in SHR, in which a significant positive correlation was found between the heart weight to body weight ratio and I(f) density. In both old WKY and old SHR, 0.1 mumol/L (-)-isoproterenol increased I(f) amplitude by shifting its activation curve toward more positive potentials. CONCLUSIONS In LVMs from both WKY and SHR, the occurrence of I(f) increases with aging. Density appears linearly related to the severity of cardiac hypertrophy and increases with beta-adrenoceptor stimulation, which suggests that I(f) may contribute to an increased propensity of the hypertrophied heart for arrhythmias.

Characterization of the hyperpolarization-activated current, I(f), in ventricular myocytes isolated from hypertensive rats.

1. Left ventricular myocytes isolated from the heart of young (2‐month‐old) and old (18‐ to 20‐month‐old) spontaneously hypertensive rats (SHRs) were studied in the whole‐cell configuration. Since multicellular preparations from old SHRs show a diastolic depolarization phase, we performed experiments to test whether it was associated with the presence of a hyperpolarization‐activated If‐like current. 2. In control Tyrode solution, a time‐dependent increasing inward current activated by hyperpolarization was recorded in myocytes from old SHRs showing a diastolic depolarization phase. A barium‐insensitive, caesium‐sensitive, time‐dependent inward current was recorded in a minority (4 of 33) of cells from young SHRs (membrane capacitance, 160 +/‐ 7 pF) but in 93% (25 of 27, P < 0.01) of myocytes from old SHRs (membrane capacitance, 355 +/‐ 19 pF, P < 0.01). 3. The current was fully activated at ‐120 mV and voltage of half‐maximal activation was ‐88.1 +/‐ 1.5 mV; it was blocked by extracellular CsCl (4 mM) in a voltage‐dependent manner. Reducing [K+]o from 25 to 5.4 mM caused a shift of the reversal potential from ‐17.3 +/‐ 3.8 to ‐25.7 +/‐ 2.7 mV and a 60% decrease of current conductance. 4. These findings suggest that an If‐like current is present in rat ventricular myocytes from old SHRs, where it might favour the occurrence of spontaneous action potentials.

Molecular basis of funny current (If) in normal and failing human heart

I(f) overexpression has been functionally demonstrated in ventricular myocytes from failing human hearts. Altered expression of I(f)-channels as a consequence of electrophysiological remodeling may represent an arrhythmogenic mechanism in heart failure; however, the molecular basis of I(f) overexpression in human cardiac disease is unknown. HCN1, 2 and 4 subtypes, which encode I(f)-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. I(f) 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, I(f) 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 beta-subunit of the I(f)-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 I(f) and may play a role in ventricular and atrial arrhythmogenesis in heart failure.

Cardiac effects of 3-iodothyronamine: a new aminergic system modulating cardiac function

3‐iodothyronamine T1AM is a novel endogenous thyroid hormone derivative that activates the G protein‐coupled receptor known as trace anime‐associated receptor 1 (TAAR1). In the isolated working rat heart and in rat cardiomyocytes, T1AM produced a reversible, dose‐dependent negative inotropic effect (e.g.,27±5, 51 ±3, and 65±2% decrease in cardiac output at 19, 25, and 38 μM concentration, respectively). An independent negative chronotropic effect was also observed. The hemodynamic effects of T1AM were remarkably increased in the presence of the tyrosine kinase inhibitor genistein, whereas they were attenuated in the presence of the tyrosine phosphatase inhibitor vanadate. No effect was produced by inhibitors of protein kinase A, protein kinase C, calcium‐calmodulin kinase II, phosphatidylinositol‐3‐kinase, or MAP kinases. Tissue cAMP levels were unchanged. In rat ventricular tissue, Western blot experiments with antiphosphotyrosine antibodies showed reduced phosphorylation of microsomal and cytosolic proteins after perfusion with synthetic T1AM;reverse transcriptase‐polymerase chain reaction experiments revealed the presence of transcripts for at least 5 TAAR subtypes; specific and saturable binding of [125I]T1AM was observed, with a dissociation constant in the low micromolar range (5 μM); and endogenous T1AM was detectable by tandem mass spectrometry. In conclusion, our findings provide evidence for the existence of a novel aminergic system modulating cardiac function.—Chiellini G., Frascarelli, S., Ghelardoni, S., Carnicelli, V., Tobias, S. C., DeBarber, A., Brogioni, S., Ronca‐Testoni, S., Cerbai, E., Grandy, D. K., Scanlan, T. S., Zucchi R. Cardiac effects of 3‐iodothyronamine: a new aminergic system modulating cardiac function. FASEB J. 21, 1597–1608 (2007)

Role of potassium currents in cardiac arrhythmias

Abnormal excitability of myocardial cells may give rise to ectopic beats and initiate re-entry around an anatomical or functional obstacle. As K(+) currents control the repolarization process of the cardiac action potential (AP), the K(+) channel function determines membrane potential and refractoriness of the myocardium. Both gain and loss of the K(+) channel function can lead to arrhythmia. The former because abbreviation of the active potential duration (APD) shortens refractoriness and wave length, and thereby facilitates re-entry and the latter because excessive prolongation of APD may lead to torsades de pointes (TdP) arrhythmia and sudden cardiac death. The pro-arrhythmic consequences of malfunctioning K(+) channels in ventricular and atrial tissue are discussed in the light of three pathophysiologically relevant aspects: genetic background, drug action, and disease-induced remodelling. In the ventricles, loss-of-function mutations in the genes encoding for K(+) channels and many drugs (mainly hERG channel blockers) are related to hereditary and acquired long-QT syndrome, respectively, that put individuals at high risk for developing TdP arrhythmias and life-threatening ventricular fibrillation. Similarly, down-regulation of K(+) channels in heart failure also increases the risk for sudden cardiac death. Mutations and polymorphisms in genes encoding for atrial K(+) channels can be associated with gain-of-function and shortened, or with loss-of-function and prolonged APs. The block of atrial K(+) channels becomes a particular therapeutic challenge when trying to ameliorate atrial fibrillation (AF). This arrhythmia has a strong tendency to cause electrical remodelling, which affects many K(+) channels. Atrial-selective drugs for the treatment of AF without affecting the ventricles could target structures such as I(Kur) or constitutively active I(K,ACh) channels.

Influence of postnatal-development on If occurrence and properties in neonatal rat ventricular myocytes

OBJECTIVE I(f) is a hyperpolarization-activated current, which plays a key role in determining the spontaneous rate of cardiac pacemaker cells. We have previously shown that I(f) 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 I(f) 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 I(f). 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 (I(f)) 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 I(f) (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 I(f) decreased with aging, being present in 64%, 48% and 32% of cells at 10, 15 and 28 days, respectively. When present, I(f) 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, I(f), is expressed in ventricular myocytes from neonatal rats and progressively disappears; when present, it shows electrophysiological properties similar to I(f) re-expressed in hypertrophied adult rat ventricular myocytes. Thus, it is likely that the occurrence of I(f) in ventricular myocytes of hypertrophied and failing hearts is due to the re-expression of a fetal gene.

Cellular electrophysiological basis for oxygen radical-induced arrhythmias. A patch-clamp study in guinea pig ventricular myocytes.

BackgroundOxygen radicals have been implicated in the pathogenesis of reperfusion arrhythmias. However, the basic electrophysiological alterations accompanying the effects of oxygen radicals on action potential (AP) are poorly understood. Methods and ResultsWe investigated the effects of oxygen radicals generated by dihydroxyfumarate (DHF, 5 mM) on AP parameters and on ionic currents in patch-clamped guinea pig ventricular myocytes. DHF consistently caused a marked prolongation of AP duration, which was already significant after 60 seconds of exposure and continued to increase over time. Within 5 minutes, the majority of cells developed early afterdepolarizations (EADs) or became unexcitable. Both AP prolongation and occurrence of EADs were completely prevented in the presence of the oxygen radical scavengers superoxide dismutase (SOD) and catalase (CAT). Prolongation of AP duration was accompanied by a marked decreased in time-dependent potassium current (IK) and calcium current (LCa). The inward rectifier K current (IKI) was unaffected, suggesting no widespread changes in membrane properties. LK and LCa alterations were also significantly reduced by SOD and CAT. In additional experiments, intracellular calcium levels were kept constantly low by addition of 200 PM ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetra-acetic acid (EGTA) to the pipette solution. Under these conditions, the effects of DHF on AP duration and the occurrence of EADs were largely prevented. However, EGTA did not prevent cells from becoming unexcitable, nor did it affect the decrease in both IK and IC. upon exposure to DHF. ConclusionsExposure to an exogenous source of oxygen radicals may induce major electrophysiological alterations in isolated myocytes, which might be related to changes in specific ionic currents and in level of intracellular calcium. These alterations occur with a time course consistent with the rapid onset of ventricular arrhythmias in reperfused hearts.