Heart disease remains a leading cause of death worldwide. Owing to the limited regenerative capacity of heart tissue, cardiac regenerative therapy has emerged as an attractive approach. Direct reprogramming of human cardiac fibroblasts (HCFs) into cardiomyocytes may hold great potential for this purpose. We reported previously that induced cardiomyocyte-like cells (iCMs) can be directly generated from mouse cardiac fibroblasts in vitro and vivo by transduction of three transcription factors: Gata4, Mef2c, and Tbx5, collectively termed GMT. In the present study, we sought to determine whether human fibroblasts also could be converted to iCMs by defined factors. Our initial finding that GMT was not sufficient for cardiac induction in HCFs prompted us to screen for additional factors to promote cardiac reprogramming by analyzing multiple cardiac-specific gene induction with quantitative RT-PCR. The addition of Mesp1 and Myocd to GMT up-regulated a broader spectrum of cardiac genes in HCFs more efficiently compared with GMT alone. The HCFs and human dermal fibroblasts transduced with GMT, Mesp1, and Myocd (GMTMM) changed the cell morphology from a spindle shape to a rod-like or polygonal shape, expressed multiple cardiac-specific proteins, increased a broad range of cardiac genes and concomitantly suppressed fibroblast genes, and exhibited spontaneous Ca 2+ oscillations. Moreover, the cells matured to exhibit action potentials and contract synchronously in coculture with murine cardiomyocytes. A 5-ethynyl-2′-deoxyuridine assay revealed that the iCMs thus generated do not pass through a mitotic cell state. These findings demonstrate that human fibroblasts can be directly converted to iCMs by defined factors, which may facilitate future applications in regenerative medicine.cell fate conversion | regeneration | cardiogenesis C ardiovascular disease remains a leading cause of death worldwide, for which current therapeutic regimens remain limited. Given that adult human hearts have little regenerative capacity after injury, the demand is high for cardiac regenerative therapy. The recent discovery of induced pluripotent stem cells (iPSCs) allows the direct generation of specific cell types from differentiated somatic cells by overexpression of lineagespecific factors.Several previous studies have demonstrated that such direct lineage reprogramming can yield a diverse range of cell types, including pancreatic β cells, neurons, neural progenitors, blood progenitors, and hepatocyte-like cells (1-5). We previously reported that a minimum mixture of three cardiac-specific transcription factors-Gata4, Mef2c, and Tbx5 (GMT)-directly induced cardiomyocyte-like cells (iCMs) from mouse fibroblasts in vitro (6). Following our report, three other groups also reported generation of functional cardiomyocytes from mouse fibroblasts with various combinations of transcription factors, either with GMT plus Hand2 (GHMT) or Mef2c, Myocd, and Tbx5 or using microRNAs (7-9). Although full reprogramming into beating cardiomyocytes was not effic...

The complex profile of amiodarone actions on the electrophysiological properties of cardiac cells reviewed in this article may be summarized as follows. As acute effects, amiodarone inhibits both inward and outward currents. The inhibition of inward Na+ and Ca2+ currents is enhanced in a use- and voltage-dependent manner, resulting in suppression of excitability and conductivity in both iNa- and iCa-dependent cardiac tissues. The inhibition is greater in the tissues stimulated at higher frequencies, and in those with less negative resting (or diastolic) membrane potentials. As outward currents, iK (iKr and iKs), iK,ACh and iK,Na are inhibited by acute amiodarone, iKl could also be inhibited at high concentrations of amiodarone. Acute effects of amiodarone on i(to) remain unclear. Previous reports on the acute effects of amiodarone on APD are conflicting, presumably because different ionic currents are responsible for the repolarization of action potential in different animal species, cardiac tissues and experimental conditions. APD would be shortened if the inhibitory action of amiodarone on the inward current is greater than on the outward current, and vice versa in the opposite case. The major and consistent chronic effect of amiodarone is a moderate APD prolongation with minimal frequency-dependence. This prolongation is most likely due to a decrease in the current density of iK and i(to). Chronic effects of amiodarone are modulated by tissue accumulation of amiodarone and DEA. Variable suppression of excitability and conductivity of the heart by chronic amiodarone might reflect direct acute effects of the parent drug and/or its active metabolite (DEA) retained at the sites of action. Chronic amiodarone was shown to cause a down-regulation of Kv1.5 mRNA in rat hearts, suggesting a drug-induced modulation of potassium channel gene expression. Electrophysiological changes in the heart induced by chronic amiodarone resemble those induced by hypothyroidism. Three mechanisms have been proposed to explain this hypothyroid-like action of amiodarone. Amiodarone and/or DEA may inhibit peripheral conversion from T4 to T3, cellular uptake of T4 and T3, and T3 binding to nuclear receptors (TR). The second and third mechanisms are considered to be more important than the first. Amiodarone or DEA could antagonize T3 action on the heart at a cellular or subcellular level. Two distinct characteristics in the cellular electropharmacology or amiodarone are different from those of other antiarrhythmic drugs. First, it acts on many different types of molecular targets including Na+, Ca2+, and K+ channels as well as adrenoceptors. Second, it may cause antiarrhythmic remodeling of cardiac cells, probably through a modulation of gene expression of ion channels and other functional proteins. We hypothesize that this remodeling is mediated most likely by cellular or subcellular T3 antagonism. Nevertheless, much remains to be studied as ot the acute and especially chronic effects of amiodarone on ionic currents, transporters, receptors...

Drug-induced block of cardiac hERG K ϩ channels causes acquired long QT syndrome. Here, we characterized the molecular mechanism of hERG block by two low-potency drugs (Nifekalant and bepridil) and two high-potency drugs 1-[2-(6-methyl-2pyridyl)ethyl]-4-(4-methylsulfonyl aminobenzoyl)piperidine (E-4031) and dofetilide). Channels were expressed in Xenopus laevis oocytes, and currents were measured using the two-microelectrode voltage-clamp technique. All four drugs progressively reduced hERG current during a 20-s depolarization to 0 mV after a 10-min pulse-free period, consistent with the preferential block of open channels. Recovery from block in response to pulses to Ϫ160 mV was observed for D540K hERG channels but not for wild-type hERG channels, suggesting that all four drugs are trapped in the central cavity by closure of the activation gate. The molecular determinants of hERG channel block were defined by using a site-directed mutagenesis approach. Mutation to alanine of three residues near the pore helix (Thr623, Ser624, and Val625) and four residues in Ser6 (Gly648, Tyr652, Phe656, and Val659) reduced channel sensitivity to block by dofetilide and E-4031, effects identical with those reported previously for two other methanesulfonanilides, (ϩ)- -499) and ibutilide. The effect of nifekalant on mutant channels was similar, except that V659A retained normal sensitivity and I655A channels were less sensitive. Finally, mutation of the three residues near the pore helix and Phe656 in the Ser6 domain reduced channel block by bepridil. We conclude that the binding site is not identical for all drugs that preferentially block hERG in the open state.Class III antiarrhythmic drugs are defined by their ability to block potassium channels and prolong the action potential duration of cardiomyocytes. Some of the most potent class III drugs such as dofetilide, E-4031, and MK-499 are structural analogs of the methanesulfonanilide sotalol, a compound with low potency. All of these drugs prolong action potentials by a relatively specific block of the rapid delayed rectifier K ϩ current, I Kr (Sanguinetti and Jurkiewicz, 1990). Sotalol is the only class III methanesulfonanilide that has been shown in clinical trials to improve prognosis; however, the potency of I Kr block by sotalol is several hundred-fold weaker than dofetilide, E-4031, or MK-499. The discrepancy between the beneficial clinical profile and a low potency for I Kr block has long been reported for sotalol and may indicate the greater importance of the ␤-adrenergic receptor blocking activity compared with I Kr block.The human I Kr channel is encoded by HERG and mutations in this gene cause long QT syndrome , a disorder of cardiomyocyte repolarization that predisposes affected individuals to an increased risk of torsades de pointes and lethal ventricular fibrillation. The most common cause of prolonged QT interval is treatment with class III antiarrhythmic agents and side effects associated with treatment with certain noncardiac medications. For this re...

Background Fibroblast proliferation and differentiation are central in atrial fibrillation (AF)–promoting remodeling. Here, we investigated fibroblast regulation by Ca2+-permeable transient receptor potential canonical-3 (TRPC3) channels. Methods and Results Freshly isolated rat cardiac fibroblasts abundantly expressed TRPC3 and had appreciable nonselective cation currents (INSC) sensitive to a selective TPRC3 channel blocker, pyrazole-3 (3 μmol/L). Pyrazole-3 suppressed angiotensin II-induced Ca2+ influx, proliferation, and α-smooth muscle actin protein expression in fibroblasts. Ca2+ removal and TRPC3 blockade suppressed extracellular signal-regulated kinase phosphorylation, and extracellular signal-regulated kinase phosphorylation inhibition reduced fibroblast proliferation. TRPC3 expression was upregulated in atria from AF patients, goats with electrically maintained AF, and dogs with tachypacing-induced heart failure. TRPC3 knockdown (based on short hairpin RNA [shRNA]) decreased canine atrial fibroblast proliferation. In left atrial fibroblasts freshly isolated from dogs kept in AF for 1 week by atrial tachypacing, TRPC3 protein expression, currents, extracellular signal-regulated kinase phosphorylation, and extracellular matrix gene expression were all significantly increased. In cultured left atrial fibroblasts from AF dogs, proliferation rates, α-smooth muscle actin expression, and extracellular signal-regulated kinase phosphorylation were increased and were suppressed by pyrazole-3. MicroRNA-26 was downregulated in canine AF atria; experimental microRNA-26 knockdown reproduced AF-induced TRPC3 upregulation and fibroblast activation. MicroRNA-26 has NFAT (nuclear factor of activated T cells) binding sites in the 5′ promoter region. NFAT activation increased in AF fibroblasts, and NFAT negatively regulated microRNA-26 transcription. In vivo pyrazole-3 administration suppressed AF while decreasing fibroblast proliferation and extracellular matrix gene expression. Conclusions TRPC3 channels regulate cardiac fibroblast proliferation and differentiation, likely by controlling the Ca2+ influx that activates extracellular signal-regulated kinase signaling. AF increases TRPC3 channel expression by causing NFAT-mediated downregulation of microRNA-26 and causes TRPC3-dependent enhancement of fibroblast proliferation and differentiation. In vivo, TRPC3 blockade prevents AF substrate development in a dog model of electrically maintained AF. TRPC3 likely plays an important role in AF by promoting fibroblast pathophysiology and is a novel potential therapeutic target.