Regeneration of the Cardiac Conduction System by Adipose Tissue-Derived Stem Cells.

BACKGROUND: Adipose tissue is one of the sources of mesenchymal stem cells, which have the potential to differentiate into various types of cells, including myocytes. Whether brown adipose tissue (BAT)-derived cells might differentiate into the cardiac pacemaking-conducting cells, and have the potential to regenerate the cardiac conduction system (CCS), is investigated in this study. METHODS AND RESULTS: BAT was isolated from the interscapular area of mice and enzymatically digested before culture. Round or fusiform cells showed spontaneous beating at 4-7 days after culturing of BAT-derived cells. Reverse transcriptase-polymerase chain reaction analysis and immunocytochemical analysis revealed that BAT-derived cells expressed several cardiomyocytes, the CCS and pacemaker (PM) cell marker genes and proteins. Patch-clamp techniques revealed that spontaneous electrical activity and the shape of the action potential showed properties of cardiac PM cells. Next, a complete atrioventricular (AV) block was created in mice and green fluorescent protein-positive (GFP (+)) BAT-derived cells were injected intramyocardially around the AV node. At 1 week after transplantation, 50% of BAT-derived cells injected mice showed a sinus rhythm or a 2:1 AV block. Immunohistochemical analysis revealed that injected GFP (+) cells were engrafted and some GFP (+) cells co-expressed several cardiac PM cell marker proteins. CONCLUSIONS: BAT-derived cells differentiate into the CCS and PM-like cells in vitro and in vivo, and may become a useful cell source for arrhythmia therapy.

Effects of a highly selective acetylcholine-activated K+ channel blocker on experimental atrial fibrillation.

BACKGROUND: The acetylcholine-activated K(+) current (I(K,ACh)) is a novel candidate for atrial-specific antiarrhythmic therapy. The present study investigates the involvement of I(K,ACh) in atrial fibrillation (AF) using NTC-801, a novel potent and selective I(K,ACh) blocker. METHODS AND RESULTS: The effects of NTC-801, substituted 4-(aralkylamino)-2,2-dimethyl-3,4-dihydro-2H-benzopyran-3-ol, on I(K,ACh) and other cardiac ionic currents (I(Na), I(CaL), I(to), I(Kur), I(Kr), I(Ks), I(Kl), I(KATP), and I(f)) and on atrial and ventricular action potentials were examined in vitro. NTC-801 potently inhibited carbachol-induced I(K,ACh) in guinea pig atrial cells and the GIRK1/4 current in Xenopus oocytes with IC(50) values of 5.7 and 0.70 nmol/L, respectively. NTC-801 selectively inhibited I(K,ACh) >1000-fold over other cardiac ionic currents. NTC-801 (10 to 100 nmol/L) reversed the action potential duration (APD(90)) shortened by carbachol or adenosine in atrial cells, whereas it did not affect APD(90) at 100 nmol/L in ventricular cells. Antiarrhythmic effects of NTC-801 were evaluated in 3 AF models in vivo. NTC-801 significantly prolonged atrial effective refractory period without affecting ventricular effective refractory period under vagal nerve stimulation. NTC-801 dose-dependently converted AF to normal sinus rhythm in both vagal nerve stimulation-induced (0.3 to 3 µg · kg(-1) · min(-1) IV) and aconitine-induced (0.01 to 0.1 mg/kg IV) models. In a rapid atrial pacing model, NTC-801 (3 µg · kg(-1) · min(-1) IV) significantly decreased AF inducibility with a prolonged atrial effective refractory period that was frequency-independent. CONCLUSIONS: A selective I(K,ACh) blockade induced by NTC-801 exerted anti-AF effects mediated by atrial-selective effective refractory period prolongation. These findings suggest that I(K,ACh) may be important in the development and maintenance of AF.

Effects of nicorandil on the cAMP-dependent Cl- current in guinea-pig ventricular cells.

In guinea-pig cardiomyocytes, a cAMP-dependent Cl(-) current (I(Cl,cAMP)) flows through a cardiac isoform of the cystic fibrosis transmembrane conductance regulator (CFTR), which belongs to a family of the ATP-binding cassette (ABC) proteins. Although several K(+)-channel openers and sulfonylurea ATP-sensitive K(+) (K(ATP))-channel blockers reportedly inhibit I(Cl,cAMP), effects of nicorandil on the Cl(-) current have not been evaluated. This study was conducted to examine the effects of nicorandil on I(Cl,cAMP) in isolated guinea-pig ventricular cells using patch clamp techniques. Nicorandil in concentrations higher than 300 microM enhanced the I(Cl,cAMP) preactivated by 0.1 microM isoproterenol. The isoproterenol-induced I(Cl,cAMP) was inhibited by 100 microM glibenclamide, but not by 100 microM pinacidil. SNAP (S-nitroso-N-acetyl-D,L-penicillamine, 10 microM), a nitric oxide (NO) donor, similarly enhanced the isoproterenol-induced I(Cl,cAMP). However, SG-86, a denitrated metabolite possessing K(+ )channel-opening action, failed to enhance the Cl(-) current. When the I(Cl,cAMP) was activated by 3-isobutyl-1-methylxanthine (IBMX, 30 microM), either nicorandil or SNAP failed to enhance the isoproterenol-induced I(Cl,cAMP). Thus, nicorandil enhances I(Cl,cAMP) in guinea-pig cardiomyocytes through an increase in intracellular cGMP, although direct modulation of I(Cl,cAMP) by NO cannot be completely excluded.

Identification and expression analysis of ras gene in silkworm, Bombyx mori.

Ras proteins play important roles in development especially for cell proliferation and differentiation in various organisms. However, their functions in the most insect species are still not clear. We identified three ras cDNAs from the silk worm, Bombyx mori. These sequences corresponded to three Ras of Drosophila melanogaster, but not to three mammalian Ras (H-Ras, K-Ras, N-Ras). Subsequently, the expression profiles of ras were investigated by quantitative real-time PCR using whole body of individuals from the embryonic to adult stages, and various tissues of 4th and 5th instar larvae. Each of three Bombyx ras showed different expression patterns. We also showed membrane localization of their products. These results indicate that the three Bombyx Ras are functional and have different roles.

New aspects for the treatment of cardiac diseases based on the diversity of functional controls on cardiac muscles: mitochondrial ion channels and cardioprotection.

Mitochondrial ATP-sensitive K(+) (mitoK(ATP)) and Ca(2+)-activated K(+) (mitoK(Ca)) channels exist in cardiac myocytes, and they play key roles in cardioprotection. We have recently reported that K(+) influx through mitoK(ATP) or mitoK(Ca) channels occurs independently of each other and confers cardioprotection in a similar manner. Activation of mitoK(ATP) channel is augmented by protein kinase C (PKC), whereas mitoK(Ca) channel is activated by protein kinase A (PKA). However, phosphatidylinositol 3-kinase (PI3-K) is linked to neither mitoK(ATP) nor mitoK(Ca) channels. We have demonstrated that bioactive substances modulate the opening of mitoK(ATP) channels via a PKC-dependent pathway or opening of mitoK(Ca) channels via a PKA-dependent pathway and thereby protecting the heart from ischemia/reperfusion injury. Several endogenous substances such as adenosine and bradykinin can reduce infarct size by activation of mitoK(ATP) channels in a PKC-dependent manner. Adrenomedullin, a potent vasodilator peptide, potentiates the opening of mitoK(Ca) channels by PKA activation. Treatment with adrenomedullin prior to ischemia results in the reduction of infarct size via a PKA-mediated activation of mitoK(Ca) channels. Thus, some endogenous substances confer cardioprotection via PKA- or PKC-mediated activation of mitoK(ATP) or mitoK(Ca) channels.

Effects of azimilide on the muscarinic acetylcholine receptor-operated K+ current and experimental atrial fibrillation in guinea-pig hearts.

Effects of azimilide, a class III antiarrhythmic drug, on the acetylcholine (ACh) receptor-operated K+ current (I K.ACh) and the delayed rectifier K+ current (IK) were examined in guinea-pig atrial cells using patch-clamp techniques. Effects of azimilide on experimental atrial fibrillation (AF) were also examined in isolated guinea-pig hearts. In single atrial myocytes, azimilide inhibited both the rapid (IKr) and slow component of IK (IKs). Azimilide inhibited the I K.ACh induced by carbachol (CCh, 1 microM), adenosine (10 microM), and intracellular loading of GTPgammaS (100 microM) in a concentration-dependent manner. The IC50 values of azimilide for inhibiting the CCh-, adenosine-, and GTPgammaS-induced I K.ACh were 1.25, 29.1, and 20.9 microM, respectively, suggesting that azimilide inhibits I K.ACh mainly by blocking the muscarinic receptors. Azimilide concentration-dependently (0.3 - 10 microM) prolonged the action potential duration (APD) in the absence and presence of muscarinic stimulation. In isolated hearts, perfusion of CCh shortened the duration of the monophasic action potential (MAP) and effective refractory period (ERP) of the left atrium and lowered the atrial fibrillation threshold (AFT). Addition of azimilide inhibited the induction of AF by prolonging the duration of MAP and ERP. The I K.ACh inhibition by azimilide may at least in part contribute to the effectiveness to prevent parasympathetic-type AF.

Inhibitory effects of AMP 579, a novel cardioprotective adenosine A1/A2A receptor agonist, on native IKr and cloned HERG current.

We investigated the effects of 1S-[1a,2b,3b,4a(S*)]-4-[7-[[1-[(3-chloro-2-thienyl)methylpropyl]propyl-amino]-3H-imidazo[4,5-b] pyridyl-3-yl]-N-ethyl-2,3-dihydroxycyclopentane carboxamide (AMP 579), a novel cardioprotective adenosine A(1)/A(2A) receptor agonist, on the rapid and slow components of the delayed rectifier K(+) current (I(Kr) and I(Ks)) in guinea-pig ventricular myocytes and on the human ether-a-go-go-related gene (HERG) channel expressed in human embryonic kidney (HEK 293) cells. Whole-cell current and membrane potential were recorded using patch-clamp techniques. In guinea-pig ventricular myocytes, AMP 579 inhibited I(Kr) in a concentration-dependent manner with IC(50) value of 15.2 microM, when I(Kr) was blocked by chromanol 293B. On the contrary, AMP 579 (10 microM) did not affect I(Ks) in the presence of the I(Kr) blocker E-4031. The former effect of AMP 579 was unaffected by either the selective adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine or the non-selective adenosine A(1)/A(2) receptor antagonist 8-sulphophenyltheophylline. Moreover, AMP 579-induced inhibition of I(Kr) was not voltage- and frequency-dependent. In HEK 293 cells expressing HERG channels, AMP 579 (10 microM) significantly blocked the HERG current at +10 mV by 34.9+/-7.0% (n=4, p<0.05), and the degree of inhibition was comparable with that observed in guinea-pig ventricular myocytes (36.8+/-6.0%, n=4). AMP 579 (10 microM) significantly inhibited the L-type Ca(2+) current (I(Ca)) by 41.0+/-6.8% (n=5, p<0.05), which was unaffected by 8-sulphophenyl-theophylline. Consequently, despite its inhibitory actions on I(Kr) or HERG current, the drug significantly shortened the action potential duration measured at 90% repolarization from 275.6+/-19.4 to 208.3+/-18.6 ms (n=4, p<0.05). Thus, AMP 579 inhibits both native I(Kr) and cloned HERG channels with additional inhibitory effect of I(Ca), and such inhibitory effects may at least partially underlie the observed antifibrillatory action of the drug during myocardial ischemia/reperfusion.

Beating is necessary for transdifferentiation of skeletal muscle-derived cells into cardiomyocytes.

Cell transplantation could be a potential therapy for heart damage. Skeletal myoblasts have been expected to be a good cell source for autologous transplantation; however, the safety and efficacy of their transplantation are still controversial. Recent studies have revealed that skeletal muscle possesses the stem cell population that is distinct from myoblasts. To elucidate whether skeletal muscle stem cells can transdifferentiate into cardiomyocytes, we cocultured skeletal muscle cells isolated from transgenic mice expressing green fluorescent protein with cardiomyocytes of neonatal rats. Skeletal muscle-derived cells expressed cardiac-specific proteins such as cardiac troponin T and atrial natriuretic peptide as well as cardiac-enriched transcription factors such as Nkx2E (formerly called Csx/Nkx2.5) and GATA4 by coculture with cardiomyocytes. Skeletal muscle-derived cells also expressed cadherin and connexin 43 at the junctions with neighboring cardiomyocytes. Cardiomyocyte-like action potentials were recorded from beating skeletal muscle-derived cells. Treatment of nifedipine or culture in Ca2+-free media suppressed contraction of cardiomyocytes and inhibited skeletal muscle cells to express cardiac-specific proteins. Cyclic stretch completely restored this inhibitory effect. These results suggest that some part of skeletal muscle cells can transdifferentiate into cardiomyocytes and that direct cell-to-cell contact and contraction of neighboring cardiomyocytes are important for the transdifferentiation.

Inhibitory effect of the class III antiarrhythmic drug nifekalant on HERG channels: mode of action.

Nifekalant is a class III antiarrhythmic drug that has been shown to be effective against ventricular tachyarrhythmias in experimental animals and humans. We examined the detailed electrophysiological effects of nifekalant on human-ether-a-go-go-related gene (HERG) channels expressed in Xenopus oocytes. Nifekalant inhibited the HERG current in a concentration-dependent manner with an IC(50) value of 7.9 microM although the drug did not inhibit the minK current in Xenopus oocytes, suggesting selective inhibition of the rapid component of the delayed rectifier K(+) current (I(Kr)) in cardiomyocytes. Nifekalant showed a higher binding affinity for the open state than for the inactive state of HERG channels. Nifekalant inhibited HERG channels in a frequency-dependent manner. The onset of the blockade was rapid but the recovery from the block was slow. Nifekalant modified the voltage dependence and kinetics of HERG channel gating. Thus, nifekalant inhibits HERG channels in a voltage-dependent and frequency-dependent manner, and the inhibitory effect may underlie the clinical efficacy of the drug against ventricular tachyarrhythmias.

Cystine transport activity of heterozygous rBAT mutants expressed in Xenopus oocytes.

The rBAT gene encodes a transport protein for cystine and dibasic amino acids. It is a candidate gene for type I cystinuria, a genetic disorder inherited as an autosomal-recessive trait. Recently, several mutations in rBAT from Japanese patients with cystinuria have been reported from our laboratory. Some of these patients were heterozygous, which appears to be inconsistent with the previous concept that mutations in rBAT are recessive. To investigate the function of heterozygous mutants, we introduced these mutations into rBAT gene and analyzed the transport activity of cystine associated with the mutants in Xenopus oocytes. Co-injection of the mutant T1037C (L346P) and the polymorphism G1854A (M6181) into Xenopus oocytes produced a transport activity of 67.9% of the wild type. Oocytes co-injected with T2017C (C673R) and wild type had a transport activity of 70.3% of the wild type. These findings indicate that the heterozygous mutants show decreased transport activity compared to wild-type rBAT. Further, some mutants in rBAT may show decreased cystine transport activity even in heterozygous condition, which may contribute to stone-forming cystinuria.

ClC-3B, a novel ClC-3 splicing variant that interacts with EBP50 and facilitates expression of CFTR-regulated ORCC.

We have cloned ClC-3B, a novel alternative splicing variant of ClC-3 (ClC-3A) that is expressed predominantly in epithelial cells. ClC-3B has a different, slightly longer C-terminal end than ClC-3A and contains a consensus motif for binding to the second PDZ (PSD95/Dlg/ZO-1) domain of the epithelium-specific scaffolding protein EBP50. Both in vitro and in vivo binding assays demonstrate interaction between ClC-3B and EBP50. C127 mouse mammary epithelial cells transfected with ClC-3B alone showed diffuse immunoreactivity for ClC-3B in the cytoplasmic region. In contrast, when EBP50 was cotransfected with ClC-3B, strong immunoreactivity for ClC-3B appeared at the leading edges of membrane ruffles. Patch-clamp experiments revealed that cotransfection of ClC-3B and EBP50 resulted in a remarkable increase in outwardly rectifying Cl- channel (ORCC) activities at the leading edges of membrane ruffles in C127 cells. The electrophysiological properties of the ClC-3B-induced ORCCs are similar to those of ORCCs described in native epithelial cells. When cystic fibrosis transmembrane conductance regulator (CFTR) was cotransfected with ClC-3B and EBP50, ClC-3B-dependent ORCCs were activated via the protein kinase A-dependent pathway. These findings indicate that ClC-3B is itself a CFTR-regulated ORCC molecule or its activator.