Leukemia inhibitory factor activates cardiac L-Type Ca2+ channels via phosphorylation of serine 1829 in the rabbit Cav1.2 subunit.

We have previously reported that leukemia inhibitory factor (LIF) gradually increased cardiac L-type Ca2+ channel current (I(CaL)), which peaked at 15 minutes in both adult and neonatal rat cardiomyocytes, and this increase was blocked by the mitogen-activated protein kinase kinase inhibitor PD98059. This study investigated the molecular basis of LIF-induced augmentation of I(CaL) in rodent cardiomyocytes. LIF induced phosphorylation of a serine residue in the alpha(1c) subunit (Ca(v)1.2) of L-type Ca2+ channels in cultured rat cardiomyocytes, and this phosphorylation was inhibited by PD98059. When constructs encoding either a wild-type or a carboxyl-terminal-truncated rabbit Ca(v)1.2 subunit were transfected into HEK293 cells, LIF induced phosphorylation of the resultant wild-type protein but not the mutant protein. Cotransfection of constitutively active mitogen-activated protein kinase kinase also resulted in phosphorylation of the Ca(v)1.2 subunit in the absence of LIF stimulation. In in-gel kinase assays, extracellular signal-regulated kinase phosphorylated a glutathione S-transferase fusion protein of the carboxyl-terminal region of Ca(v)1.2 (residues 1700 through 1923), which contains the consensus sequence Pro-Leu-Ser-Pro. A point mutation within this consensus sequence, which results in a substitution of alanine for serine at residue 1829 (S1829A), was sufficient to abolish the LIF-induced phosphorylation. LIF increased I(CaL) in HEK cells transfected with wild-type Ca(v)1.2 but not with the mutated version. These results provide direct evidence that LIF phosphorylates the serine residue at position 1829 of the Ca(v)1.2 subunit via the actions of extracellular signal-regulated kinase and that this phosphorylation increases I(CaL) in cardiomyocytes.

Ameliorating effect of FK614, a novel nonthiazolidinedione peroxisome proliferator-activated receptor gamma agonist, on insulin resistance in Zucker fatty rat.

Effect of 3-(2,4-dichlorobenzyl)-2-methyl-N-(pentylsulfonyl)-3H-benzimidazole-5-carboxamide (FK614), a novel nonthiazolidinedione peroxisome proliferator-activated receptor (PPAR) gamma agonist, on glucose tolerance and insulin resistance in peripheral tissues and in liver using Zucker fatty rats (genetically obese and insulin-resistant) was evaluated and compared to other insulin sensitizers. FK614 (0.32, 1 and 3.2 mg/kg), two thiazolidinedione PPAR gamma agonists, rosiglitazone (0.1, 0.32, 1 and 3.2 mg/kg) and pioglitazone (1, 3.2 and 10 mg/kg), and a biguanide, metformin (320 and 1000 mg/kg), were orally administered to Zucker fatty rats once a day for 14 days. Zucker fatty rats treated with FK614 and rosiglitazone were subjected to evaluation by oral glucose tolerance test. Ameliorating effect of each compound on peripheral and hepatic insulin resistance was evaluated using a euglycemic-hyperinsulineamic clamp procedure. FK614 and rosiglitazone dose-dependently improved impaired glucose tolerance in Zucker fatty rats. In addition, FK614 dose-dependently ameliorated peripheral and hepatic insulin resistance in Zucker fatty rats, with the degree of its effect in peripheral tissues almost equivalent to that in liver when compared at each dose tested. Similar data indicating ameliorating effects on insulin resistance was obtained for rosiglitazone and pioglitazone. Metformin showed less potent effects than other insulin sensitizers and its effect in liver tended to be greater than that in peripheral tissues. These findings suggest clinical potential for FK614 as a treatment of type 2 diabetes, acting by ameliorating insulin resistance both in peripheral tissues and liver.

Pharmacological characteristics of a novel nonthiazolidinedione insulin sensitizer, FK614.

We evaluated antidiabetic effects of 3-(2,4-dichlorobenzyl)-2-methyl-N-(pentylsulfonyl)-3 H-benzimidazole-5-carboxamide (FK614), a benzimidazole derivative without a thiazolidinedione structure, which was obtained using C57BL/KsJ-db/db mice (db/db mice). In db/db mice, the potency of FK614 for hypoglycemic effect was comparable to that of rosiglitazone and approximately 15-fold greater than that of pioglitazone. FK614 also showed a potent attenuating effect on hypertriglyceridemia in db/db mice, as well as rosiglitazone and pioglitazone. In C57BL/6J-ob/ob mice (ob/ob mice), ED(50) values of FK614 and pioglitazone for hypoinsulinemic effect were 1.3 and 11.8 mg/kg, respectively. FK614 also improved the impaired glucose tolerance in ob/ob mice. In normal rats, FK614 did not influence plasma glucose and insulin levels but significantly decreased both plasma triglyceride and nonesterified fatty acid levels. FK614 was found to activate peroxisome proliferator-activated receptor (PPAR)gamma-mediated transcriptional activity in the reporter gene assay as well as thiazolidinedione derivatives, although its maximum effect was less than that of thiazolidinedione derivatives. In rat toxicity studies, hemodilution effects for FK614 were less than that for rosiglitazone. Overall, these studies suggest that FK614 improves insulin resistance in such animal models through activation of PPARgamma-mediated transcriptional activity and that it would be a new therapeutic candidate with potential for the treatment of type 2 diabetic patients.

A mathematical model of phase 2 reentry: role of L-type Ca current.

Phase 2 reentry (P2R) is known to be one of the mechanisms of malignant ventricular arrhythmias, especially those associated with Brugada syndrome. However, little is known about the underlying mechanism for P2R. Our aim in this study was to simulate P2R in a mathematical model to enable us to understand its mechanism and identify a potential therapeutic target. A mathematical model of the L-type Ca current was composed according to whole cell current data from guinea pig ventricular myocytes recorded at 37 degrees C. Our mathematical model was incorporated into the modified Luo-Rudy phase 2 model. We set a dispersion in transient outward current (I(to)) density within the theoretical fiber, composed of 80 serially arranged epicardial cells with gap junctions and then observed the P2R. The dispersion in I(to) density within an only 0.8-cm epicardial theoretical fiber generated P2R with our Ca channel but not with the original model. When the P2R developed in the theoretical fiber, the calculated extracellular field potential showed coved-type ST segment elevation. We succeeded in generating P2R in our model for the first time. The local epicardial P2R may contribute the genesis of coved-type ST segment elevation in the Brugada syndrome.

Can the life span of human marrow stromal cells be prolonged by bmi-1, E6, E7, and/or telomerase without affecting cardiomyogenic differentiation?

BACKGROUND: Cell transplantation has recently been challenged to improve cardiac function of severe heart failure. Human mesenchymal stem cells (hMSCs) are multipotent cells that can be isolated from adult marrow stroma, but because of their limited life span, it is difficult to study them further. To overcome this problem, we attempted to prolong the life span of hMSCs and investigate whether the hMSCs modified with cell-cycle-associated genes can differentiate into cardiomyocytes in vitro. METHODS: We attempted to prolong the life span of hMSCs by infecting retrovirus encoding bmi-1, human papillomavirus E6 and E7, and/or human telomerase reverse transcriptase genes. To determine whether the hMSCs with an extended life span could differentiate into cardiomyocytes, 5-azacytidine-treated hMSCs were co-cultured with fetal cardiomyocytes in vitro. RESULT: The established hMSCs proliferated over 150 population doublings. On day 3 of co-cultivation, the hMSCs became elongated, like myotubes, began spontaneously beating, and acquired automaticity. Their rhythm clearly differed from that of the surrounding fetal mouse cardiomyocytes. The number of beating cardiomyocytes increased until 3 weeks. hMSCs clearly exhibited differentiated cardiomyocyte phenotypes in vitro as revealed by immunocytochemistry, RT-PCR, and action potential recording. CONCLUSIONS: The life span of hMSCs was prolonged without interfering with cardiomyogenic differentiation. hMSCs with an extended life span can be used to produce a good experimental model of cardiac cell transplantation and may serve as a highly useful cell source for cardiomyocytic transplantation.