Morihiko Takeda

Identification of Cardiac-Specific Myosin Light Chain Kinase

Two myosin light chain (MLC) kinase (MLCK) proteins, smooth muscle (encoded by mylk1 gene) and skeletal (encoded by mylk2 gene) MLCK, have been shown to be expressed in mammals. Even though phosphorylation of its putative substrate, MLC2, is recognized as a key regulator of cardiac contraction, a MLCK that is preferentially expressed in cardiac muscle has not yet been identified. In this study, we characterized a new kinase encoded by a gene homologous to mylk1 and -2, named cardiac MLCK, which is specifically expressed in the heart in both atrium and ventricle. In fact, expression of cardiac MLCK is highly regulated by the cardiac homeobox protein Nkx2-5 in neonatal cardiomyocytes. The overall structure of cardiac MLCK protein is conserved with skeletal and smooth muscle MLCK; however, the amino terminus is quite unique, without significant homology to other known proteins, and its catalytic activity does not appear to be regulated by Ca2+/calmodulin in vitro. Cardiac MLCK is phosphorylated and the level of phosphorylation is increased by phenylephrine stimulation accompanied by increased level of MLC2v phosphorylation. Both overexpression and knockdown of cardiac MLCK in cultured cardiomyocytes revealed that cardiac MLCK is likely a new regulator of MLC2 phosphorylation, sarcomere organization, and cardiomyocyte contraction.

Protective Role of Endogenous Erythropoietin System in Nonhematopoietic Cells Against Pressure Overload–Induced Left Ventricular Dysfunction in Mice

Background— Erythropoietin (Epo) receptors (EpoRs) are expressed in the heart. We have recently demonstrated that the endogenous Epo-EpoR system plays an important protective role in myocardial ischemia in mice and humans. In the present study, we tested our hypothesis that the endogenous Epo-EpoR system in nonhematopoietic cells also plays a protective role against pressure overload-induced cardiac dysfunction in vivo. Methods and Results— Transgene-rescued EpoR-null mutant mice (EpoR−/−rescued) that express EpoR exclusively in the hematopoietic cells were subjected to transverse aortic constriction (TAC). At 1 week after TAC, left ventricular weight and lung weight were significantly increased in EpoR−/−rescued mice compared with wild-type mice, although the fibrotic area was comparably increased after TAC in the 2 genotypes. In the EpoR−/−rescued mice with TAC, left ventricular end-diastolic diameter was significantly increased, left ventricular fractional shortening was significantly decreased, and survival rate was significantly decreased compared with wild-type mice with TAC. Phosphorylation of STAT3 at 5 hours and 1 week after TAC and that of p38 at 5 hours after TAC were significantly increased in wild-type mice but not in EpoR−/−rescued mice. Vascular endothelial growth factor protein expression and capillary density in left ventricular myocardium were significantly decreased in EpoR−/−rescued mice with TAC compared with wild-type mice with TAC. Conclusions— These results suggest that the endogenous Epo-EpoR system in the nonhematopoietic cells plays an important protective role against pressure overload-induced cardiac dysfunction in vivo.

Perinatal Loss of Nkx2-5 Results in Rapid Conduction and Contraction Defects

Homeobox transcription factor Nkx2-5, highly expressed in heart, is a critical factor during early embryonic cardiac development. In this study, using tamoxifen-inducible Nkx2-5 knockout mice, we demonstrate the role of Nkx2-5 in conduction and contraction in neonates within 4 days after perinatal tamoxifen injection. Conduction defect was accompanied by reduction in ventricular expression of the cardiac voltage-gated Na+ channel pore-forming &agr;-subunit (Nav1.5-&agr;), the largest ion channel in the heart responsive for rapid depolarization of the action potential, which leads to increased intracellular Ca2+ for contraction (conduction–contraction coupling). In addition, expression of ryanodine receptor 2, through which Ca2+ is released from sarcoplasmic reticulum, was substantially reduced in Nkx2-5 knockout mice. These results indicate that Nkx2-5 function is critical not only during cardiac development but also in perinatal hearts, by regulating expression of several important gene products involved in conduction and contraction.

Enhanced Rho-Kinase Activity in Circulating Neutrophils of Patients With Vasospastic Angina: A Possible Biomarker for Diagnosis and Disease Activity Assessment

OBJECTIVES The aim of this study was to examine whether Rho-kinase activity is systemically enhanced in patients with vasospastic angina (VSA) and, if so, whether a noninvasive diagnostic method could be developed to improve practice. BACKGROUND The activated Rho-kinase pathway plays a central role in the molecular mechanism of coronary vasospasm in animal models and patients with VSA. Recently, it has been reported that Rho-kinase activity in circulating leukocytes is associated with various diseases. METHODS Fifty-three consecutive patients with chest pain who underwent acetylcholine provocation testing for coronary spasm were examined. Patients were divided into 2 groups depending on their response to the test: VSA (n = 33) and non-VSA (n = 20) groups. Venous blood samples were collected to measure Rho-kinase activity in circulating neutrophils, determined by the extent of phosphorylation of myosin-binding subunit (MBS), a substrate of Rho-kinase. RESULTS Rho-kinase activity was significantly higher in the VSA group than in the non-VSA group (phosphorylated MBS/total MBS ratio 1.33 ± 0.37 vs. 0.95 ± 0.22, p < 0.001). In the VSA group, no correlation was noted between Rho-kinase activity and high-sensitivity C-reactive protein, smoking, or accumulated number of coronary risk factors. After the 3-month medical treatment, Rho-kinase activity in the VSA group was significantly decreased to 1.08 ± 0.31 (p < 0.001). On receiver-operating characteristic curve analysis, a phosphorylated MBS ratio of 1.18 was identified as the best cutoff level to predict the diagnosis of VSA. CONCLUSIONS These results indicate that Rho-kinase activity in circulating neutrophils is enhanced in patients with VSA and may be a useful biomarker for diagnosis and disease activity assessment of the vasospastic disorder.

Slow progressive conduction and contraction defects in loss of Nkx2-5 mice after cardiomyocyte terminal differentiation

Mutations in homeoprotein NKX2-5 are linked to human congenital heart disease, resulting in various cardiac anomalies, as well as in postnatal progressive conduction defects and occasional left ventricular dysfunction; yet the function of Nkx2-5 in the postnatal period is largely unexplored. In the heart, the majority of cardiomyocytes are believed to complete cell-cycle withdrawal shortly after birth, which is generally accompanied by a re-organization of chromatin structure shown in other tissues. We reasoned that the effects of the loss of Nkx2-5 in mice may be different after cell-cycle withdrawal compared with those of the perinatal loss of Nkx2-5, which results in rapid conduction and contraction defects within 4 days after the deletion of Nkx2-5 alleles (Circ Res. 2008;103:580). In this study, floxed-Nkx2-5 alleles were deleted using tamoxifen-inducible Cre transgene (Cre-ER) beginning at 2 weeks of age. The loss of Nkx2-5 beginning at 2 weeks of age resulted in conduction and contraction defects similar to the perinatal loss of Nkx2-5, however, with a substantially slower disease progression shown by 1° atrioventricular block at 6 weeks of age (4 weeks after tamoxifen injections) and heart enlargement after 12 weeks of age (10 weeks after tamoxifen injections). The phenotypes were accompanied by a slower and smaller degree of reduction of several critical Nkx2-5 downstream targets that were observed in mice with a perinatal loss of Nkx2-5. These results suggest that Nkx2-5 is necessary for proper conduction and contraction after 2 weeks of age, but with a substantially distinct level of necessity at 2 weeks of age compared with that in the perinatal period.

Cardiac shock wave therapy ameliorates left ventricular remodeling after myocardial ischemia–reperfusion injury in pigs in vivo

ObjectivesLeft ventricular (LV) remodeling after acute myocardial infarction (AMI) is associated with a poor prognosis and an impaired quality of life. We have shown earlier that low-energy extracorporeal cardiac shock wave (SW) therapy improves chronic myocardial ischemia in pigs and humans and also ameliorates LV remodeling in a pig model of AMI induced by permanent coronary ligation. However, in the current clinical setting, most of the patients with AMI receive reperfusion therapy. Thus, in this study we examined whether our SW therapy also ameliorates LV remodeling after myocardial ischemia–reperfusion (I/R) injury in pigs in vivo. MethodsPigs were subjected to a 90-min ischemia and reperfusion using a balloon catheter and were randomly assigned to two groups with or without SW therapy to the ischemic border zone (0.09 mJ/mm2, 200 pulses/spot, 9 spots/animal, three times in the first week) (n=15 each). ResultsFour weeks after I/R, compared with the control group, the SW group showed significantly ameliorated LV remodeling in terms of LV enlargement (131±9 vs. 100±7 ml), reduced LV ejection fraction (28±2 vs. 36±3%), and elevated left ventricular end-diastolic pressure (11±2 vs. 4±1 mmHg) (all P<0.05, n=8 each). The SW group also showed significantly increased regional myocardial blood flow (−0.06±0.11 vs. 0.36±0.13 ml/min/g, P<0.05), capillary density (1.233±31 vs. 1.560±60/mm2, P<0.001), and endothelial nitric oxide synthase activity (0.24±0.03 vs. 0.41±0.05, P<0.05) in the ischemic border zone compared with the control group (n=7 each). ConclusionThese results indicate that our SW therapy is also effective in ameliorating LV remodeling after myocardial I/R injury in pigs in vivo.

AAV-mediated knockdown of phospholamban leads to improved contractility and calcium handling in cardiomyocytes.

Reduced contractility due to dysregulation of intracellular calcium (Ca2+) is a common pathologic feature of chronic heart failure. Calcium stores in the sarcoplasmic reticulum play a major role in regulating cardiac contractility. Several animal models of heart failure have been treated by altering the regulation of the sarcoplamic reticulum ATPase through ablation or down‐regulation of its inhibitor peptide, phospholamban (PLN).

Depressed contractile reserve and impaired calcium handling of cardiac myocytes from chronically unloaded hearts are ameliorated with the administration of physiological treatment dose of T3 in rats.

Aim:  Chronic cardiac unloading causes a time‐dependent upregulation of phospholamban (PLB) and depression of myocyte contractility in normal rat hearts. As thyroid hormone is known to decrease PLB expression, we examined whether thyroid hormone restores the depressed contractile performance of myocytes from chronically unloaded hearts.

Nonselective endothelin receptor antagonist initiated soon after the onset of myocardial infarction may deteriorate 24-hour survival.

&NA; To investigate the effects of endothelin blockade initiated immediately after the onset of myocardial infarction on survival and left ventricular remodeling, treatment with the nonselective receptor antagonist TAK‐044 (n = 22) or saline (n = 19) for 3 weeks was initiated immediately after coronary ligation in rats. The 24‐h survival rate was significantly lower in the TAK‐044 group than in the saline group. The systolic blood pressure 24 h after the onset of myocardial infarction was similar in the saline and TAK‐044 groups, although it was significantly lower in the TAK‐044 group during the 3‐week protocol. Heart weight/tibial length was significantly increased in the TAK‐044 group compared with the saline group. As all deaths in the TAK‐044 group occurred within 24 h after myocardial infarction, we performed additional experiments using a separate group of rats 12–16 h after myocardial infarction. Plasma and myocardial endothelin‐1 levels were significantly increased, and a bolus injection of TAK‐044 significantly reduced left ventricular dP/dtmax in these rats that had had a myocardial infarction compared with sham‐operated rats. Endothelin receptor blockade initiated immediately after the onset of myocardial infarction may deteriorate acute‐phase survival and left ventricular remodeling. Inhibition of the positive inotropic action of endothlin‐1 may partially explain the increased 24‐h mortality.

Gene expression, cellular localization, and enzymatic activity of diacylglycerol kinase isozymes in rat ovary and placenta

Female reproductive organs show remarkable cyclic changes in morphology and function in response to a combination of hormones. Evidence has accumulated suggesting that phosphoinositide turnover and the consequent diacylglycerol (DG) protein kinase C (PKC) pathway are intimately involved in these mechanisms. The present study has been performed to investigate the gene expression, cellular localization, and enzymatic activity of the DG kinase (DGK) isozymes that control the DG-PKC pathway. Gene expression for DGKα, -ε, -ζ, and -ι was detected in the ovary and placenta. Intense expression signals for DGKζ and -α were observed in the theca cells and moderate signals in the interstitium and corpora lutea of the ovary. On the other hand, signals for DGKε were seen more intensely in granulosa cells. In the placenta, signals for DGKα and -ι were observed in the junctional zone, whereas those for DGKζ were detected in the labyrinthine zone. At higher magnification, the signals for DGKα were mainly discerned in giant cytotrophoblasts, and those for DGKι were found in small cytotrophoblasts of the junctional zone. DGKζ signals were observed in all cellular components of the labyrinthine zone, including mesenchyme, trabecular trophoblasts, and cytotrophoblasts. DGKε signals were detected in the junctional zone on day 13 and 15 of pregnancy and were diffusely distributed both in the labyrinthine and junctional zones at later stages. The present study reveals distinct patterns of mRNA localization for DGK isozymes in the rat ovary and placenta, suggesting that each isozyme plays a unique role in distinct cell types in these organs.