[show abstract][hide abstract] ABSTRACT: BACKGROUND: The efficacy of pharmacological interventions to prevent sudden arrhythmic death in patients with chronic heart failure remains limited. Evidence now suggests increased ventricular expression of hyperpolarization-activated cation (HCN) channels in hypertrophied and failing hearts contributes to their arrythmicity. Still, the role of induced HCN channel expression in the enhanced arrhythmicity associated with heart failure and the capacity of HCN channel blockade to prevent lethal arrhythmias remains undetermined. METHODS AND RESULTS: We examined the effects of ivabradine, a specific HCN channel blocker, on survival and arrhythmicity in transgenic mice (dnNRSF-Tg) expressing a cardiac-specific dominant-negative form of neuron-restrictive silencer factor, a useful mouse model of dilated cardiomyopathy leading to sudden death. Ivabradine (7 mg/kg per day orally) significantly reduced ventricular tachyarrhythmias and improved survival among dnNRSF-Tg mice while having no significant effect on heart rate or cardiac structure or function. Ivabradine most likely prevented the increase in automaticity otherwise seen in dnNRSF-Tg ventricular myocytes. Moreover, cardiac-specific overexpression of HCN2 in mice (HCN2-Tg) made hearts highly susceptible to arrhythmias induced by chronic β-adrenergic stimulation. Indeed, ventricular myocytes isolated from HCN2-Tg mice were highly susceptible to β-adrenergic stimulation-induced abnormal automaticity, which was inhibited by ivabradine. CONCLUSIONS: HCN channel blockade by ivabradine reduces lethal arrhythmias associated with dilated cardiomyopathy in mice. Conversely, cardiac-specific overexpression of HCN2 channels increases arrhythmogenicity of β-adrenergic stimulation. Our findings demonstrate the contribution of HCN channels to the increased arrhythmicity seen in failing hearts and suggest HCN channel blockade is a potentially useful approach to preventing sudden death in patients with heart failure.
Journal of the American Heart Association. 01/2013; 2(3):e000150.
[show abstract][hide abstract] ABSTRACT: The bioactive lysophospholipid mediator sphingosine-1-phosphate (S1P) promotes the egress of newly formed T cells from the thymus and the release of immature B cells from the bone marrow. It has remained unclear, however, where and how S1P is released. Here, we show that in mice, the S1P transporter spinster homolog 2 (Spns2) is responsible for the egress of mature T cells and immature B cells from the thymus and bone marrow, respectively. Global Spns2-KO mice exhibited marked accumulation of mature T cells in thymi and decreased numbers of peripheral T cells in blood and secondary lymphoid organs. Mature recirculating B cells were reduced in frequency in the bone marrow as well as in blood and secondary lymphoid organs. Bone marrow reconstitution studies revealed that Spns2 was not involved in S1P release from blood cells and suggested a role for Spns2 in other cells. Consistent with these data, endothelia-specific deletion of Spns2 resulted in defects of lymphocyte egress similar to those observed in the global Spns2-KO mice. These data suggest that Spns2 functions in ECs to establish the S1P gradient required for T and B cells to egress from their respective primary lymphoid organs. Furthermore, Spns2 could be a therapeutic target for a broad array of inflammatory and autoimmune diseases.
The Journal of clinical investigation 03/2012; 122(4):1416-26. · 15.39 Impact Factor
[show abstract][hide abstract] ABSTRACT: Podocytes play an important role in maintaining normal glomerular function. A podocyte-specific conditional knockout technology has been established by the use of transgenic mice expressing a podocyte-specific Cre recombinase to clarify the role of genes expressed in the podocytes. However, it may be difficult to examine the role of genes in certain pathologic conditions using conventional podocyte-specific knockout mice because they may be embryonically lethal or exhibit congenital renal abnormality.
To introduce a temporal control in the genetic experiments targeting the podocyte, we constructed tamoxifen-inducible Cre recombinase (CreER(T2)) transgenic mice under the control of podocyte-specific promoter, 2.5-kb fragment of the human podocin (NPHS2) gene. The specificity and efficiency of Cre activity were examined by crossing NPHS2-CreER(T2) with the ROSA26 reporter (R26R) mouse in which a floxed-stop cassette has been placed upstream of the beta-galactosidase gene. Four-week-old double-mutant mice (NPHS2-CreER(T2)/R26R) were intraperitoneally administered with 0.5 mg of 4-hydroxytamoxifen (4-OHT) for three consecutive days.
NPHS2-CreER(T2)/R26R treated with 4-OHT expressed beta-galactosidase specifically in 85% of the podocytes in glomeruli. Expression of Cre recombinase mRNA was mostly restricted to the kidney, especially in glomeruli.
In conclusion, we have successfully generated podocyte-specific inducible Cre transgenic mice by tamoxifen administration. These mice allow us to disrupt the genes specifically in the podocytes after birth.
[show abstract][hide abstract] ABSTRACT: It is known that the transcriptional coactivator p300 is crucially involved in the differentiation and growth of cardiac myocytes during development. However, the physiological function of p300 in the postnatal hearts remains to be characterized.
We have now investigated the physiological function of p300 in adult hearts.
We analyzed transgenic mice exhibiting cardiac-specific overexpression of a dominant-negative p300 mutant lacking the C/H3 domain (p300DeltaC/H3 transgenic [TG] mice). p300DeltaC/H3 significantly inhibited p300-induced activation of GATA- and myocyte enhancer factor 2-dependent promoters in cultured ventricular myocytes, and p300DeltaC/H3-TG mice showed cardiac dysfunction that was lethal by 20 weeks of age. The numbers of mitochondria in p300DeltaC/H3-TG myocytes were markedly increased, but the mitochondria were diminished in size. Moreover, cardiac mitochondrial gene expression, mitochondrial membrane potential and ATP contents were all significantly disrupted in p300DeltaC/H3-TG hearts, suggesting that mitochondrial dysfunction contributes to the progression of the observed cardiomyopathy. Transcription of peroxisome proliferator-activated receptor gamma coactivator (PGC)-1alpha, a master regulator of mitochondrial gene expression, and its target genes was significantly downregulated in p300DeltaC/H3-TG mice, and p300DeltaC/H3 directly repressed myocyte enhancer factor 2C-dependent PGC-1alpha promoter activity and disrupted the transcriptional activity of PGC-1alpha in cultured ventricular myocytes. In addition, myocytes showing features of autophagy were observed in p300DeltaC/H3-TG hearts.
Collectively, our findings suggest that p300 is essential for the maintenance of mitochondrial integrity and for myocyte survival in the postnatal left ventricular myocardium.
Circulation Research 10/2009; 105(8):746-54. · 11.86 Impact Factor
[show abstract][hide abstract] ABSTRACT: Pharmacological interventions for prevention of sudden arrhythmic death in patients with chronic heart failure remain limited. Accumulating evidence suggests increased ventricular expression of T-type Ca(2+) channels contributes to the progression of heart failure. The ability of T-type Ca(2+) channel blockade to prevent lethal arrhythmias associated with heart failure has never been tested, however.
We compared the effects of efonidipine and mibefradil, dual T- and L-type Ca(2+) channel blockers, with those of nitrendipine, a selective L-type Ca(2+) channel blocker, on survival and arrhythmogenicity in a cardiac-specific, dominant-negative form of neuron-restrictive silencer factor transgenic mice (dnNRSF-Tg), which is a useful mouse model of dilated cardiomyopathy leading to sudden death. Efonidipine, but not nitrendipine, substantially improved survival among dnNRSF-Tg mice. Arrhythmogenicity was dramatically reduced in dnNRSF-Tg mice treated with efonidipine or mibefradil. Efonidipine acted by reversing depolarization of the resting membrane potential otherwise seen in ventricular myocytes from dnNRSF-Tg mice and by correcting cardiac autonomic nervous system imbalance. Moreover, the R(-)-isomer of efonidipine, a recently identified, highly selective T-type Ca(2+) channel blocker, similarly improved survival among dnNRSF-Tg mice. Efonidipine also reduced the incidence of sudden death and arrhythmogenicity in mice with acute myocardial infarction.
T-type Ca(2+) channel blockade reduced arrhythmias in a mouse model of dilated cardiomyopathy by repolarizing the resting membrane potential and improving cardiac autonomic nervous system imbalance. T-type Ca(2+) channel blockade also prevented sudden death in mice with myocardial infarction. Our findings suggest T-type Ca(2+) channel blockade is a potentially useful approach to preventing sudden death in patients with heart failure.
[show abstract][hide abstract] ABSTRACT: Hcn4, which encodes the hyperpolarization-activated, cyclic nucleotide-sensitive channel (I(h)), is a well-established marker of the cardiac sino-atrial node. We aimed to identify cis-elements in the genomic locus of the Hcn4 gene that regulate the transcription of Hcn4.
We screened evolutionarily conserved non-coding sequences (CNSs) that are often involved in the regulation of gene expression. The VISTA Enhancer Browser identified 16 regions, termed CNS 1-16, within the Hcn4 locus. Using the luciferase reporter assay in primary neonatal rat cardiomyocytes, we found that CNS13 conferred a prominent enhancer activity (more than 30-fold) on the Hcn4 promoter. Subsequent mutation analysis revealed that the Hcn4 enhancer function was dependent on myocyte enhancer factor-2 (MEF2) and activator protein-1 (AP1) binding sequences located in CNS13. Electrophoretic mobility shift assay and chromatin immunoprecipitation confirmed that MEF2 and AP1 proteins bound CNS13. Furthermore, overexpression of a dominant negative MEF2 mutant inhibited the enhancer activity of CNS13, decreased Hcn4 mRNA expression and also decreased the amplitude of I(h) current in myocytes isolated from the inflow tract of embryonic heart.
These results suggest that the novel enhancer CNS13 and MEF2 may play a critical role in the transcription of Hcn4 in the heart.
Cardiovascular research 07/2009; 83(4):682-7. · 5.80 Impact Factor
[show abstract][hide abstract] ABSTRACT: Ventricular myocytes are known to show increased expression of the cardiac hormones atrial and brain natriuretic peptide (ANP and BNP, respectively) in response to pathological stress on the heart, but their function during the progression of nonischemic dilated cardiomyopathy remains unclear. In this study, we crossed a mouse model of dilated cardiomyopathy and sudden death, which we generated by cardioselectively overexpressing a dominant-negative form of the transcriptional repressor neuron-restrictive silencer factor (dnNRSF Tg mice), with mice lacking guanylyl cyclase-A (GC-A), a common receptor for ANP and BNP, to assess the effects of endogenously expressed natriuretic peptides during progression of the cardiomyopathy seen in dnNRSF Tg mice. We found that dnNRSF Tg;GC-A(-/-) mice were born normally, but then most died within 4 wk. The survival rates among dnNRSF Tg;GC-A(+/-) and dnNRSF Tg mice were comparable, but dnNRSF Tg;GC-A(+/-) mice showed greater systolic dysfunction and a more severe cardiomyopathic phenotype than dnNRSF Tg mice. Collectively, our findings suggest that endogenous ANP/BNP protects the heart against the death and progression of pathological remodeling in a mouse model of dilated cardiomyopathy and sudden death.
[show abstract][hide abstract] ABSTRACT: Mice lacking guanylyl cyclase-A (GC-A), a natriuretic peptide receptor, have pressure-independent cardiac hypertrophy. However, the mechanism underlying GC-A-mediated inhibition of cardiac hypertrophy remains to be elucidated. In the present report, we examined the role of regulator of G-protein signaling subtype 4 (RGS4), a GTPase activating protein for G(q) and G(i), in the antihypertrophic effects of GC-A.
In cultured cardiac myocytes, treatment of atrial natriuretic peptide stimulated the binding of guanosine 3',5'-cyclic monophosphate-dependent protein kinase (PKG) I-alpha to RGS4, PKG-dependent phosphorylation of RGS4, and association of RGS4 and Galpha(q). In contrast, blockade of GC-A by an antagonist, HS-142-1, attenuated the phosphorylation of RGS4 and association of RGS4 and Galpha(q). Moreover, overexpressing a dominant negative form of RGS4 diminished the inhibitory effects of atrial natriuretic peptide on endothelin-1-stimulated inositol 1,4,5-triphosphate production, [(3)H]leucine incorporation, and atrial natriuretic peptide gene expression. Furthermore, expression and phosphorylation of RGS4 were significantly reduced in the hearts of GC-A knockout (GC-A-KO) mice compared with wild-type mice. For further investigation, we constructed cardiomyocyte-specific RGS4 transgenic mice and crossbred them with GC-A-KO mice. The cardiac RGS4 overexpression in GC-A-KO mice significantly reduced the ratio of heart to body weight (P<0.001), cardiomyocyte size (P<0.01), and ventricular calcineurin activity (P<0.05) to 80%, 76%, and 67% of nontransgenic GC-A-KO mice, respectively. It also significantly suppressed the augmented cardiac expression of hypertrophy-related genes in GC-A-KO mice.
These results provide evidence that GC-A activates cardiac RGS4, which attenuates Galpha(q) and its downstream hypertrophic signaling, and that RGS4 plays important roles in GC-A-mediated inhibition of cardiac hypertrophy.
[show abstract][hide abstract] ABSTRACT: Grb2-associated binder (Gab) family of scaffolding adaptor proteins coordinate signaling cascades downstream of growth factor and cytokine receptors. In the heart, among EGF family members, neuregulin-1beta (NRG-1beta, a paracrine factor produced from endothelium) induced remarkable tyrosine phosphorylation of Gab1 and Gab2 via erythroblastic leukemia viral oncogene (ErbB) receptors. We examined the role of Gab family proteins in NRG-1beta/ErbB-mediated signal in the heart by creating cardiomyocyte-specific Gab1/Gab2 double knockout mice (DKO mice). Although DKO mice were viable, they exhibited marked ventricular dilatation and reduced contractility with aging. DKO mice showed high mortality after birth because of heart failure. In addition, we noticed remarkable endocardial fibroelastosis and increase of abnormally dilated vessels in the ventricles of DKO mice. NRG-1beta induced activation of both ERK and AKT in the hearts of control mice but not in those of DKO mice. Using DNA microarray analysis, we found that stimulation with NRG-1beta upregulated expression of an endothelium-stabilizing factor, angiopoietin 1, in the hearts of control mice but not in those of DKO mice, which accounted for the pathological abnormalities in the DKO hearts. Taken together, our observations indicated that in the NRG-1beta/ErbB signaling, Gab1 and Gab2 of the myocardium are essential for both maintenance of myocardial function and stabilization of cardiac capillary and endocardial endothelium in the postnatal heart.
Journal of Clinical Investigation 08/2007; 117(7):1771-81. · 12.81 Impact Factor
[show abstract][hide abstract] ABSTRACT: Cardiogenesis can be induced in vitro in ES cells, though it is difficult to distinguish cardiac-specific genes, since embryoid bodies simultaneously differentiate into multiple lineages. In the present study, transient serum removal during culture greatly enhanced cardiogenesis, and reduced generation of endothelial and hematopoietic cells. Using DNA microarray analysis of 24 differentiated sample cultures including cardiogenesis-enhanced cells, we successfully selected genes up-regulated in embryoid bodies that had undergone cardiogenic differentiation. Besides contractile protein genes, cardiac transcriptional regulatory genes, such as Nkx2-5, Gata4/5, Mef2c, and Myocd, were primary constituents of the first 100 genes chosen as cardiogenesis-associated genes. Further, whole mount in situ hybridization analysis of 13 genes containing non-characterized ones confirmed that most of them were specifically expressed in the heart region of mouse embryos from E9.5-10.5. Based on our results, we consider that the present profiling method may be useful to identify novel genes important for cardiac development.
Biochemical and Biophysical Research Communications 04/2007; 355(1):47-53. · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: Vascular endothelial cadherin (VE-cadherin) is expressed on vascular endothelial cells, which are involved in developmental vessel formation. However, it remains elusive how VE-cadherin-expressing cells function in postnatal neovascularization. To trace VE-cadherin-expressing cells, we developed mice expressing either green fluorescent protein or LacZ driven by VE-cadherin promoter using Cre-loxP system. Although VE-cadherin promoter is less active after birth than during embryogenesis in blood vessels, it is reactivated on cardiac ischemia. Both types of reporter-positive cells are found in the vasculature and in the infarcted myocardium. Those found in the vasculature were pre-existing endothelial cells and incorporated endothelial progenitor cells derived from extracardiac tissue. In addition to the vasculature, VE-cadherin promoter-activated cells were positive for CD45 in the bone marrow cells of the infarcted mice. VE-cadherin promoter-reactivated CD45-positive leukocytes were also found in the infarcted area. In addition, VE-cadherin promoter was activated in the bone marrow vessels of the infarcted mice. Collectively, our findings reveal a new ischemia-induced neovascularization mechanism involving VE-cadherin; the re-expressed VE-cadherin-mediated cell adhesion between cells may be involved not only in homing of bone marrow-derived cells to ischemic area but also mobilization from bone marrow.
Circulation Research 05/2006; 98(7):897-904. · 11.86 Impact Factor
[show abstract][hide abstract] ABSTRACT: Ghrelin, a 28-amino acid acylated peptide, displays strong GH-releasing activity in concert with GHRH. The fatty acid modification of ghrelin is essential for the actions, and des-acyl ghrelin, which lacks the modification, has been assumed to be devoid of biological effects. Some recent reports, however, indicate that des-acyl ghrelin has effects on cell proliferation and survival. In the present study, we generated two lines of transgenic mice bearing the preproghrelin gene under the control of chicken beta-actin promoter. Transgenic mice overexpressed des-acyl ghrelin in a wide variety of tissues, and plasma des-acyl ghrelin levels reached 10- and 44-fold of those in control mice. They exhibited lower body weights and shorter nose-to-anus lengths, compared with control mice. The serum GH levels tended to be lower, and the serum IGF-I levels were significantly lower in both male and female transgenic mice than control mice. The responses of GH to administered GHRH were normal, whereas those to administered ghrelin were reduced, especially in female transgenic mice, compared with control mice. These data suggest that overexpressed des-acyl ghrelin may modulate the GH-IGF-I axis and result in small phenotype in transgenic mice.
[show abstract][hide abstract] ABSTRACT: Reactivation of the fetal cardiac gene program is a characteristic feature of hypertrophied and failing hearts that correlates with impaired cardiac function and poor prognosis. However, the mechanism governing the reversible expression of fetal cardiac genes remains unresolved. Here we show that neuron-restrictive silencer factor (NRSF), a transcriptional repressor, selectively regulates expression of multiple fetal cardiac genes, including those for atrial natriuretic peptide, brain natriuretic peptide and alpha-skeletal actin, and plays a role in molecular pathways leading to the re-expression of those genes in ventricular myocytes. Moreover, transgenic mice expressing a dominant-negative mutant of NRSF in their hearts exhibit dilated cardiomyopathy, high susceptibility to arrhythmias and sudden death. We demonstrate that genes encoding two ion channels that carry the fetal cardiac currents I(f) and I(Ca,T), which are induced in these mice and are potentially responsible for both the cardiac dysfunction and the arrhythmogenesis, are regulated by NRSF. Our results indicate NRSF to be a key transcriptional regulator of the fetal cardiac gene program and suggest an important role for NRSF in maintaining normal cardiac structure and function.
The EMBO Journal 01/2004; 22(23):6310-21. · 9.82 Impact Factor