Toru Oka

Genetic Manipulation of Periostin Expression Reveals a Role in Cardiac Hypertrophy and Ventricular Remodeling

The cardiac extracellular matrix is a dynamic structural support network that is both influenced by, and a regulator of, pathological remodeling and hypertrophic growth. In response to pathologic insults, the adult heart reexpresses the secreted extracellular matrix protein periostin (Pn). Here we show that Pn is critically involved in regulating the cardiac hypertrophic response, interstitial fibrosis, and ventricular remodeling following long-term pressure overload stimulation and myocardial infarction. Mice lacking the gene encoding Pn (Postn) were more prone to ventricular rupture in the first 10 days after a myocardial infarction, but surviving mice showed less fibrosis and better ventricular performance. Pn−/− mice also showed less fibrosis and hypertrophy following long-term pressure overload, suggesting an intimate relationship between Pn and the regulation of cardiac remodeling. In contrast, inducible overexpression of Pn in the heart protected mice from rupture following myocardial infarction and induced spontaneous hypertrophy with aging. With respect to a mechanism underlying these alterations, Pn−/− hearts showed an altered molecular program in fibroblast function. Indeed, fibroblasts isolated from Pn−/− hearts were less effective in adherence to cardiac myocytes and were characterized by a dramatic alteration in global gene expression (7% of all genes). These are the first genetic data detailing the function of Pn in the adult heart as a regulator of cardiac remodeling and hypertrophy.

Cardiac-Specific Deletion of Gata4 Reveals Its Requirement for Hypertrophy, Compensation, and Myocyte Viability

The transcription factor GATA4 is a critical regulator of cardiac gene expression where it controls embryonic development, cardiomyocyte differentiation, and stress responsiveness of the adult heart. Traditional deletion of Gata4 caused embryonic lethality associated with endoderm defects and cardiac malformations, precluding an analysis of the role of GATA4 in the adult myocardium. To address the function of GATA4 in the adult heart, Gata4-loxP–targeted mice (Gata4fl/fl) were crossed with mice containing a β-myosin heavy chain (β-MHC) or α-MHC promoter-driven Cre transgene, which produced viable mice that survived into adulthood despite a 95% and 70% loss of GATA4 protein, respectively. However, cardiac-specific deletion of Gata4 resulted in a progressive and dosage-dependent deterioration in cardiac function and dilation in adulthood. Moreover, pressure overload stimulation induced rapid decompensation and heart failure in cardiac-specific Gata4-deleted mice. More provocatively, Gata4-deleted mice were compromised in their ability to hypertrophy following pressure overload or exercise stimulation. Mechanistically, cardiac-specific deletion of Gata4 increased cardiomyocyte TUNEL at baseline in embryos and adults as they aged, as well as dramatically increased TUNEL following pressure overload stimulation. Examination of gene expression profiles in the heart revealed a number of profound alterations in known GATA4-regulated structural genes as well as genes with apoptotic implications. Thus, GATA4 is a necessary regulator of cardiac gene expression, hypertrophy, stress-compensation, and myocyte viability.

Bone Morphogenetic Proteins Induce Cardiomyocyte Differentiation through the Mitogen-Activated Protein Kinase Kinase Kinase TAK1 and Cardiac Transcription Factors Csx/Nkx-2.5 and GATA-4

ABSTRACT Bone morphogenetic proteins (BMPs) have been shown to induce ectopic expression of cardiac transcription factors and beating cardiomyocytes in nonprecardiac mesodermal cells in chicks, suggesting that BMPs are inductive signaling molecules that participate in the development of the heart. However, the precise molecular mechanisms by which BMPs regulate cardiac development are largely unknown. In the present study, we examined the molecular mechanisms by which BMPs induce cardiac differentiation by using the P19CL6 in vitro cardiomyocyte differentiation system, a clonal derivative of P19 embryonic teratocarcinoma cells. We established a permanent P19CL6 cell line, P19CL6noggin, which constitutively overexpresses the BMP antagonist noggin. Although almost all parental P19CL6 cells differentiate into beating cardiomyocytes when treated with 1% dimethyl sulfoxide, P19CL6noggin cells did not differentiate into beating cardiomyocytes nor did they express cardiac transcription factors or contractile protein genes. The failure of differentiation was rescued by overexpression of BMP-2 or addition of BMP protein to the culture media, indicating that BMPs were indispensable for cardiomyocyte differentiation in this system. Overexpression of TAK1, a member of the mitogen-activated protein kinase kinase kinase superfamily which transduces BMP signaling, restored the ability of P19CL6noggin cells to differentiate into cardiomyocytes and concomitantly express cardiac genes, whereas overexpression of the dominant negative form of TAK1 in parental P19CL6 cells inhibited cardiomyocyte differentiation. Overexpression of both cardiac transcription factors Csx/Nkx-2.5 and GATA-4 but not of Csx/Nkx-2.5 or GATA-4 alone also induced differentiation of P19CL6noggin cells into cardiomyocytes. These results suggest that TAK1, Csx/Nkx-2.5, and GATA-4 play a pivotal role in the cardiogenic BMP signaling pathway.

Targeted Disruption of Na+/Ca2+ Exchanger Gene Leads to Cardiomyocyte Apoptosis and Defects in Heartbeat

Ca2+, which enters cardiac myocytes through voltage-dependent Ca2+channels during excitation, is extruded from myocytes primarily by the Na+/Ca2+ exchanger (NCX1) during relaxation. The increase in intracellular Ca2+ concentration in myocytes by digitalis treatment and after ischemia/reperfusion is also thought to result from the reverse mode of the Na+/Ca2+ exchange mechanism. However, the precise roles of the NCX1 are still unclear because of the lack of its specific inhibitors. We generated Ncx1-deficient mice by gene targeting to determine the in vivo function of the exchanger. Homozygous Ncx1-deficient mice died between embryonic days 9 and 10. Their hearts did not beat, and cardiac myocytes showed apoptosis. No forward mode or reverse mode of the Na+/Ca2+ exchange activity was detected in null mutant hearts. The Na+-dependent Ca2+ exchange activity as well as protein content of NCX1 were decreased by ∼50% in the heart, kidney, aorta, and smooth muscle cells of the heterozygous mice, and tension development of the aortic ring in Na+-free solution was markedly impaired in heterozygous mice. These findings suggest that NCX1 is required for heartbeats and survival of cardiac myocytes in embryos and plays critical roles in Na+-dependent Ca2+ handling in the heart and aorta.

Peroxisome Proliferator–Activated Receptor Activators Inhibit Lipopolysaccharide-Induced Tumor Necrosis Factor-α Expression in Neonatal Rat Cardiac Myocytes

Abstract —Peroxisome proliferator–activated receptors (PPARs) are transcription factors belonging to the nuclear receptor superfamily. Recently, PPAR activators have been shown to inhibit the production of proinflammatory cytokines in macrophages or vascular smooth muscle cells. It has been reported that tumor necrosis factor-α (TNF-α) expression is elevated in the failing heart and that TNF-α has a negative inotropic effect on cardiac myocytes. Therefore, we examined the effects of PPARα and PPARγ activators on expression of TNF-α in neonatal rat cardiac myocytes. Northern blot analysis revealed expression of PPARα and PPARγ mRNA in cardiac myocytes. Immunofluorescent staining demonstrated that both PPARα and PPARγ were expressed in the nuclei of cells. When cardiac myocytes were transfected with PPAR responsive element (PPRE)-luciferase reporter plasmid, both PPARα and PPARγ activators increased the promoter activity. Cardiomyocytes were stimulated with lipopolysaccharide (LPS), and the levels of TNF-α in the medium were measured by ELISA. After exposure to LPS, the levels of TNF-α significantly increased. However, pretreatment of myocytes with PPARα or PPARγ activators decreased LPS-induced expression of TNF-α in the medium. Both PPARα and PPARγ activators also inhibited LPS-induced increase in TNF-α mRNA in myocytes. In addition, electrophoretic mobility shift assays demonstrated that PPAR activators reduced LPS-induced nuclear factor-κB activation. These results suggest that both PPARα and PPARγ activators inhibit cardiac expression of TNF-α in part by antagonizing nuclear factor-κB activity and that treatment with PPAR activators may lead to improvement in congestive heart failure.

Cardiomyocyte GATA4 functions as a stress-responsive regulator of angiogenesis in the murine heart

The transcription factor GATA4 is a critical regulator of cardiac gene expression, modulating cardiomyocyte differentiation and adaptive responses of the adult heart. We report what we believe to be a novel function for GATA4 in murine cardiomyocytes as a nodal regulator of cardiac angiogenesis. Conditional overexpression of GATA4 within adult cardiomyocytes increased myocardial capillary and small conducting vessel densities and increased coronary flow reserve and perfusion-dependent cardiac contractility. Coculture of HUVECs with either GATA4-expressing cardiomyocytes or with myocytes expressing a dominant-negative form of GATA4 enhanced or reduced HUVEC tube formation, respectively. Expression of GATA4 in skeletal muscle by adenoviral gene transfer enhanced capillary densities and hindlimb perfusion following femoral artery ablation. Deletion of Gata4 specifically from cardiomyocytes reduced myocardial capillary density and prevented pressure overload-augmented angiogenesis in vivo. GATA4 induced the angiogenic factor VEGF-A, directly binding the Vegf-A promoter and enhancing transcription. GATA4-overexpressing mice showed increased levels of cardiac VEGF-A, while Gata4-deleted mice demonstrated decreased VEGF-A levels. The induction of HUVEC tube formation in GATA4-overexpressing cocultured myocytes was blocked with a VEGF receptor antagonist. Pressure overload-induced dysfunction in Gata4-deleted hearts was partially rescued by adenoviral gene delivery of VEGF and angiopoietin-1. To our knowledge, these results demonstrate [corrected] a previously unrecognized function for GATA4 as a regulator of cardiac angiogenesis through a nonhypoxic, load, and/or disease-responsive mechanism.

THE RHO FAMILY G PROTEINS PLAY A CRITICAL ROLE IN MUSCLE DIFFERENTIATION

ABSTRACT The Rho family GTP-binding proteins play a critical role in a variety of cytoskeleton-dependent cell functions. In this study, we examined the role of Rho family G proteins in muscle differentiation. Dominant negative forms of Rho family proteins and RhoGDI, a GDP dissociation inhibitor, suppressed transcription of muscle-specific genes, while mutationally activated forms of Rho family proteins strongly activated their transcription. C2C12 cells overexpressing RhoGDI (C2C12RhoGDI cells) did not differentiate into myotubes, and expression levels of myogenin, MRF4, and contractile protein genes but not MyoD and myf5 genes were markedly reduced in C2C12RhoGDI cells. The promoter activity of the myogenin gene was suppressed by dominant negative mutants of Rho family proteins and was reduced in C2C12RhoGDI cells. Expression of myocyte enhancer binding factor 2 (MEF2), which has been reported to be required for the expression of the myogenin gene, was reduced at the mRNA and protein levels in C2C12RhoGDI cells. These results suggest that the Rho family proteins play a critical role in muscle differentiation, possibly by regulating the expression of the myogenin and MEF2 genes.

Complement C1q Activates Canonical Wnt Signaling and Promotes Aging-Related Phenotypes

Wnt signaling plays critical roles in development of various organs and pathogenesis of many diseases, and augmented Wnt signaling has recently been implicated in mammalian aging and aging-related phenotypes. We here report that complement C1q activates canonical Wnt signaling and promotes aging-associated decline in tissue regeneration. Serum C1q concentration is increased with aging, and Wnt signaling activity is augmented during aging in the serum and in multiple tissues of wild-type mice, but not in those of C1qa-deficient mice. C1q activates canonical Wnt signaling by binding to Frizzled receptors and subsequently inducing C1s-dependent cleavage of the ectodomain of Wnt coreceptor low-density lipoprotein receptor-related protein 6. Skeletal muscle regeneration in young mice is inhibited by exogenous C1q treatment, whereas aging-associated impairment of muscle regeneration is restored by C1s inhibition or C1qa gene disruption. Our findings therefore suggest the unexpected role of complement C1q in Wnt signal transduction and modulation of mammalian aging.

Calcineurin Plays a Critical Role in Pressure Overload–Induced Cardiac Hypertrophy

BACKGROUND Cardiac hypertrophy is a fundamental adaptive response to hemodynamic overload; how mechanical load induces cardiac hypertrophy, however, remains elusive. It was recently reported that activation of a calcium-dependent phosphatase, calcineurin, induces cardiac hypertrophy. In the present study, we examined whether calcineurin plays a critical role in pressure overload-induced cardiac hypertrophy. METHODS AND RESULTS Pressure overload produced by constriction of the abdominal aorta increased the activity of calcineurin in the rat heart and induced cardiac hypertrophy, including reprogramming of gene expression. Treatment of rats with a calcineurin inhibitor, FK506, inhibited the activation of calcineurin and prevented the pressure overload-induced cardiac hypertrophy and fibrosis without change of hemodynamic parameters. Load-induced expression of immediate-early-response genes and fetal genes was also suppressed by the FK506 treatment. CONCLUSIONS The present results suggest that the calcineurin signaling pathway plays a pivotal role in load-induced cardiac hypertrophy and may pave the way for a novel pharmacological approach to prevent cardiac hypertrophy.

Angiogenesis and Cardiac Hypertrophy Maintenance of Cardiac Function and Causative Roles in Heart Failure

Cardiac hypertrophy is an adaptive response to physiological and pathological overload. In response to the overload, individual cardiac myocytes become mechanically stretched and activate intracellular hypertrophic signaling pathways to re-use embryonic transcription factors and to increase the synthesis of various proteins, such as structural and contractile proteins. These hypertrophic responses increase oxygen demand and promote myocardial angiogenesis to dissolve the hypoxic situation and to maintain cardiac contractile function; thus, these responses suggest crosstalk between cardiac myocytes and microvasculature. However, sustained pathological overload induces maladaptation and cardiac remodeling, resulting in heart failure. In recent years, specific understanding has increased with regard to the molecular processes and cell–cell interactions that coordinate myocardial growth and angiogenesis. In this review, we summarize recent advances in understanding the regulatory mechanisms of coordinated myocardial growth and angiogenesis in the pathophysiology of cardiac hypertrophy and heart failure.