ß1 integrin gene excision in the adult murine cardiac myocyte causes defective mechanical and signaling responses.

How mechanical signals are transmitted in the cardiac myocyte is poorly understood. In this study, we produced a tamoxifen-inducible mouse model in which ß1 integrin could be reduced specifically in the adult cardiomyocyte, so that the function of this integrin could be assessed in the postnatal and mechanically stressed heart. The expression of ß1 integrin was reduced to 35% of control levels, but function remained normal at baseline. With aortic constriction, the knockout mice survived but had a blunted hypertrophic response. Integrin knockout myocytes, in contrast to controls, showed reduced integrin-linked kinase expression both at baseline and after hemodynamic stress; focal adhesion kinase expression was reduced after stress. Alterations in multiple signaling pathways were detected in the integrin knockout group after acute and chronic hemodynamic stress. Most remarkably, when we challenged the knockout mice with short-term loading, the robust responses of several kinases (extracellular signal-regulated kinase 1/2, p38, and Akt) evident in control mice were essentially abolished in the knockout mice. We also found that reduction of myocyte ß1 integrin expression modified adrenergic-mediated signaling through extracellular signal-regulated kinase, p38, and Akt. Reduction of ß1 integrin expression in the mature cardiac myocyte leads to a varied response compared with when this protein is reduced during either the embryonic or perinatal period. These results show that ß1 integrin expression is required for proper mechanotransductive and adrenergic responses of the adult heart.

Trends in incidence and mortality rates of ventricular septal rupture during acute myocardial infarction.

Since the introduction of reperfusion in the treatment of acute myocardial infarction (AMI), rates of ventricular septal rupture (VSR) and associated mortality have decreased, but it is not known if incidence and mortality have continued to decrease. We describe trends in incidence and mortality rates of patients with postinfarction VSR during the previous 2 decades and identify risk factors that predict the development and mortality of this rare but catastrophic complication. We analyzed occurrence and mortality rates in patients with first AMI with (n = 408) and without VSR (n = 148,473) who were hospitalized from 1990 to 2007 using the New Jersey Myocardial Infarction Data Acquisition System (MIDAS) database. The annual rate of VSR in AMI was 0.25% to 0.31%. Compared to patients with AMI without VSR, patients with VSR were older, more likely to be women, had increased rate of chronic renal disease, congestive heart failure, and cardiogenic shock, and were less likely to be hypertensive or diabetic (all p values < 0.0001). During the 18-year study period, we found no change in hospital and 1-year mortalities, which were 41% and 60% in 1990 to 1992 and 44% and 56% in 2005 to 2007, respectively. The survival benefit associated with VSR surgical repair was seen only in hospital (hazard ratio 0.66, 95% confidence interval 0.45 to 0.95) but not at 30 days or 1 year. In conclusion, despite improvement in medical treatment and revascularization techniques, the rate of VSR complicating AMI has not changed during the previous 2 decades, and the mortality associated with VSR has remained high and relatively constant.

T0901317, an LXR agonist, augments PKA-induced vascular cell calcification.

We examined the effect of liver X receptor (LXR) agonists on vascular calcification, prevalent in atherosclerotic lesions. T0901317, an LXR agonist, augmented protein kinase A (PKA)-induced mineralization and alkaline phosphatase (ALP) activity in aortic smooth muscle cells isolated from wild-type, but not from Lxrbeta(-/-)mice. A six-hour T0901317 treatment augmented the PKA-induced expression of the phosphate transporter Pit-1, a positive regulator of mineralization, suggesting a direct role. A ten-day T0901317 treatment attenuated PKA-induced expression of mineralization inhibitors, osteopontin and ectonucleotide pyrophosphatase/phosphodiesterase-1, suggesting an indirect role. The effects of T0901317 were attenuated by inhibition of ALP, Pit-1 and Rho-associated kinase, but not by inhibition of PKA. These results suggest that T0901317-augmented mineralization occurs downstream of PKA, involving both direct and indirect LXR-mediated pathways.

Phosphate and pyrophosphate mediate PKA-induced vascular cell calcification.

Vascular calcification is associated with increased cardiovascular risk and occurs by osteochondrogenic differentiation of vascular cells. Many of the same regulatory factors that control skeletal mineralization, including the complex metabolic pathway controlling levels of the activator, inorganic phosphate, and the potent inhibitor, pyrophosphate, also govern vascular calcification. We previously found that the cAMP/PKA signaling pathway mediates in vitro vascular cell calcification induced by inflammatory factors including tumor necrosis factor-alpha 1 and oxidized phospholipids. In this report, we tested whether this signaling pathway modulates phosphate and pyrophosphate metabolism. Treatment of primary murine aortic cells with the PKA activator, forskolin, significantly induced osteoblastic differentiation markers, including alkaline phosphatase (ALP), osteopontin, and osteocalcin as well as the pyrophosphate generator, ectonucleotide-pyrophosphatase/phosphodiesterase-1 (Enpp1) and the pyrophosphate transporter, ankylosis protein, but not the sodium/phosphate cotransporter, Pit-1. In the presence of a substrate for ALP, beta-glycerophosphate, which generates inorganic phosphate, forskolin also enhanced matrix mineralization. Inhibitors of ALP or Pit-1 abrogated forskolin-induced osteopontin expression and mineralization but not forskolin-induced osteocalcin or ALP. These results suggest that phosphate is necessary for PKA-induced calcification of vascular cells and that the extent of PKA-induced calcification is controlled by feedback induction of the inhibitor, pyrophosphate.

Hyperlipidemia impairs osteoanabolic effects of PTH.

Epidemiological and in vitro studies have suggested that hyperlipidemia/oxidized phospholipids adversely affect bone. We recently found that oxidized phospholipids attenuate PTH-induced cAMP and immediate-early gene (IEG) expression in MC3T3-E1 cells, raising concerns that clinical hyperlipidemia may attenuate osteoanabolic effects of PTH in vivo. Thus, we studied whether intermittent PTH treatment has differential osteoanabolic effects in wildtype (C57BL/6) and hyperlipidemic (LDLR(-/-)) mice. Consistent with our previous in vitro studies, induction of IEGs in calvarial tissue, 45 min after a single dose of recombinant hPTH(1-34), was attenuated in LDLR(-/-) mice compared with C57BL/6 mice. Daily hPTH(1-34) injections for 5 wk significantly increased total and cortical BMD and BMC, assessed by pQCT, in C57BL/6 mice. However, this induction was completely abrogated in LDLR(-/-) mice. Similarly, PTH(1-34) failed to increase BMD in another hyperlipidemic mouse model, ApoE(-/-) mice. Histomorphometric analysis showed that trabecular bone of both mice responded similarly to PTH(1-34). Structural parameters improved significantly in response to PTH(1-34) in both mouse strains, although to a lesser degree in LDLR(-/-) mice. With PTH(1-34) treatment, osteoblast surface trended toward an increase in C57BL/6 mice and increased significantly in LDLR(-/-) mice. PTH(1-34) did not alter resorption parameters significantly, except for the eroded surface (ES/BS), which was reduced in the C57BL/6 but not in the LDLR(-/-) mice. These results show that PTH(1-34) has adverse effects on cortical bones of the hyperlipidemic mice, suggesting that the therapeutic effects of PTH may be compromised in the presence of hyperlipidemia.

Combined deficiency of dystrophin and beta1 integrin in the cardiac myocyte causes myocardial dysfunction, fibrosis and calcification.

The dystrophin-glycoprotein complex is a large complex of membrane-associated proteins linking the cytoskeleton to the extracellular matrix in muscle. Transmembrane heterodimeric (alphabeta) integrins serve also as cellular adhesion molecules and mechanotransducers. In the animal model for Duchenne muscular dystrophy, the mdx mouse, loss of dystrophin causes more severe abnormalities in skeletal than in cardiac muscle. We hypothesized that ablation of cardiac myocyte integrins in the mdx background would lead to a severe cardiomyopathic phenotype. Mdx mice were crossed to ones with cardiac myocyte-specific deletion of beta1 integrin (beta1KO) to generate beta1KOmdx. Unstressed beta1KOmdx mice were viable and had normal cardiac function; however, high mortality was seen in peri- and postpartum females by 6 months of age, when severe myocardial necrosis and fibrosis and extensive dystrophic calcification was seen. Decreased ventricular function and blunted adrenergic responsiveness was found in the beta1KOmdx mice compared with control (Lox/Lox, no Cre), beta1KO, and mdx. Similarly, adult beta1KOmdx males were more prone to isoproterenol-induced heart failure and death compared with control groups. Given the extensive calcification, we analyzed transcript levels of genes linked to fibrosis and calcification and found matrix gamma-carboxyglutamic acid protein, decorin, periostin, and the osteoblast transcription factor Runx2/Cbfa1 significantly increased in beta1KOmdx cardiac muscle. Our data show that combined deficiency of dystrophin and integrins in murine cardiac myocytes results in more severe cardiomyopathic changes in the stressed myocardium than reduction of either dystrophin or integrins alone and predisposes to myocardial calcification.

Atherogenic phospholipids attenuate osteogenic signaling by BMP-2 and parathyroid hormone in osteoblasts.

Cardiovascular disease, such as atherosclerosis, has been associated with reduced bone mineral density and fracture risk. A major etiologic factor in atherogenesis is believed to be oxidized phospholipids. We previously found that these phospholipids inhibit spontaneous osteogenic differentiation of marrow stromal cells, suggesting that they may account for the clinical link between atherosclerosis and osteoporosis. Currently, anabolic agents that promote bone formation are increasingly used as a new treatment for osteoporosis. It is not known, however, whether atherogenic phospholipids alter the effects of bone anabolic agents, such as bone morphogenetic protein (BMP)-2 and parathyroid hormone (PTH). Therefore we investigated the effects of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (ox-PAPC) on osteogenic signaling induced by BMP-2 and PTH in MC3T3-E1 cells. Results showed that ox-PAPC attenuated BMP-2 induction of osteogenic markers alkaline phosphatase and osteocalcin. Ox-PAPC also inhibited both spontaneous and BMP-induced expression of PTH receptor. Consistently, pretreatment of cells with ox-PAPC inhibited PTH-induced cAMP production and expression of immediate early genes Nurr1 and IL-6. Results from immunofluorescence and Western blot analyses showed that inhibitory effects of ox-PAPC on BMP-2 signaling were associated with inhibition of SMAD 1/5/8 but not p38-MAPK activation. These effects appear to be due to ox-PAPC activation of the ERK pathway, as the ERK inhibitor PD98059 reversed ox-PAPC inhibitory effects on BMP-2-induced alkaline phosphatase activity, osteocalcin expression, and SMAD activation. These results suggest that atherogenic lipids inhibit osteogenic signaling induced by BMP-2 and PTH, raising the possibility that hyperlipidemia and atherogenic phospholipids may interfere with anabolic therapy.

Role of the DRM and CMT3 methyltransferases in RNA-directed DNA methylation.

RNA interference is a conserved process in which double-stranded RNA is processed into 21-25 nucleotide siRNAs that trigger posttranscriptional gene silencing. In addition, plants display a phenomenon termed RNA-directed DNA methylation (RdDM) in which DNA with sequence identity to silenced RNA is de novo methylated at its cytosine residues. This methylation is not only at canonical CpG sites but also at cytosines in CpNpG and asymmetric sequence contexts. In this report, we study the role of the DRM and CMT3 DNA methyltransferase genes in the initiation and maintenance of RdDM. Neither drm nor cmt3 mutants affected the maintenance of preestablished RNA-directed CpG methylation. However, drm mutants showed a nearly complete loss of asymmetric methylation and a partial loss of CpNpG methylation. The remaining asymmetric and CpNpG methylation was dependent on the activity of CMT3, showing that DRM and CMT3 act redundantly to maintain non-CpG methylation. These DNA methyltransferases appear to act downstream of siRNAs, since drm1 drm2 cmt3 triple mutants show a lack of non-CpG methylation but elevated levels of siRNAs. Finally, we demonstrate that DRM activity is required for the initial establishment of RdDM in all sequence contexts including CpG, CpNpG, and asymmetric sites.

DNA methylation controls histone H3 lysine 9 methylation and heterochromatin assembly in Arabidopsis.

We propose a model for heterochromatin assembly that links DNA methylation with histone methylation and DNA replication. The hypomethylated Arabidopsis mutants ddm1 and met1 were used to investigate the relationship between DNA methylation and chromatin organization. Both mutants show a reduction of heterochromatin due to dispersion of pericentromeric low-copy sequences away from heterochromatic chromocenters. DDM1 and MET1 control heterochromatin assembly at chromocenters by their influence on DNA maintenance (CpG) methylation and subsequent methylation of histone H3 lysine 9. In addition, DDM1 is required for deacetylation of histone H4 lysine 16. Analysis of F(1) hybrids between wild-type and hypomethylated mutants revealed that DNA methylation is epigenetically inherited and represents the genomic imprint that is required to maintain pericentromeric heterochromatin.