Protease-activated receptor 2 deficiency reduces cardiac ischemia/reperfusion injury.

OBJECTIVE: To investigate the effect of protease-activated receptor (PAR) 2 deficiency on ischemia/reperfusion (I/R) injury-induced infarct size, inflammation, heart remodeling, and cardiac function. METHODS AND RESULTS: PAR-2 signaling enhances inflammation in different diseases. The effect of PAR-2 deficiency in cardiac I/R injury is unknown. PAR-2(-/-) mice and wild-type littermates were subjected to 30 minutes of ischemia and up to 4 weeks of reperfusion. Infarct size, oxidative/nitrative stress, phosphorylation of mitogen-activated protein kinases, and inflammatory gene expression were assessed 2 hours after reperfusion. Changes in heart size and function were measured by echocardiography up to 4 weeks after reperfusion. Infarct size was significantly reduced in hearts of PAR-2(-/-) mice compared with wild-type littermates. In addition, oxidative/nitrative stress, phosphorylation of mitogen-activated protein kinase, and expression of proinflammatory genes were significantly attenuated in injured hearts of PAR-2(-/-) mice. Finally, PAR-2(-/-) mice were protected from postinfarction remodeling and showed less impairment in heart function compared with wild-type littermates up to 4 weeks after I/R injury. CONCLUSIONS: PAR-2 deficiency reduces myocardial infarction and heart remodeling after I/R injury.

Identification of Nogo as a novel indicator of heart failure.

Numerous genetically engineered animal models of heart failure (HF) exhibit multiple characteristics of human HF, including aberrant beta-adrenergic signaling. Several of these HF models can be rescued by cardiac-targeted expression of the Gbetagamma inhibitory carboxy-terminus of the beta-adrenergic receptor kinase (betaARKct). We recently reported microarray analysis of gene expression in multiple animal models of HF and their betaARKct rescue, where we identified gene expression patterns distinct and predictive of HF and rescue. We have further investigated the muscle LIM protein knockout model of HF (MLP-/-), which closely parallels human dilated cardiomyopathy disease progression and aberrant beta-adrenergic signaling, and their betaARKct rescue. A group of known and novel genes was identified and validated by quantitative real-time PCR whose expression levels predicted phenotype in both the larger HF group and in the MLP-/- subset. One of these novel genes is herein identified as Nogo, a protein widely studied in the nervous system, where it plays a role in regeneration. Nogo expression is altered in HF and normalized with rescue, in an isoform-specific manner, using left ventricular tissue harvested from both animal and human subjects. To investigate cell type-specific expression of Nogo in the heart, immunofluorescence and confocal microscopy were utilized. Nogo expression appears to be most clearly associated with cardiac fibroblasts. To our knowledge, this is the first report to demonstrate the relationship between Nogo expression and HF, including cell-type specificity, in both mouse and human HF and phenotypic rescue.

Protease-activated receptor-1 contributes to cardiac remodeling and hypertrophy.

BACKGROUND: Protease-activated receptor-1 (PAR-1) is the high-affinity receptor for the coagulation protease thrombin. It is expressed by a variety of cell types in the heart, including cardiomyocytes and cardiac fibroblasts. We have shown that tissue factor (TF) and thrombin contribute to infarct size after cardiac ischemia-reperfusion (I/R) injury. Moreover, in vitro studies have shown that PAR-1 signaling induces hypertrophy of cardiomyocytes and proliferation of cardiac fibroblasts. The purpose of the present study was to investigate the role of PAR-1 in infarction, cardiac remodeling, and hypertrophy after I/R injury. In addition, we analyzed the effect of overexpression of PAR-1 on cardiomyocytes. METHODS AND RESULTS: We found that PAR-1 deficiency reduced dilation of the left ventricle and reduced impairment of left ventricular function 2 weeks after I/R injury. Activation of ERK1/2 was increased in injured PAR-1(-/-) mice compared with wild-type mice; however, PAR-1 deficiency did not affect infarct size. Cardiomyocyte-specific overexpression of PAR-1 in mice induced eccentric hypertrophy (increased left ventricular dimension and normal left ventricular wall thickness) and dilated cardiomyopathy. Deletion of the TF gene in cardiomyocytes reduced the eccentric hypertrophy in mice overexpressing PAR-1. CONCLUSIONS: Our results demonstrate that PAR-1 contributes to cardiac remodeling and hypertrophy. Moreover, overexpression of PAR-1 on cardiomyocytes induced eccentric hypertrophy. Inhibition of PAR-1 after myocardial infarction may represent a novel therapy to reduce hypertrophy and heart failure in humans.

Altered PKC expression and phosphorylation in response to the nature, direction, and magnitude of mechanical stretch.

Protein kinase C (PKC) isozymes have been shown to play a role in mechanotransduction in a variety of cell types. We sought to identify the PKC isozymes involved in transducing mechanical (cyclic vs. static), direction and intensity of stretch by examining changes in protein expression and phosphorylation. We used a 3-dimensional culture system with aligned neonatal rat cardiac myocytes on silastic membranes. Myocytes were subjected to either cyclic stretch at 5 cycles/min or static stretch for a period of 24 h at intensities of 0%, 2.5%, 5%, or 10% of full membrane length. Stretch was applied in perpendicular or parallel directions to myocyte alignment. PKC delta was most sensitive to stretch applied perpendicular to myocyte alignment regardless of the nature of stretch, while phospho PKC delta T505 increased in response to static-perpendicular stretch. PKC epsilon expression was altered by cyclic stretch but not static stretch, while phospho PKC epsilon S719 remained unchanged. PKC alpha expression was not altered by stretch; however, phospho PKC alpha S657 increased in a dose-dependent manner following cyclic-perpendicular stretch. Our results indicate that changes in PKC expression and phosphorylation state may be a mechanism for cardiac myocytes to discriminate between the nature, direction, and intensity of mechanical stretch.

Down-regulation of ERK but not MEK phosphorylation in cultured endothelial cells by repeated changes in cyclic stretch.

OBJECTIVE: Effects of cyclic stretch on endothelial cells are studied usually by exposing cells cultured under stretch-free conditions to some levels of cyclic stretch, but in vivo these cells experience both increase and decrease in stretch. Experiments were designed to study how endothelial cells maintained under certain levels of cyclic stretch responded to shifts in stretch frequencies and amplitudes. METHODS: Confluent endothelial cells cultured on flexible silicone membranes with or without pre-stretching for 2-12 h were exposed to various levels of stretch amplitude or frequency and assayed for extracellular signal-regulated kinase 1/2 (ERK) phosphorylation. RESULTS: When endothelial cells without pre-stretching were cyclically stretched, ERK phosphorylation increased, peaking approximately 15 min and slowly decreased. In contrast, when pre-stretched cells were exposed to either higher or lower stretch condition, ERK phosphorylation transiently decreased within 5 min, indicating that some mechanism which down-regulated ERK phosphorylation was activated. Because phosphorylation of ERK kinase (MEK) was not inhibited in these cells, this mechanism targeted ERK directly, not the upstream kinases of the Ras-Raf-MEK-ERK cascade. Furthermore, this ERK down-regulation in pre-stretched cells was not induced by agonists, was inhibited by Na(3)VO(4) but not okadaic acid, and was detected in the cytosolic fraction. Repeated shifts in stretch conditions induced continuous down-regulation of ERK but not MEK phosphorylation. CONCLUSIONS: Endothelial cells are capable of down-regulating ERK phosphorylation in a cyclic stretch- and tyrosine phosphatase-dependent manner. Frequent changes in stretch conditions constitutively activated this ability, which could play some role in regulating ERK activity in endothelial cells in vivo.

Phospholipase C epsilon modulates beta-adrenergic receptor-dependent cardiac contraction and inhibits cardiac hypertrophy.

Phospholipase C (PLC) epsilon is a recently identified enzyme regulated by a wide range of molecules including Ras family small GTPases, Rho A, Galpha(12/13), and Gbetagamma with primary sites of expression in the heart and lung. In a screen for human signal transduction genes altered during heart failure, we found that PLCepsilon mRNA is upregulated. Two murine models of cardiac hypertrophy confirmed upregulation of PLCepsilon protein expression or PLCepsilon RNA. To identify a role for PLCepsilon in cardiac function and pathology, a PLCepsilon-deficient mouse strain was created. Echocardiography indicated PLCepsilon(-/-) mice had decreased cardiac function, and direct measurements of left ventricular contraction demonstrated that PLCepsilon(-/-) mice had a decreased contractile response to acute isoproterenol administration. Cardiac myocytes isolated from PLCepsilon(-/-) mice had decreased beta-adrenergic receptor (betaAR)-dependent increases in Ca2+ transient amplitudes, likely accounting for the contractile deficiency in vivo. This defect appears to be independent from the ability of the betaAR system to produce cAMP and regulation of sarcoplasmic reticulum Ca2+ pool size. To address the significance of these functional deficits to cardiac pathology, PLCepsilon(-/-) mice were subjected to a chronic isoproterenol model of hypertrophic stress. PLCepsilon(-/-) mice were more susceptible than wild-type littermates to development of hypertrophy than wild-type littermates. Together, these data suggest a novel PLC-dependent component of betaAR signaling in cardiac myocytes responsible for maintenance of maximal contractile reserve and loss of PLCepsilon signaling sensitizes the heart to development of hypertrophy in response to chronic cardiac stress.

The expression and role of protein kinase C in neonatal cardiac myocyte attachment, cell volume, and myofibril formation is dependent on the composition of the extracellular matrix.

The extracellular matrix (ECM) is a dynamic component of tissues that influences cellular phenotype and behavior. We sought to determine the role of specific ECM substrates in the regulation of protein kinase C (PKC) isozyme expression and function in cardiac myocyte attachment, cell volume, and myofibril formation. PKC isozyme expression was ECM substrate specific. Increasing concentrations of the PKC delta inhibitor rottlerin attenuated myocyte attachment to randomly organized collagen (1, 5, and 10 microM), laminin (5 and 10 microM), aligned collagen (5 and 10 microM), and fibronectin (10 microM). Rottlerin significantly decreased cell volume on laminin and randomly organized collagen, and inhibited myofibril formation on laminin. The PKC alpha inhibitor Gö 6976 inhibited attachment to randomly organized collagen at 6 nM but did not affect cell volume. The general PKC inhibitor Bisindolylmalemide I (10 and 30 microM) did not affect myocyte attachment; however, it significantly decreased cell volume on randomly organized collagen. Our data indicate that PKC isozymes are expressed and utilized by neonatal cardiac myocytes during attachment, cell growth, and myofibril formation. Specifically, it appears that PKC delta and/or its downstream effectors play an important role in the interaction between cardiac myocytes and laminin, providing further evidence that the ECM influences cardiac myocyte behavior.

Effects of platelet-derived growth factor-AA and -BB on embryonic cardiac development.

Several studies have shown that disruption of the normal expression patterns of platelet-derived growth factor (PDGF) ligands and receptors during development results in gross cardiac defects and embryonic or neonatal death. However, little is known about the specific role that PDGF plays in the differentiation of cardiac myocytes. In experiments complementing studies that utilized naturally-occurring Patch mice lacking the PDGFr alpha, or knockout animals lacking a PDGF ligand or receptor, we used rat and mouse whole-embryo culture (WEC) techniques to increase the exposure of embryos to the PDGF-AA or -BB ligands. Following a 48-hr culture period, we analyzed heart growth and cardiac myocyte differentiation. Exposure of rat embryos to 50 ng/ml of PDGF-AA resulted in a 42% increase in total protein levels in the heart, but did not result in a significant increase in heart growth, as determined by measurements of the atrioventricular length and the left ventricular length and width. Exposure of embryos to 50 ng/ml of PDGF-BB resulted in a 77% increase in total protein levels and a significant (P < 0.05) 8-15% increase in the measured heart parameters. Although a comparison of control and PDGF-AA-treated embryos showed no increase in the overall size of the heart, confocal microscopy showed an increase in the size and number of myofibrillar bundles in the developing myocardium. In addition, transmission electron microscopy (TEM) revealed an increase in the presence of sarcomeres, indicating that myofibrils were more highly differentiated in these areas of the treated embryos. In PDGF-BB-treated embryos, the compact zone of the myocardium was thicker and, as shown by confocal microscopy and TEM, f-actin and well-developed sarcomeres were more prevalent, indicating that the myofibrils were more differentiated in the treated embryos than in the control embryos. These studies indicate that increased exposure of embryonic hearts to PDGF-AA or -BB increases the rate of myocardial development.