Previous studies have established that cardiomyocytes express protease-activated receptor (PAR)-1, a high-affinity receptor for thrombin, which is also activated by the tethered-ligand domain sequence (SFLLRN) and which promotes inositol trisphosphate accumulation, stimulates extracellular signal-regulated protein kinase, and modulates contractile function. A single previous report identified PAR-1 as a hypertrophic stimulus, but there have been no subsequent investigations of the mechanism. This study reveals the coexpression of PAR-1 and PAR-2 (a second PAR, which is activated by trypsin/tryptase but not thrombin) by Northern blot analysis and compares their signaling properties in neonatal rat ventricular cardiomyocytes. SFLLRN and SLIGRL (an agonist peptide for PAR-2) promote inositol trisphosphate accumulation, stimulate mitogen-activated protein kinases (extracellular signal-regulated protein kinase and p38-mitogen-activated protein kinase), elevate calcium concentration, and increase spontaneous automaticity. SFLLRN (but not SLIGRL) also activates c-Jun NH(2)-terminal kinase and AKT. In keeping with their linkage to pathways that have been associated with growth and/or survival, SFLLRN and SLIGRL both induce hypertrophy. However, PAR agonists promote cell elongation, a morphology that is distinct from the uniform increase in cell dimension induced by alpha(1)-adrenergic receptor activation. These studies provide novel evidence that cardiomyocytes coexpress 2 functional PARs, which link to a common set of signals that culminate in changes in contractile function and hypertrophic growth. PAR actions may assume clinical importance in the border zone surrounding an infarction, where local proteolysis of PARs by serine proteases generated during inflammatory or thrombogenic pathways would elevate calcium concentration (setting the stage for arrhythmias), promote hypertrophic growth, and/or influence cardiomyocyte survival.

The physiological function of beta 2-adrenergic receptors in the neonatal and adult heart is incompletely understood, and possible age-dependent differences in beta 2-receptor actions have not been considered. We used isoproterenol (mixed beta 1- and beta 2-receptor agonist) and zinterol (beta 2-selective agonist) to compare beta-receptor subtype actions in neonatal and adult rat ventricular myocytes. When delivered as a bolus at a final concentration of 10(-7) mol/L, both isoproterenol and zinterol increased the amplitude and hastened the kinetics of the calcium and cell-shortening transients in neonatal myocytes. Under identical experimental conditions, isoproterenol increased the amplitude and accelerated the kinetics of the calcium transient and the twitch in adult myocytes, whereas zinterol did not. In the presence of CGP 20712A (beta 1-receptor blocker), a 100-fold higher concentration of zinterol increased the amplitude but prolonged the duration of the twitch in adult myocytes. To probe the mechanism for this age-dependent difference in beta 2-receptor responsiveness, we compared beta-receptor expression and stimulation of cAMP accumulation in neonatal and adult myocytes. beta-Receptor density was 44,339 +/- 5178 sites per cell in neonatal myocytes and 186,346 +/- 13,356 sites per cell in adult myocytes; the relative proportion of beta 2-receptors was comparable in each (16.7 +/- 2.3% and 16.9 +/- 0.9%, respectively). Isoproterenol induced a large increase in cAMP accumulation in neonatal and adult myocytes (20.0 +/- 1.0- and 20.6 +/- 2.6-fold over basal). In contrast, zinterol evoked a substantial increase in cAMP accumulation in neonatal myocytes but only a minor increase in adult myocytes. These studies provide evidence that at low agonist concentrations, beta 2-receptor activation contributes to the positive inotropic response by increasing cAMP and increasing the amplitude and hastening the kinetics of the twitch in neonatal, but not adult, myocytes. Moreover, these results suggest that age-dependent differences in beta 2-receptor coupling to more distal elements in the signaling cascade can influence myocyte beta 2-receptor responsiveness.

Obesity has more than doubled in children and tripled in adolescents in the past 30 yr. The association between metabolic disorders in offspring of obese mothers with diabetes has long been known; however, a growing body of research indicates that fathers play a significant role through presently unknown mechanisms. Recent observations have shown that changes in paternal diet may result in transgenerational inheritance of the insulin-resistant phenotype. Although diet-induced epigenetic reprogramming via paternal lineage has recently received much attention in the literature, the effect of paternal physical activity on offspring metabolism has not been adequately addressed. In the current study, we investigated the effects of long-term voluntary wheel-running in C57BL/6J male mice on their offspring's predisposition to insulin resistance. Our observations revealed that fathers subjected to wheel-running for 12 wk produced offspring that were more susceptible to the adverse effects of a high-fat diet, manifested in increased body weight and adiposity, impaired glucose tolerance, and elevated insulin levels. Long-term paternal exercise also altered expression of several metabolic genes, including Ogt, Oga, Pdk4, H19, Glut4, and Ptpn1, in offspring skeletal muscle. Finally, prolonged exercise affected gene methylation patterns and micro-RNA content in the sperm of fathers, providing a potential mechanism for the transgenerational inheritance. These findings suggest that paternal exercise produces offspring with a thrifty phenotype, potentially via miRNA-induced modification of sperm.

Recent observations demonstrated that translation of mRNAs may occur in axonal processes at sites that are long distances away from the neuronal perikaria. While axonal protein synthesis has been documented in several studies, the mechanism of its regulation remains unclear. The aim of this study was to investigate whether RNA interference (RNAi) may be one of the pathways that control local protein synthesis in axons. Here we show that sciatic nerve contains Argonaute2 nuclease, fragile X mental retardation protein, p100 nuclease, and Gemin3 helicase-components of the RNA-induced silencing complex (RISC). Application of short-interfering RNAs against neuronal beta-tubulin to the sciatic nerve initiated RISC formation, causing a decrease in levels of neuronal beta-tubulin III mRNA and corresponding protein, as well as a significant reduction in retrograde labeling of lumbar motor neurons. Our observations indicate that RNAi is functional in peripheral mammalian axons and is independent from the neuronal cell body or Schwann cells. We introduce a concept of local regulation of axonal translation via RNAi.

Embryonic stem (ES) cells have been investigated in various animal models of neurodegenerative disease; however, few studies have examined the ability of ES cells to improve functional outcome following traumatic brain injury (TBI). The purpose of the present study was to examine the ability of pre-differentiated murine ES cells (neuronal and glial precursors) to improve functional outcome. Rats were prepared with a unilateral controlled cortical impact injury or sham and then transplanted 7 days later with 100K ES cells (WW6G) (~30% neurons) or media. Two days following transplantation rats were tested on a battery of behavioral tests. It was found that transplantation of ES cells improved behavioral outcome by reducing the initial magnitude of the deficit on the bilateral tactile removal and locomotor placing tests. ES cells also induced almost complete recovery on the vibrissae --> forelimb placing test, whereas, media-transplanted rats failed to show recovery. Acquisition of a reference memory task in the Morris water maze was not improved by transplantation of ES cells. Histological analysis revealed a large number of surviving ES cells in the lesion cavity and showed migration of ES cells into subcortical structures. It was found that transplantation of ES cells prevented the occurrence of multiple small necrotic cavities that were seen in the cortex adjacent to the lesion cavity in media transplanted rats. Additionally, ES cells transplants also significantly reduced lesion size. Results of this study suggest that ES cells that have been pre-differentiated into neuronal precursors prior to transplantation have therapeutic potential.

Cathepsin G is a neutrophil-derived serine protease that contributes to tissue damage at sites of inflammation. The actions of cathepsin G are reported to be mediated by protease-activated receptor (PAR)-4 (a thrombin receptor) in human platelets. This study provides the first evidence that cathepsin G promotes inositol 1,4,5-trisphosphate accumulation, activates ERK, p38 MAPK, and AKT, and decreases contractile function in cardiomyocytes. Because some cathepsin G responses mimic cardiomyocyte activation by thrombin, a role for PARs was considered. Cathepsin G markedly activates phospholipase C and p38 MAPK in cardiomyocytes from PAR-1 ؊/؊ mice, but it fails to activate phospholipase C, ERK, p38 MAPK, or AKT in PAR-1-or PAR-4-expressing PAR-1 ؊/؊ fibroblasts (which display robust responses to thrombin). These results argue that PAR-1 does not mediate the actions of cathepsin G in cardiomyocytes, and neither PAR-1 nor PAR-4 mediates the actions of cathepsin G in fibroblasts. Of note, prolonged incubation of cardiomyocytes with cathepsin G results in the activation of caspase-3, cleavage of FAK and AKT, sarcomeric disassembly, cell rounding, cell detachment from underlying matrix, and morphologic features of apoptosis. Inhibition of Src family kinases or caspases (with PP1 or benzyloxycarbonyl-VAD-fluoromethyl ketone, respectively) delays FAK and AKT cleavage and cardiomyocyte detachment from substrate. Collectively, these studies describe novel cardiac actions of cathepsin G that do not require PARs and are predicted to assume functional importance at sites of interstitial inflammation in the heart.

Abstract-Genetically altered mouse models constitute unique systems to delineate the role of adrenergic receptor (AR) signaling mechanisms as modulators of cardiomyocyte function. The interpretation of results from these models depends on knowledge of the signaling properties of endogenous ARs in mouse cardiomyocytes. In the present study, we identify for the first time several defects in AR signaling in cardiomyocytes cultured from mouse ventricles. ␤ 1 -ARs induce robust increases in cAMP accumulation and the amplitude of the calcium and cell motion transients in mouse cardiomyocytes. Selective ␤ 2 -AR stimulation increases the amplitude of calcium and motion transients, with only a trivial rise in cAMP accumulation in comparison. ␤ 2 -AR responses are not influenced by pertussis toxin in cultured mouse cardiomyocytes. ␣ 1 -ARs fail to activate phospholipase C, the extracellular signal-regulated protein kinase, p38-MAPK, or stimulate hypertrophy in mouse cardiomyocytes. Control experiments establish that this is not due to a lesion in distal elements in the signaling machinery, because these responses are induced by protease-activated receptor-1 agonists and phospholipase C is activated by Pasteurella multocida toxin (a G q ␣-subunit agonist). Surprisingly, norepinephrine activates p38-MAPK via ␤-ARs in mouse cardiomyocytes, but ␤-AR activation of p38-MAPK alone is not sufficient to induce cardiomyocyte hypertrophy. Collectively, these results identify a generalized defect in ␣ 1 -AR signaling and a defect in ␤ 2 -AR linkage to cAMP (although not to an inotropic response) in cultured mouse cardiomyocytes. These naturally occurring vagaries in AR signaling in mouse cardiomyocytes provide informative insights into the requirements for hypertrophic signaling and impact on the value of mouse cardiomyocytes as a reconstitution system to investigate AR signaling in the heart.

Cardiomyocyte ␤ 2 -adrenergic receptors (␤-ARs) provide a source of inotropic support and influence the evolution of heart failure. Recent studies identify distinct mechanisms for ␤ 2 -AR actions in neonatal and adult rat cardiomyocytes. This study examines whether ontogenic changes in cardiac ␤ 2 -AR actions can be attributed to altered Gi expression or changes in the spatial organization of the ␤ 2 -AR complex in membrane subdomains (caveolae). We show that ␤ 2 -ARs increase cAMP, calcium, and contractile amplitude in a pertussis toxin (PTX)-insensitive manner in neonatal cardiomyocytes. This is not caused by lack of Gi; G␣i expression is higher in neonatal cardiomyocytes than in those of adult rats. ␤ 2 -ARs provide inotropic support without detectably increasing cAMP, in adult cardiomyocytes. This cannot be attributed to dual coupling of ␤ 2 -ARs to Gs and Gi, because ␤ 2 -ARs do not promote cAMP accumulation in PTX-pretreated adult cardiomyocytes. Spatial segregation of ␤ 2 -ARs, G␣s/G␣i, and adenylyl cyclase to distinct membrane subdomains also is not a factor, because all of these proteins copurify in caveolin-3-enriched vesicles isolated from adult cardiomyocytes. However, these studies demonstrate that enzyme-based protocols routinely used to isolate ventricular cardiomyocytes lead to proteolysis of ␤-ARs. The functional consequences of this limited ␤-AR proteolysis is uncertain, because truncated ␤ 1 -ARs promote cAMP accumulation and truncated ␤ 2 -ARs provide inotropic support in adult cardiomyocytes. Collectively, these studies indicate that components of the ␤ 2 -AR signaling complex compartmentalize to restricted membrane subdomains in adult rat cardiomyocytes. Neither compartmentalization nor changes in Gi expression fully explain the ontogenic changes in ␤ 2 -AR responsiveness in the rat ventricle.