PARM-1 Is an Endoplasmic Reticulum Molecule Involved in Endoplasmic Reticulum Stress-Induced Apoptosis in Rat Cardiac Myocytes

To identify novel transmembrane and secretory molecules expressed in cardiac myocytes, signal sequence trap screening was performed in rat neonatal cardiac myocytes. One of the molecules identified was a transmembrane protein, prostatic androgen repressed message-1 (PARM-1). While PARM-1 has been identified as a gene induced in prostate in response to castration, its function is largely unknown. Our expression analysis revealed that PARM-1 was specifically expressed in hearts and skeletal muscles, and in the heart, cardiac myocytes, but not non-myocytes expressed PARM-1. Immunofluorescent staining showed that PARM-1 was predominantly localized in endoplasmic reticulum (ER). In Dahl salt-sensitive rats, high-salt diet resulted in hypertension, cardiac hypertrophy and subsequent heart failure, and significantly stimulated PARM-1 expression in the hearts, with a concomitant increase in ER stress markers such as GRP78 and CHOP. In cultured cardiac myocytes, PARM-1 expression was stimulated by proinflammatory cytokines, but not by hypertrophic stimuli. A marked increase in PARM-1 expression was observed in response to ER stress inducers such as thapsigargin and tunicamycin, which also induced apoptotic cell death. Silencing PARM-1 expression by siRNAs enhanced apoptotic response in cardiac myocytes to ER stresses. PARM-1 silencing also repressed expression of PERK and ATF6, and augmented expression of CHOP without affecting IRE-1 expression and JNK and Caspase-12 activation. Thus, PARM-1 expression is induced by ER stress, which plays a protective role in cardiac myocytes through regulating PERK, ATF6 and CHOP expression. These results suggested that PARM-1 is a novel ER transmembrane molecule involved in cardiac remodeling in hypertensive heart disease

MURC, a Muscle-Restricted Coiled-Coil Protein That Modulates the Rho/ROCK Pathway, Induces Cardiac Dysfunction and Conduction Disturbance▿

We identified a novel muscle-restricted putative coiled-coil protein, MURC, which is evolutionarily conserved from frog to human. MURC was localized to the cytoplasm with accumulation in the Z-line of the sarcomere in the murine adult heart. MURC mRNA expression in the heart increased during the developmental process from the embryonic stage to adulthood. In response to pressure overload, MURC mRNA expression increased in the hypertrophied heart. Using the yeast two-hybrid system, we identified the serum deprivation response (SDPR) protein, a phosphatidylserine-binding protein, as a MURC-binding protein. MURC induced activation of the RhoA/ROCK pathway, which modulated serum response factor-mediated atrial natriuretic peptide (ANP) expression and myofibrillar organization. SDPR augmented MURC-induced transactivation of the ANP promoter in cardiomyocytes, and RNA interference of SDPR attenuated the action of MURC on the ANP promoter. Transgenic mice expressing cardiac-specific MURC (Tg-MURC) exhibited cardiac contractile dysfunction and atrioventricular (AV) conduction disturbances with atrial chamber enlargement, reduced thickness of the ventricular wall, and interstitial fibrosis. Spontaneous episodes of atrial fibrillation and AV block were observed in Tg-MURC mice. These findings indicate that MURC modulates RhoA signaling and that MURC plays an important role in the development of cardiac dysfunction and conduction disturbance with increased vulnerability to atrial arrhythmias

Cholesterol crystal depth in coronary atherosclerotic plaques: A novel index of plaque vulnerability using optical frequency domain imaging.

The involvement of cholesterol crystals (CCs) in plaque progression and destabilization of atherosclerotic plaques has been recently recognized. This study aimed to evaluate the association between the intraplaque localization of CCs and plaque vulnerability.We investigated 55 acute coronary syndrome (ACS) and 80 stable angina pectoris (stable AP) lesions using optical frequency domain imaging (OFDI) prior to percutaneous coronary intervention. The distance between CCs and the luminal surface of coronary plaques was defined as CC depth.Although the incidence of CCs had similar frequencies in the ACS and stable AP groups (95% vs. 89%, p = 0.25), CC depth was significantly less in patients with ACS than in those with stable AP (median [25th to 75th percentile]: 68 μm [58 to 92 μm] vs. 152 μm [115 to 218 μm]; p < 0.001). The incidences of plaque rupture, thrombus, lipid-rich plaques, and thin-cap fibroatheroma were significantly greater in patients with ACS than in those with stable AP (62% vs. 18%, p < 0.001; 67% vs. 16%, p < 0.001; 84% vs. 57%, p < 0.01; and 56% vs. 19%, p < 0.001, respectively).OFDI analysis revealed that CCs were found in the more superficial layers within the coronary atherosclerotic plaques in patients with ACS than in those with stable AP, suggesting that CC depth is associated with plaque vulnerability. CC depth, a novel OFDI-derived parameter, could be potentially used as an alternative means of evaluating plaque vulnerability in coronary arteries

PARM-1 silencing decreases PERK and ATF6, and increases CHOP expression in ER stress condition.

<p>Cultured neonatal rat cardiac myocytes were transfected with siRNAs, and, after 72 hours, were treated with TM or TG for 48 hours. <i>A</i>: Immunoblot analysis was performed with antibody against GRP78, phospho-PERK, PERK, ATF6, IRE-1 or GAPDH. XBP-1 mRNA splicing was assessed by PCR. <i>B</i>: Immunoblot analysis was performed with antibody against CHOP, Caspase-12, phospho-JNK, JNK or GAPDH. <i>C</i>: Densitometric analysis was carried out using ImageJ software. The results were normalized against GAPDH, and expressed as percentages of the maximum. *<i>P</i><0.05 versus control siRNA (Si cont) of respective treatment with TM or TG.</p

PARM-1 expression was induced by inflammatory cytokines and ER stress inducers specifically in cardiac myocytes.

<p><i>A</i>: Cultured rat neonatal cardiac myocytes were stimulated by hypertrophic stimuli such as 100 µmol/l phenylephrine (PE), 1000 U/ml leukemia inhibitory factor (LIF) and 10 µmol/l isoproterenol (ISP), or inflammatory cytokines such as 100 ng/ml TNF-α, 5 ng/ml IL-1β and 4 ng/ml TGF-β. PARM-1 expression was analyzed 48 hours after stimulation. <i>B</i>: Cardiac myocytes were treated with 0.5 µmol/l thapsigargin (TG) or 0.1 µg/ml tunicamycin (TM) for the indicated periods of time, and GRP78, CHOP and PARM-1 expression was analyzed by kinetic real time PCR. <i>C</i>: Cardiac myocytes and fibroblasts were treated with TG or TM for 48 hours at the indicated concentration, and PARM-1 expression was analyzed. <i>D</i>: Cardiac myocytes were treated with TG or TM as indicated, and cell viability was assessed by WST-8 assay. *<i>P</i><0.05 versus non-treated control cells.</p

PARM-1 is an ER protein expressed in cardiac myocytes.

<p><i>A, B, C</i>: PARM-1 expression was assessed by kinetic real-time RT-PCR in various tissues of adult mice (A), cultured rat neonatal cardiac myocytes and non-myocytes (B), and developmental mouse hearts (C), respectively. *<i>P</i><0.05 versus cardiac myocytes (B) or embryonic hearts (C). <i>D</i>: Cellular localization of PARM-1 was analyzed by immunostaining of cultured neonatal rat cardiac myocytes expressing flag-tagged PARM-1 with anti-Flag antibody (left), and anti-GRP78 antibody or MitoTacker (center). Nuclei were stained by DAPI.</p