The actions of catecholamines on adrenergic receptors (ARs) induce sympathetic responses, and sustained activation of the sympathetic nervous system results in disrupted circulatory homeostasis. In cardiomyocytes, α1-ARs localize to flask-shaped membrane microdomains known as “caveolae.” Caveolae require both caveolin and cavin proteins for their biogenesis and function. However, the functional roles and molecular interactions of caveolar components in cardiomyocytes are poorly understood. Here, we showed that muscle-restricted coiled-coil protein (MURC)/Cavin-4 regulated α1-AR–induced cardiomyocyte hypertrophy through enhancement of ERK1/2 activation in caveolae. MURC/Cavin-4 was expressed in the caveolae and T tubules of cardiomyocytes. MURC/Cavin-4 overexpression distended the caveolae, whereas MURC/Cavin-4 was not essential for their formation. MURC/Cavin-4 deficiency attenuated cardiac hypertrophy induced by α1-AR stimulation in the presence of caveolae. Interestingly, MURC/Cavin-4 bound to α1A- and α1B-ARs as well as ERK1/2 in caveolae, and spatiotemporally modulated MEK/ERK signaling in response to α1-AR stimulation. Thus, MURC/Cavin-4 facilitates ERK1/2 recruitment to caveolae and efficient α1-AR signaling mediated by caveolae in cardiomyocytes, which provides a unique insight into the molecular mechanisms underlying caveola-mediated signaling in cardiac hypertrophy.
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.
Mutations in the PTRF/Cavin-1 gene cause congenital generalized lipodystrophy type 4 (CGL4) associated with myopathy. Additionally, long-QT syndrome and fatal cardiac arrhythmia are observed in patients with CGL4 who have homozygous PTRF/Cavin-1 mutations. PTRF/Cavin-1 deficiency shows reductions of caveolae and caveolin-3 (Cav3) protein expression in skeletal muscle, and Cav3 deficiency in the heart causes cardiac hypertrophy with loss of caveolae. However, it remains unknown how loss of PTRF/Cavin-1 affects cardiac morphology and function. Here, we present a characterization of the hearts of PTRF/Cavin-1-null (PTRF−/−) mice. Electron microscopy revealed the reduction of caveolae in cardiomyocytes of PTRF−/− mice. PTRF−/− mice at 16 weeks of age developed a progressive cardiomyopathic phenotype with wall thickening of left ventricles and reduced fractional shortening evaluated by echocardiography. Electrocardiography revealed that PTRF−/− mice at 24 weeks of age had low voltages and wide QRS complexes in limb leads. Histological analysis showed cardiomyocyte hypertrophy accompanied by progressive interstitial/perivascular fibrosis. Hypertrophy-related fetal gene expression was also induced in PTRF−/− hearts. Western blotting analysis and quantitative RT-PCR revealed that Cav3 expression was suppressed in PTRF−/− hearts compared with that in wild-type (WT) ones. ERK1/2 was activated in PTRF−/− hearts compared with that in WT ones. These results suggest that loss of PTRF/Cavin-1 protein expression is sufficient to induce a molecular program leading to cardiomyocyte hypertrophy and cardiomyopathy, which is partly attributable to Cav3 reduction in the heart.
Muscle-restricted coiled-coil protein (MURC), also referred to as cavin-4, is a member of the cavin family that works cooperatively with caveolins in caveola formation and function. Cavins are cytoplasmic proteins with coiled-coil domains and form heteromeric complexes, which are recruited to caveolae in cells expressing caveolins. Among caveolins, caveolin-3 (Cav3) is exclusively expressed in muscle cells, similar to MURC/cavin-4. In the heart, Cav3 overexpression contributes to cardiac protection, and its deficiency leads to progressive cardiomyopathy. Mutations in the MURC/cavin-4 gene have been identified in patients with dilated cardiomyopathy. In the present study, we show the role of MURC/cavin-4 as a caveolar component in the heart. In H9c2 cells, MURC/cavin-4 was localized at the plasma membrane, whereas a MURC/cavin-4 mutant lacking the coiled-coil domain (ΔCC) was primarily localized to the cytoplasm. ΔCC bound to Cav3 and impaired membrane localization of Cav3 in cardiomyocytes. Additionally, although ΔCC did not alter Cav3 mRNA expression, ΔCC decreased the Cav3 protein level. MURC/cavin-4 and ΔCC similarly induced cardiomyocyte hypertrophy; however, ΔCC showed higher hypertrophy-related fetal gene expression than MURC/cavin-4. ΔCC induced ERK activation in cardiomyocytes. Transgenic mice expressing ΔCC in the heart (ΔCC-Tg mice) showed impaired cardiac function accompanied by cardiomyocyte hypertrophy and marked interstitial fibrosis. Hearts from ΔCC-Tg mice showed a reduction of the Cav3 protein level and activation of ERK. These results suggest that MURC/cavin-4 requires its coiled-coil domain to target the plasma membrane and to stabilize Cav3 at the plasma membrane of cardiomyocytes and that MURC/cavin-4 functions as a crucial caveolar component to regulate cardiac function.
Pulmonary arterial hypertension (PAH) is a refractory disease characterized by uncontrolled vascular remodeling and elevated pulmonary arterial pressure. Although synthetic inhibitors of some tyrosine kinases have been used to treat PAH, their therapeutic efficacies and safeties remain controversial. Thus, the establishment of novel therapeutic targets based on the molecular pathogenesis underlying PAH is a clinically urgent issue. In the present study, we demonstrated that proline-rich tyrosine kinase 2 (Pyk2), a nonreceptor type protein tyrosine kinase, plays a crucial role in the pathogenesis of pulmonary hypertension (PH) using an animal model of hypoxia-induced PH. Resistance to hypoxia-induced PH was markedly higher in Pyk2-deficient mice than in wild-type mice. Pathological investigations revealed that medial thickening of the pulmonary arterioles, which is a characteristic of hypoxia-induced PH, was absent in Pyk2-deficient mice, suggesting that Pyk2 is involved in the hypoxia-induced aberrant proliferation of vascular smooth muscle cells in hypoxia-induced PH. In vitro experiments using human pulmonary smooth muscle cells showed that hypoxic stress increased the proliferation and migration of cells in a Pyk2-dependent manner. We also demonstrated that Pyk2 plays a crucial role in ROS generation during hypoxic stress and that this Pyk2-dependent generation of ROS is necessary for the activation of hypoxia-inducible factor-1α, a key molecule in the pathogenesis of hypoxia-induced PH. In summary, the results of the present study reveal that Pyk2 plays an important role in the pathogenesis of hypoxia-induced PH. Therefore, Pyk2 may represent a promising therapeutic target for PAH in a clinical setting.
Emerging evidence suggests that caveolin-1 (Cav1) is associated with pulmonary arterial hypertension. MURC (also called Cavin-4) is a member of the cavin family, which regulates caveolar formation and functions together with caveolins. Here, we show that hypoxia increased Murc mRNA expression in the mouse lung, and that Murc-null mice exhibited attenuation of hypoxia-induced pulmonary hypertension (PH) accompanied by reduced ROCK activity in the lung. Conditional knockout mice lacking Murc in smooth muscle also resist hypoxia-induced PH. MURC regulates the proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) through Rho/ROCK signalling. Cav1 suppresses RhoA activity in PASMCs, which is reversed by MURC. MURC binds to Cav1 and inhibits the association of Cav1 with the active form of Gα13, resulting in the facilitated association of the active form of Gα13 with p115RhoGEF. These results reveal that MURC has a function in the development of PH through modulating Rho/ROCK signalling.
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