Liam M. Casey

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.

Jag2-Notch1 signaling regulates oral epithelial differentiation and palate development

During mammalian palatogenesis, palatal shelves initially grow vertically from the medial sides of the paired maxillary processes flanking the developing tongue and subsequently elevate and fuse with each other above the tongue to form the intact secondary palate. Pathological palate–mandible or palate–tongue fusions have been reported in humans and other mammals, but the molecular and cellular mechanisms that prevent such aberrant adhesions during normal palate development are unknown. We previously reported that mice deficient in Jag2, which encodes a cell surface ligand for the Notch family receptors, have cleft palate associated with palate–tongue fusions. In this report, we show that Jag2 is expressed throughout the oral epithelium and is required for Notch1 activation during oral epithelial differentiation. We show that Notch1 is normally highly activated in the differentiating oral periderm cells covering the developing tongue and the lateral oral surfaces of the mandibular and maxillary processes during palate development. Oral periderm activation of Notch1 is significantly attenuated during palate development in the Jag2 mutants. Further molecular and ultrastructural analyses indicate that oral epithelial organization and periderm differentiation are disrupted in the Jag2 mutants. Moreover, we show that the Jag2 mutant tongue fused to wild‐type palatal shelves in recombinant explant cultures. These data indicate that Jag2‐Notch1 signaling is spatiotemporally regulated in the oral epithelia during palate development to prevent premature palatal shelf adhesion to other oral tissues and to facilitate normal adhesion between the elevated palatal shelves. Developmental Dynamics 235:1830–1844, 2006.

Small Molecule Disruption of Gβγ Signaling Inhibits the Progression of Heart Failure

Rationale: Excess signaling through cardiac G&bgr;&ggr; subunits is an important component of heart failure (HF) pathophysiology. They recruit elevated levels of cytosolic G protein–coupled receptor kinase (GRK)2 to agonist-stimulated &bgr;-adrenergic receptors (&bgr;-ARs) in HF, leading to chronic &bgr;-AR desensitization and downregulation; these events are all hallmarks of HF. Previous data suggested that inhibiting G&bgr;&ggr; signaling and its interaction with GRK2 could be of therapeutic value in HF. Objective: We sought to investigate small molecule G&bgr;&ggr; inhibition in HF. Methods and Results: We recently described novel small molecule G&bgr;&ggr; inhibitors that selectively block G&bgr;&ggr;-binding interactions, including M119 and its highly related analog, gallein. These compounds blocked interaction of G&bgr;&ggr; and GRK2 in vitro and in HL60 cells. Here, we show they reduced &bgr;-AR–mediated membrane recruitment of GRK2 in isolated adult mouse cardiomyocytes. Furthermore, M119 enhanced both adenylyl cyclase activity and cardiomyocyte contractility in response to &bgr;-AR agonist. To evaluate their cardiac-specific effects in vivo, we initially used an acute pharmacological HF model (30 mg/kg per day isoproterenol, 7 days). Concurrent daily injections prevented HF and partially normalized cardiac morphology and GRK2 expression in this acute HF model. To investigate possible efficacy in halting progression of preexisting HF, calsequestrin cardiac transgenic mice (CSQ) with extant HF received daily injections for 28 days. The compound alone halted HF progression and partially normalized heart size, morphology, and cardiac expression of HF marker genes (GRK2, atrial natriuretic factor, and &bgr;-myosin heavy chain). Conclusions: These data suggest a promising therapeutic role for small molecule inhibition of pathological G&bgr;&ggr; signaling in the treatment of HF.Rationale: Excess signaling through cardiac Gβγ subunits is an important component of heart failure (HF) pathophysiology. They recruit elevated levels of cytosolic G protein–coupled receptor kinase (GRK)2 to agonist-stimulated β-adrenergic receptors (β-ARs) in HF, leading to chronic β-AR desensitization and downregulation; these events are all hallmarks of HF. Previous data suggested that inhibiting Gβγ signaling and its interaction with GRK2 could be of therapeutic value in HF. Objective: We sought to investigate small molecule Gβγ inhibition in HF. Methods and Results: We recently described novel small molecule Gβγ inhibitors that selectively block Gβγ-binding interactions, including M119 and its highly related analog, gallein. These compounds blocked interaction of Gβγ and GRK2 in vitro and in HL60 cells. Here, we show they reduced β-AR–mediated membrane recruitment of GRK2 in isolated adult mouse cardiomyocytes. Furthermore, M119 enhanced both adenylyl cyclase activity and cardiomyocyte contractility in response to β-AR agonist. To evaluate their cardiac-specific effects in vivo, we initially used an acute pharmacological HF model (30 mg/kg per day isoproterenol, 7 days). Concurrent daily injections prevented HF and partially normalized cardiac morphology and GRK2 expression in this acute HF model. To investigate possible efficacy in halting progression of preexisting HF, calsequestrin cardiac transgenic mice (CSQ) with extant HF received daily injections for 28 days. The compound alone halted HF progression and partially normalized heart size, morphology, and cardiac expression of HF marker genes (GRK2, atrial natriuretic factor, and β-myosin heavy chain). Conclusions: These data suggest a promising therapeutic role for small molecule inhibition of pathological Gβγ signaling in the treatment of HF.

Small molecule disruption of G beta gamma signaling inhibits the progression of heart failure.

Rationale: Excess signaling through cardiac G&bgr;&ggr; subunits is an important component of heart failure (HF) pathophysiology. They recruit elevated levels of cytosolic G protein–coupled receptor kinase (GRK)2 to agonist-stimulated &bgr;-adrenergic receptors (&bgr;-ARs) in HF, leading to chronic &bgr;-AR desensitization and downregulation; these events are all hallmarks of HF. Previous data suggested that inhibiting G&bgr;&ggr; signaling and its interaction with GRK2 could be of therapeutic value in HF. Objective: We sought to investigate small molecule G&bgr;&ggr; inhibition in HF. Methods and Results: We recently described novel small molecule G&bgr;&ggr; inhibitors that selectively block G&bgr;&ggr;-binding interactions, including M119 and its highly related analog, gallein. These compounds blocked interaction of G&bgr;&ggr; and GRK2 in vitro and in HL60 cells. Here, we show they reduced &bgr;-AR–mediated membrane recruitment of GRK2 in isolated adult mouse cardiomyocytes. Furthermore, M119 enhanced both adenylyl cyclase activity and cardiomyocyte contractility in response to &bgr;-AR agonist. To evaluate their cardiac-specific effects in vivo, we initially used an acute pharmacological HF model (30 mg/kg per day isoproterenol, 7 days). Concurrent daily injections prevented HF and partially normalized cardiac morphology and GRK2 expression in this acute HF model. To investigate possible efficacy in halting progression of preexisting HF, calsequestrin cardiac transgenic mice (CSQ) with extant HF received daily injections for 28 days. The compound alone halted HF progression and partially normalized heart size, morphology, and cardiac expression of HF marker genes (GRK2, atrial natriuretic factor, and &bgr;-myosin heavy chain). Conclusions: These data suggest a promising therapeutic role for small molecule inhibition of pathological G&bgr;&ggr; signaling in the treatment of HF.Rationale: Excess signaling through cardiac Gβγ subunits is an important component of heart failure (HF) pathophysiology. They recruit elevated levels of cytosolic G protein–coupled receptor kinase (GRK)2 to agonist-stimulated β-adrenergic receptors (β-ARs) in HF, leading to chronic β-AR desensitization and downregulation; these events are all hallmarks of HF. Previous data suggested that inhibiting Gβγ signaling and its interaction with GRK2 could be of therapeutic value in HF. Objective: We sought to investigate small molecule Gβγ inhibition in HF. Methods and Results: We recently described novel small molecule Gβγ inhibitors that selectively block Gβγ-binding interactions, including M119 and its highly related analog, gallein. These compounds blocked interaction of Gβγ and GRK2 in vitro and in HL60 cells. Here, we show they reduced β-AR–mediated membrane recruitment of GRK2 in isolated adult mouse cardiomyocytes. Furthermore, M119 enhanced both adenylyl cyclase activity and cardiomyocyte contractility in response to β-AR agonist. To evaluate their cardiac-specific effects in vivo, we initially used an acute pharmacological HF model (30 mg/kg per day isoproterenol, 7 days). Concurrent daily injections prevented HF and partially normalized cardiac morphology and GRK2 expression in this acute HF model. To investigate possible efficacy in halting progression of preexisting HF, calsequestrin cardiac transgenic mice (CSQ) with extant HF received daily injections for 28 days. The compound alone halted HF progression and partially normalized heart size, morphology, and cardiac expression of HF marker genes (GRK2, atrial natriuretic factor, and β-myosin heavy chain). Conclusions: These data suggest a promising therapeutic role for small molecule inhibition of pathological Gβγ signaling in the treatment of HF.

Small Molecule Disruption of Gβγ Signaling Inhibits the Progression of Heart FailureNovelty and Significance

Rationale: Excess signaling through cardiac G&bgr;&ggr; subunits is an important component of heart failure (HF) pathophysiology. They recruit elevated levels of cytosolic G protein–coupled receptor kinase (GRK)2 to agonist-stimulated &bgr;-adrenergic receptors (&bgr;-ARs) in HF, leading to chronic &bgr;-AR desensitization and downregulation; these events are all hallmarks of HF. Previous data suggested that inhibiting G&bgr;&ggr; signaling and its interaction with GRK2 could be of therapeutic value in HF. Objective: We sought to investigate small molecule G&bgr;&ggr; inhibition in HF. Methods and Results: We recently described novel small molecule G&bgr;&ggr; inhibitors that selectively block G&bgr;&ggr;-binding interactions, including M119 and its highly related analog, gallein. These compounds blocked interaction of G&bgr;&ggr; and GRK2 in vitro and in HL60 cells. Here, we show they reduced &bgr;-AR–mediated membrane recruitment of GRK2 in isolated adult mouse cardiomyocytes. Furthermore, M119 enhanced both adenylyl cyclase activity and cardiomyocyte contractility in response to &bgr;-AR agonist. To evaluate their cardiac-specific effects in vivo, we initially used an acute pharmacological HF model (30 mg/kg per day isoproterenol, 7 days). Concurrent daily injections prevented HF and partially normalized cardiac morphology and GRK2 expression in this acute HF model. To investigate possible efficacy in halting progression of preexisting HF, calsequestrin cardiac transgenic mice (CSQ) with extant HF received daily injections for 28 days. The compound alone halted HF progression and partially normalized heart size, morphology, and cardiac expression of HF marker genes (GRK2, atrial natriuretic factor, and &bgr;-myosin heavy chain). Conclusions: These data suggest a promising therapeutic role for small molecule inhibition of pathological G&bgr;&ggr; signaling in the treatment of HF.Rationale: Excess signaling through cardiac Gβγ subunits is an important component of heart failure (HF) pathophysiology. They recruit elevated levels of cytosolic G protein–coupled receptor kinase (GRK)2 to agonist-stimulated β-adrenergic receptors (β-ARs) in HF, leading to chronic β-AR desensitization and downregulation; these events are all hallmarks of HF. Previous data suggested that inhibiting Gβγ signaling and its interaction with GRK2 could be of therapeutic value in HF. Objective: We sought to investigate small molecule Gβγ inhibition in HF. Methods and Results: We recently described novel small molecule Gβγ inhibitors that selectively block Gβγ-binding interactions, including M119 and its highly related analog, gallein. These compounds blocked interaction of Gβγ and GRK2 in vitro and in HL60 cells. Here, we show they reduced β-AR–mediated membrane recruitment of GRK2 in isolated adult mouse cardiomyocytes. Furthermore, M119 enhanced both adenylyl cyclase activity and cardiomyocyte contractility in response to β-AR agonist. To evaluate their cardiac-specific effects in vivo, we initially used an acute pharmacological HF model (30 mg/kg per day isoproterenol, 7 days). Concurrent daily injections prevented HF and partially normalized cardiac morphology and GRK2 expression in this acute HF model. To investigate possible efficacy in halting progression of preexisting HF, calsequestrin cardiac transgenic mice (CSQ) with extant HF received daily injections for 28 days. The compound alone halted HF progression and partially normalized heart size, morphology, and cardiac expression of HF marker genes (GRK2, atrial natriuretic factor, and β-myosin heavy chain). Conclusions: These data suggest a promising therapeutic role for small molecule inhibition of pathological Gβγ signaling in the treatment of HF.