Alessandra Matavel

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.

Molecular Basis of Decreased Kir4.1 Function in SeSAME/EAST Syndrome

SeSAME/EAST syndrome is a channelopathy consisting of a hypokalemic, hypomagnesemic, metabolic alkalosis associated with seizures, sensorineural deafness, ataxia, and developmental abnormalities. This disease links to autosomal recessive mutations in KCNJ10, which encodes the Kir4.1 potassium channel, but the functional consequences of these mutations are not well understood. In Xenopus oocytes, all of the disease-associated mutant channels (R65P, R65P/R199X, G77R, C140R, T164I, and A167V/R297C) had decreased K(+) current (0 to 23% of wild-type levels). Immunofluorescence demonstrated decreased surface expression of G77R, C140R, and A167V expressed in HEK293 cells. When we coexpressed mutant and wild-type subunits to mimic the heterozygous state, R199X, C140R, and G77R currents decreased to 55, 40, and 20% of wild-type levels, respectively, suggesting that carriers of these mutations may present with an abnormal phenotype. Because Kir4.1 subunits can form heteromeric channels with Kir5.1, we coexpressed the aforementioned mutants with Kir5.1 and found that currents were reduced at least as much as observed when we expressed mutants alone. Reduction of pH(i) from approximately 7.4 to 6.8 significantly decreased currents of all mutants except R199X but did not affect wild-type channels. In conclusion, perturbed pH gating may underlie the loss of channel function for the disease-associated mutant Kir4.1 channels and may have important physiologic consequences.

Protein kinase A modulates PLC-dependent regulation and PIP2-sensitivity of K+ channels.

Neurotransmitter and hormone regulation of cellular function can result from a concomitant stimulation of different signaling pathways. Signaling cascades are strongly regulated during disease and are often targeted by commonly used drugs. Crosstalk of different signaling pathways can have profound effects on the regulation of cell excitability. Members of all the three main structural families of potassium channels: inwardly-rectifiers, voltage-gated and 2-P domain, have been shown to be regulated by direct phosphorylation and Gq-coupled receptor activation. Here we test members of each of the three families, Kir3.1/Kir3.4, KCNQ1/KCNE1 and TREK-1 channels, all of which have been shown to be regulated directly by phosphatidylinositol bisphosphate (PIP2). The three channels are inhibited by activation of Gq-coupled receptors and are differentially regulated by protein kinase A (PKA). We show that Gq-coupled receptor regulation can be physiologically modulated directly through specific channel phosphorylation sites. Our results suggest that PKA phosphorylation of these channels affects Gq-coupled receptor inhibition through modulation of the channel sensitivity to PIP2.

PKA and PKC partially rescue long QT type 1 phenotype by restoring channel-PIP2 interactions.

Long-QT syndrome causes torsade de pointes arrhythmia, ventricular fibrillation, and sudden death. The most commonly inherited form of long-QT syndrome, LQT1, is due to mutations on the potassium channel gene KCNQ1, which forms one of the main repolarizing cardiac K+ channels, IKs. IKs has been shown to be regulated by both β-adrenergic receptors, via protein kinase A (PKA), and by Gq protein coupled receptors (GqPCR), via protein kinase C (PKC) and phosphatidylinositol 4,5-bisphosphate (PIP2). These regulatory pathways were shown to crosstalk, with PKA phosphorylation increasing the apparent affinity of IKs to PIP2. Here we study the effects of LQT1 mutations in putative PIP2-KCNQ1 interaction sites on regulation of IKs by PKA and GqPCR. The effect of the LQT1 mutations on IKs regulation was tested for mutations in conserved, positively charged amino acids, located in four distinct cytoplamic domains of the KCNQ1 subunit: R174C (S2-S3), R243C (S4-S5), R366Q (proximal c-terminus) and R555C (distal c-terminus). Mutations in the c-terminus of IKs (both proximal and distal) enhanced channel sensitivity to changes in membrane PIP2 levels, consistent with a decrease in apparent channel-PIP2 affinity. These mutant channels were more sensitive to inhibition caused by receptor mediated PIP2-depletion and more sensitive to stimulation of PIP2 production, by overexpression of phosphatidylinositol-4-phosphate-5-kinase (PI5-kinase). In addition, c-terminus mutants showed a potentiated regulation by PKA. On the other hand, for the two cytoplasmic-loop mutations, an impaired activation by PKA was observed. The effects of the mutations on PKC stimulation of the channel paralleled the effects on PKA stimulation, suggesting that both regulatory inputs are similarly affected by the mutations. We tested whether PKC-mediated activation of IKs, similarly to the PKA-mediated activation, can regulate the channel response to PIP2. After PKC activation, channel was less sensitive to changes in membrane PIP2 levels, consistent with an increase in apparent channel-PIP2 affinity. PKC-activated channel was less sensitive to inhibition caused by block of synthesis of PIP2 by the lipid kinase inhibitor wortmannin and less sensitive to stimulation of PIP2 production. Our data indicates that stimulation by PKA and PKC can partially rescue LQT1 mutant channels with weakened response to PIP2 by strengthening channel interactions with PIP2.

PKC activation and PIP2 depletion underlie biphasic regulation of IKs by Gq-coupled receptors

KCNQ1 is co-assembled with KCNE1 subunits in the heart to form the cardiac delayed rectifier K(+) current (IKs), which is one of the main currents responsible for myocyte repolarization. The most commonly inherited form of cardiac arrhythmias, long-QT syndrome type 1 (LQT1), is due to mutations on KCNQ1. Gq-coupled receptors (GqPCRs) are known to mediate positive inotropism in human ventricular myocardium. The mechanism of IKs current modulation by GqPCRs remains incompletely understood. Here we studied the molecular mechanisms underlying Gq regulation of the IKs channel. Heterologously expressed IKs (human KCNQ1/KCNE1 subunits) was measured in Xenopus oocytes, expressed together with GqPCRs. Our data from several GqPCRs shows that IKs is regulated in a biphasic manner, showing both an activation and an inhibition phase. Receptor-mediated inhibition phase was irreversible when recycling of agonist-sensitive pools of phosphatidylinositol-4,5-bisphosphate (PIP2) was blocked by the lipid kinase inhibitor wortmannin. In addition, stimulation of PIP(2) production, by overexpression of phosphatidylinositol-4-phosphate-5-kinase (PIP5-kinase), decreased receptor-mediated inhibition. The receptor-mediated activation phase was inhibited by the PKC inhibitor calphostin C and by a mutation in a putative PKC phosphorylation site in the KCNE1 subunit. Our results indicate that the depletion of membrane PIP(2) underlies receptor-mediated inhibition of IKs and that phosphorylation by PKC of the KCNE1 subunit underlies the GqPCR-mediated channel activation.

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.