Rationale: Excess signaling through cardiac G␤␥ 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 ␤-adrenergic receptors (␤-ARs) in HF, leading to chronic ␤-AR desensitization and downregulation; these events are all hallmarks of HF. Previous data suggested that inhibiting G␤␥ signaling and its interaction with GRK2 could be of therapeutic value in HF. Objective: We sought to investigate small molecule G␤␥ inhibition in HF. Methods and Results: We recently described novel small molecule G␤␥ inhibitors that selectively block G␤␥-binding interactions, including M119 and its highly related analog, gallein. These compounds blocked interaction of G␤␥ and GRK2 in vitro and in HL60 cells. Here, we show they reduced ␤-AR-mediated membrane recruitment of GRK2 in isolated adult mouse cardiomyocytes. Furthermore, M119 enhanced both adenylyl cyclase activity and cardiomyocyte contractility in response to ␤-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 ␤-myosin heavy chain). Conclusions: These data suggest a promising therapeutic role for small molecule inhibition of pathological G␤␥ signaling in the treatment of HF. (Circ Res. 2010;107:532-539.)Key Words: G proteins Ⅲ adrenergic receptor Ⅲ G protein-coupled receptor kinases Ⅲ cardiomyopathy Ⅲ heart failure Ⅲ cardiomyocyte H eart failure (HF) is a devastating disease with poor prognosis, and remains a leading cause of death worldwide. 1,2 Excess signaling through cardiac G protein G␤␥ subunits is an important component of HF pathophysiology. In particular, they recruit elevated levels of cytosolic G protein-coupled receptor kinase 2 (GRK2) (␤-adrenergic receptor kinase [␤-ARK]1) to agonist-stimulated ␤-ARs in HF, 3 leading to the chronic ␤-AR desensitization, downregulation and pathological signaling that are hallmarks of HF. 4,5 Increasing evidence suggests a critical role for G␤␥-mediated signaling in HF. In particular, GRK2 is significantly upregulated in cardiomyocytes of animal models of HF and human HF patients; this elevates G␤␥-GRK2 interactions and contributes to chronic desensitization of ␤-AR signaling 6,7 ; interestingly, levels of GRK2 appear to correlate with the severity of HF. 6,8 Enhancing G␤␥-GRK2 interaction by cardiac targeted overexpression of GRK2(s) can directly cause HF in experimental animal models 9 ; its genetic ablation has generally proven to be...

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 ϩ 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.

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: inward-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.

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 beta-adrenergic receptors, via protein kinase A (PKA), and by Gq protein coupled receptors (GqPCR), via protein kinase C (PKC) and phosphatidylinositol 4,5-bisphosphate (PIP(2)). These regulatory pathways were shown to crosstalk, with PKA phosphorylation increasing the apparent affinity of IKs to PIP(2). Here we study the effects of LQT1 mutations in putative PIP(2)-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 PIP(2) levels, consistent with a decrease in apparent channel-PIP(2) affinity. These mutant channels were more sensitive to inhibition caused by receptor mediated PIP(2)-depletion and more sensitive to stimulation of PIP(2) 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 PIP(2). After PKC activation, channel was less sensitive to changes in membrane PIP(2) levels, consistent with an increase in apparent channel-PIP(2) affinity. PKC-activated channel was less sensitive to inhibition caused by block of synthesis of PIP(2) by the lipid kinase inhibitor wortmannin and less sensitive to stimulation of PIP(2) production. Our data indicates that stimulation by PKA and PKC can partially rescue LQT1 mutant channels with weakened response to PIP(2) by strengthening channel interactions with PIP(2).

PnPP-19 potentiates erection in vivo and ex vivo via the nitric oxide/cyclic guanosine monophosphate pathway. It does not affect sodium channels or rat hearts and shows no toxicity and low immunogenicity. These findings make it a promising candidate as a novel drug in the therapy of erectile dysfunction.