Background: Inflammation is associated with cardiac remodeling and heart failure, but how it is initiated in response to non-ischemic interventions in the absence of cell death is not known. We tested the hypothesis that activation of CaMKIIδ in cardiomyocytes in response to pressure overload elicits inflammatory responses leading to adverse remodeling. Methods: Mice in which CaMKIIδ was selectively deleted from cardiomyocytes (CMs) (Cardiac specific knockout; CKO) and floxed control (CTL) mice were subjected to transverse aortic constriction (TAC). The effects of CM-specific CaMKIIδ deletion on inflammatory gene expression, inflammasome activation, macrophage accumulation and fibrosis were assessed by qPCR and histochemistry and ventricular remodeling by echocardiography. Results: TAC induced increases in cardiac mRNA levels for pro-inflammatory chemokines and cytokines within 3 days and these responses were significantly blunted when cardiomyocyte CaMKIIδ was deleted. Apoptotic and necrotic cell death were absent at this time. CMs isolated from TAC hearts mirrored these robust increases in gene expression which were markedly attenuated in CKO. Priming and activation of the NLRP3 inflammasome, assessed by measuring IL-1β and NLRP3 mRNA levels, caspase-1 activity and IL-18 cleavage, were increased at day 3 post-TAC in CTL hearts and in CMs isolated from these hearts. These responses were dependent on CaMKIIδ and associated with activation of NFkB and ROS. Accumulation of macrophages observed at day 7–14 post-TAC was diminished in CKO and by blocking MCP-1 signaling, deletion of CM MCP-1 or inhibition of inflammasome activation. Fibrosis was also attenuated by these interventions and in the CKO heart. Ventricular dilation and contractile dysfunction observed at day 42 post-TAC were diminished in the CKO. Inhibition of CaMKII, NFkB, inflammasome or MCP-1 signaling in the first one or two weeks after TAC decreased remodeling but inhibition of CaMKII after two weeks did not. Conclusions: Activation of CaMKIIδ in response to pressure overload triggers inflammatory gene expression and activation of the NLRP3 inflammasome in CMs. These responses provide signals for macrophage recruitment, fibrosis and myocardial dysfunction in the heart. Our work suggests the importance of targeting early inflammatory responses induced by cardiomyocyte CaMKIIδ signaling to prevent progression to heart failure.

Nine membrane-bound adenylyl cyclase (AC) isoforms catalyze the production of the second messenger cyclic AMP (cAMP) in response to various stimuli. Reduction of AC activity has well documented benefits, including benefits for heart disease and pain. These roles have inspired development of isoform-selective AC inhibitors, a lack of which currently limits exploration of functions and/or treatment of dysfunctions involving AC/cAMP signaling. However, inhibitors described as AC5-or AC1-selective have not been screened against the full panel of AC isoforms. We have measured pharmacological inhibitor profiles for all transmembrane AC isoforms. We found that 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22,536), 2-amino-7-(furanyl)-7,8-dihydro-5 (6H)-quinazolinone (NKY80), and adenine 9-b-D-arabinofuranoside (Ara-A), described as supposedly AC5-selective, do not discriminate between AC5 and AC6, whereas the putative AC1-selective inhibitor 5-[[2-(6-amino-9H-purin-9-yl)ethyl]amino]-1-pentanol (NB001) does not directly target AC1 to reduce cAMP levels. A structure-based virtual screen targeting the ATP binding site of AC was used to identify novel chemical structures that show some preference for AC1 or AC2. Mutation of the AC2 forskolin binding pocket does not interfere with inhibition by SQ22,536 or the novel AC2 inhibitor, suggesting binding to the catalytic site. Thus, we show that compounds lacking the adenine chemical signature and targeting the ATP binding site can potentially be used to develop AC isoformspecific inhibitors, and discuss the need to reinterpret literature using AC5/6-selective molecules SQ22,536, NKY80, and Ara-A.

Adenylyl cyclase type 9 (AC9) is found tightly associated with the scaffolding protein Yotiao and the IKs ion channel in heart. But apart from potential IKs regulation, physiological roles for AC9 are unknown. We show that loss of AC9 in mice reduces less than 3% of total AC activity in heart but eliminates Yotiao-associated AC activity. AC9−/− mice exhibit no structural abnormalities but show a significant bradycardia, consistent with AC9 expression in sinoatrial node. Global changes in PKA phosphorylation patterns are not altered in AC9−/− heart, however, basal phosphorylation of heat shock protein 20 (Hsp20) is significantly decreased. Hsp20 binds AC9 in a Yotiao-independent manner and deletion of AC9 decreases Hsp20-associated AC activity in heart. In addition, expression of catalytically inactive AC9 in neonatal cardiomyocytes decreases isoproterenol-stimulated Hsp20 phosphorylation, consistent with an AC9-Hsp20 complex. Phosphorylation of Hsp20 occurs largely in ventricles and is vital for the cardioprotective effects of Hsp20. Decreased Hsp20 phosphorylation suggests a potential baseline ventricular defect for AC9−/−. Doppler echocardiography of AC9−/− displays a decrease in the early ventricular filling velocity and ventricular filling ratio (E/A), indicative of grade 1 diastolic dysfunction and emphasizing the importance of local cAMP production in the context of macromolecular complexes.

Cardiac ischemia/reperfusion, loss of blood flow and its subsequent restoration, causes damage to the heart. Oxidative stress from ischemia/reperfusion leads to dysfunction and death of cardiomyocytes, increasing the risk of progression to heart failure. Alterations in mitochondrial dynamics, in particular mitochondrial fission, have been suggested to play a role in cardioprotection from oxidative stress. We tested the hypothesis that activation of RhoA regulates mitochondrial fission in cardiomyocytes. Our studies show that expression of constitutively active RhoA in cardiomyocytes increases phosphorylation of Dynamin-related protein 1 (Drp1) at serine-616, and leads to localization of Drp1 at mitochondria. Both responses are blocked by inhibition of Rho-associated Protein Kinase (ROCK). Endogenous RhoA activation by the GPCR agonist sphingosine-1-phosphate (S1P) also increases Drp1 phosphorylation and its mitochondrial translocation in a RhoA and ROCK dependent manner. Consistent with the role of mitochondrial Drp1 in fission, RhoA activation in cardiomyocytes leads to formation of smaller mitochondria and this is attenuated by inhibition of ROCK, by siRNA knockdown of Drp1 or by expression of a phosphorylation-deficient Drp1 S616A mutant. In addition, activation of RhoA prevents cell death in cardiomyocytes challenged by oxidative stress and this protection is blocked by siRNA knockdown of Drp1 or by Drp1 S616A expression. Taken together our findings demonstrate that RhoA activation can regulate Drp1 to induce mitochondrial fission and subsequent cellular protection, implicating regulation of fission as a novel mechanism contributing to RhoA-mediated cardioprotection.

Subversion of host organism cAMP signaling is an efficient and widespread mechanism of microbial pathogenesis. Bartonella effector protein A (BepA) of vasculotumorigenic Bartonella henselae protects the infected human endothelial cells against apoptotic stimuli by elevation of cellular cAMP levels by an as yet unknown mechanism. Here, adenylyl cyclase (AC) and the α-subunit of the AC-stimulating G protein (Gαs) were identified as potential cellular target proteins for BepA by gel-free proteomics. Results of the proteomics screen were evaluated for physical and functional interaction by: (i) a heterologous in vivo coexpression system, where human AC activity was reconstituted under the regulation of Gαs and BepA in Escherichia coli; (ii) in vitro AC assays with membrane-anchored fulllength human AC and recombinant BepA and Gαs; (iii) surface plasmon resonance experiments; and (iv) an in vivo fluorescence bimolecular complementation-analysis. The data demonstrate that BepA directly binds host cell AC to potentiate the Gαs-dependent cAMP production. As opposed to the known microbial mechanisms, such as ADP ribosylation of G protein α-subunits by cholera and pertussis toxins, the fundamentally different BepA-mediated elevation of host cell cAMP concentration appears subtle and is dependent on the stimulus of a G protein-coupled receptor-released Gαs. We propose that this mechanism contributes to the persistence of Bartonella henselae in the chronically infected vascular endothelium.bacterial infection | apoptosis | tumorigenesis | type IV secretion

Multiple neurotransmitter systems in the striatum converge to regulate the excitability of striatal neurons by activating several G protein coupled receptors (GPCRs) that signal to the type 5 adenylyl cyclase (AC5), the key effector enzyme that produces the intracellular second messenger cyclic adenosine monophosphate (cAMP). Plasticity of the cAMP signaling in the striatum is thought to play an essential role in the development of drug addiction. Here, we show that the complex between ninth regulator of G protein signaling (RGS9-2) and type 5 G protein β subunit (Gβ5), critically controls dopamine and opioid GPCRs signaling to AC5 in the striatum. We found that RGS9-2/Gβ5 suppressed basal and Gβγ sensitized activity of AC5 by a mechanism that involved both a direct RGS9-2/Gβ5-AC5 protein-protein interaction and the GTPase stimulatory action of the RGS9-2/Gβ5 complex. Furthermore, RGS9-2/Gβ5 increased the deactivation rate of the inhibitory G protein signaling on AC5. Mice lacking Gβ5-RGS complexes showed increased cAMP production and enhanced sensitization of AC5 upon withdrawal from opioid administration. Our findings establish RGS9-2/Gβ5 complexes as regulators of three key aspects of cAMP signaling: the basal activity, sensitization and temporal kinetics of adenylyl cyclase, thus highlighting the role of these RGS proteins in regulating both inhibitory and stimulatory GPCRs that shape cAMP signaling in the striatum.

Adenylyl cyclase (AC) isoforms are implicated in several physiologic processes and disease states, but advancements in the therapeutic targeting of AC isoforms have been limited by the lack of potent and isoform-selective small-molecule modulators. The discovery of AC isoform-selective small molecules is expected to facilitate the validation of AC isoforms as therapeutic targets and augment the study of AC isoform function in vivo. Identification of chemical probes for AC2 is particularly important because there are no published genetic deletion studies and few small-molecule modulators. The present report describes the development and implementation of an intact-cell, small-molecule screening approach and subsequent validation paradigm for the discovery of AC2 inhibitors.The NIH clinical collections I and II were screened for inhibitors of AC2 activity using PMA-stimulated cAMP accumulation as a functional readout. Active compounds were subsequently confirmed and validated as direct AC2 inhibitors using orthogonal and counterscreening assays. The screening effort identified SKF-83566 [8-bromo-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepin-7-ol hydrobromide] as a selective AC2 inhibitor with superior pharmacological properties for selective modulation of AC2 compared with currently available AC inhibitors. The utility of SKF-83566 as a small-molecule probe to study the function of endogenous ACs was demonstrated in C2C12 mouse skeletal muscle cells and human bronchial smooth muscle cells.

Membrane-bound adenylyl cyclase (AC) isoforms have distinct regulatory mechanisms that contribute to their signaling specificity and physiologic roles. Although insight into the physiologic relevance of AC9 has progressed, the understanding of AC9 regulation is muddled with conflicting studies. Currently, modes of AC9 regulation include stimulation by Gas, protein kinase C (PKC) bII, or calcium-calmodulin kinase II (CaMKII) and inhibition by Gai/o, novel PKC isoforms, or calcium-calcineurin. Conversely, the original cloning of human AC9 reported that AC9 is insensitive to Gai inhibition. The purpose of our study was to clarify which proposed regulators of AC9 act directly or indirectly, particularly with respect to Gai/o. The proposed regulators, including G proteins (Gas, Gai, Gao, Gbg), protein kinases (PKCbII, CaMKII), and forskolin, were systematically evaluated using classic in vitro AC assays and cell-based cAMP accumulation assays in COS-7 cells. Our studies show that AC9 is directly regulated by Gas with weak conditional activation by forskolin; other modes of proposed regulation either occur indirectly or possibly require additional scaffolding proteins to facilitate regulation. We also show that AC9 contributes to basal cAMP production; knockdown or knockout of endogenous AC9 reduces basal AC activity in COS-7 cells and splenocytes. Importantly, although AC9 is not directly inhibited by Gai/o, it can heterodimerize with Gai/o-regulated isoforms, AC5 and AC6.