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Mitophagy, a mitochondria-specific form of autophagy, removes dysfunctional mitochondria and is hence an essential process contributing to mitochondrial quality control. PTEN-induced kinase 1 (PINK1) and the E3 ubiquitin ligase Parkin are critical molecules involved in stress-induced mitophagy, but the intracellular signaling mechanisms by which this pathway is regulated are unclear. We tested the hypothesis that signaling through RhoA, a small GTPase, induces mitophagy via modulation of the PINK1/Parkin pathway as a protective mechanism against ischemic stress. We demonstrate that expression of constitutively active RhoA as well as sphingosine-1-phosphate induced activation of endogenous RhoA in cardiomyocytes result in an accumulation of PINK1 at mitochondria. This is accompanied by translocation of Parkin to mitochondria and ubiquitination of mitochondrial proteins leading to recognition of mitochondria by autophagosomes and their lysosomal degradation. Expression of RhoA in cardiomyocytes confers protection against ischemia, and this cardioprotection is attenuated by siRNA-mediated PINK1 knockdown. In vivo myocardial infarction elicits increases in mitochondrial PINK1, Parkin, and ubiquitinated mitochondrial proteins. AAV9-mediated RhoA expression potentiates these responses and a concurrent decrease in infarct size is observed. Interestingly, induction of mitochondrial PINK1 accumulation in response to RhoA signaling is neither mediated through its transcriptional upregulation nor dependent on depolarization of the mitochondrial membrane, the canonical mechanism for PINK1 accumulation. Instead, our results reveal that RhoA signaling inhibits PINK1 cleavage, thereby stabilizing PINK1 protein at mitochondria. We further show that active RhoA localizes at mitochondria and interacts with PINK1, and that the mitochondrial localization of RhoA is regulated by its downstream effector protein kinase D. These findings demonstrate that RhoA activation engages a unique mechanism to regulate PINK1 accumulation, induce mitophagy and protect against ischemic stress, and implicates regulation of RhoA signaling as a potential strategy to enhance mitophagy and confer protection under stress conditions.

Cardiac injury triggers an acute immune response that drives tissue healing and remodeling via the activation of compensatory mechanisms. Over time, remodeling and inflammation become chronic and have adverse effects that lead to a depression of cardiac function and eventual heart failure. Cardiac inflammation is characterized by dynamic spatial and temporal crosstalk between the resident cells of the heart and recruitment of circulating leukocytes. Until recently, the cardiomyocyte has not been accepted as a direct contributor to cardiac inflammation. It has now emerged as a key initiator of the acute immune response via its ability to produce cytokines and may also synchronize leukocyte recruitment post-injury. This review will focus on the role of the cardiomyocyte in the acute immune response to ischemic and non-ischemic injury and the mechanisms by which it may influence the course of cardiac remodeling and failure.

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The low molecular weight G protein RhoA (rat sarcoma virus homolog family member A) serves as a node for transducing signals through G protein–coupled receptors (GPCRs). Activation of RhoA occurs through coupling of G proteins, most prominently, G12/13, to Rho guanine nucleotide exchange factors. The GPCR ligands that are most efficacious for RhoA activation include thrombin, lysophosphatidic acid, sphingosine-1-phosphate, and thromboxane A2. These ligands also stimulate proliferation, differentiation, and inflammation in a variety of cell and tissues types. The molecular events underlying these responses are the activation of transcription factors, transcriptional coactivators, and downstream gene programs. This review describes the pathways leading from GPCRs and RhoA to the regulation of activator protein-1, NFκB (nuclear factor κ-light-chain-enhancer of activated B cells), myocardin-related transcription factor A, and Yes-associated protein. We also focus on the importance of two prominent downstream transcriptional gene targets, the inflammatory mediator cyclooxygenase 2, and the matricellular protein cysteine-rich angiogenic inducer 61 (CCN1). Finally, we describe the importance of GPCR-induced activation of these pathways in the pathophysiology of cancer, fibrosis, and cardiovascular disease.

The molecular pharmacology of the G protein–coupled receptors for sphingosine 1-phosphate (S1P) provides important insight into established and new therapeutic targets. A new, potent bitopic S1P3antagonist, SPM-354, with in vivo activity, has been used, together with S1P3-knockin and S1P3-knockout mice to define the spatial and functional properties of S1P3in regulating cardiac conduction. We show that S1P3is a key direct regulator of cardiac rhythm both in vivo and in isolated perfused hearts. 2-Amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol in vivo and S1P in isolated hearts induced a spectrum of cardiac effects, ranging from sinus bradycardia to complete heart block, as measured by a surface electrocardiogram in anesthetized mice and in volume-conducted Langendorff preparations. The agonist effects on complete heart block are absent in S1P3-knockout mice and are reversed in wild-type mice with SPM-354, as characterized and described here. Homologous knockin of S1P3-mCherry is fully functional pharmacologically and is strongly expressed by immunohistochemistry confocal microscopy in Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 4 (HCN4)-positive atrioventricular node and His-Purkinje fibers, with relative less expression in the HCN4-positive sinoatrial node. In Langendorff studies, at constant pressure, SPM-354 restored sinus rhythm in S1P-induced complete heart block and fully reversed S1P-mediated bradycardia. S1P3distribution and function in the mouse ventricular cardiac conduction system suggest a direct mechanism for heart block risk that should be further studied in humans. A richer understanding of receptor and ligand usage in the pacemaker cells of the cardiac system is likely to be useful in understanding ventricular conduction in health, disease, and pharmacology.

Introduction:Calcium-calmodulin-dependent protein kinase II (CaMKII) has emerged as a central mediator of cardiac stress responses which may serve several critical roles in the regulation of cardiac rhythm, cardiac contractility and growth. Sustained and excessive activation of CaMKII during cardiac disease has, however, been linked to arrhythmias, and maladaptive cardiac remodeling, eventually leading to heart failure (HF) and sudden cardiac death.Areas covered:In the current review, the authors describe the unique structural and biochemical properties of CaMKII and focus on its physiological effects in cardiomyocytes. Furthermore, they provide evidence for a role of CaMKII in cardiac pathologies, including arrhythmogenesis, myocardial ischemia and HF development. The authors conclude by discussing the potential for CaMKII as a target for inhibition in heart disease.Expert opinion:CaMKII provides a promising nodal point for intervention that may allow simultaneous prevention of HF progression and development of arrhythmias. For future studies and drug development there is a strong rationale for the development of more specific CaMKII inhibitors. In addition, an improved understanding of the differential roles of CaMKII subtypes is required.

-Adrenergic receptor (-AR) activation can provoke cardiac arrhythmias mediated by cAMP-dependent alterations of Ca2signaling. However, cAMP can activate both protein kinase A and an exchange protein directly activated by cAMP (Epac), but their functional interaction is unclear. In heart, selective Epac activation can induce potentially arrhythmogenic sarcoplasmic reticulum (SR) Ca2release that involves Ca2calmodulin-dependent protein kinase II (CaMKII) effects on the ryanodine receptor (RyR).

Acute activation of calcium/calmodulin-dependent protein kinase (CaMKII) in permeabilized phospholamban knockout (PLN-KO) mouse myocytes phosphorylates ryanodine receptors (RyRs) and activates spontaneous local sarcoplasmic reticulum (SR) Ca release events (Ca sparks) even at constant SR Ca load. To assess how CaMKII regulates SR Ca release in intact myocytes (independent of SR Ca content changes or PLN effects), we compared Ca sparks in PLN-KO versus mice, which also have transgenic cardiac overexpression of CaMKIIδCin the PLN-KO background (KO/TG). Compared with PLN-KO mice, these KO/TG cardiomyocytes exhibited 1), increased twitch Ca transient and fractional release (both by ∼35%), but unaltered SR Ca load; 2), increased resting Ca spark frequency (300%) despite a lower diastolic [Ca]i, which also slowed twitch [Ca]idecline (suggesting CaMKII-dependent RyR Ca sensitization); 3), elevated Ca spark amplitude and rate of Ca release (which might indicate that more RyR channels participate in a single spark); 4), prolonged Ca spark rise time (which implies that CaMKII either delays RyR closure or prolongs the time when openings can occur); 5), more frequent repetitive sparks at single release sites. Analysis of repetitive sparks from individual Ca release sites indicates that CaMKII enhanced RyR Ca sensitivity, but did not change the time course of SR Ca refilling. These results demonstrate that there are dramatic CaMKII-mediated effects on RyR Ca release that occur via regulation of both RyR activation and termination processes.

The molecular events associated with the development of pathological hypertrophy have been shown to be stimulated through G-protein-coupled receptors that activate Gq signaling pathways in neonatal cardiomyocytes and in transgenic (TG) and knockout mice. We demonstrated that CaMKII, a multifunctional Ca2+-regulated protein kinase, was activated through G-protein-coupled receptor and inositol trisphosphate-mediated Ca2+release and suggested that CaMKII was a downstream mediator of Gq-coupled hypertrophic signaling. This was supported by the demonstration of CaMKII activation by pressure overload [(transverse aortic constriction (TAC)] and induction of hypertrophy by TG CaMKII expression. CaMKII also phosphorylates Ca2+handling proteins including the ryanodine receptor (RyR2), phosphorylation of which markedly increases sarcoplasmic reticulum Ca2+leak. Increased RyR2 phosphorylation is associated with heart failure development in CaMKII TG mice, and mice genetically deleted for CaMKII (KO) have attenuated RyR2 phosphorylation, sarcoplasmic reticulum Ca2+leak, and heart failure development after long-term TAC. Genetic ablation of CaMKII also decreases development of heart failure in Gq TG mice and decreases infarct size, while improving functional recovery in mice subject to ischemia/reperfusion and preventing adverse remodeling after coronary artery occlusion. The underlying mechanisms are currently under study.

Transgenic (TG) Ca/calmodulin-dependent protein kinase II (CaMKII)Cmice have heart failure and isoproterenol (ISO)-inducible arrhythmias. We hypothesized that CaMKII contributes to arrhythmias and underlying cellular events and that inhibition of CaMKII reduces cardiac arrhythmogenesis in vitro and in vivo.

Lysophosphatidic acid (LPA), a bioactive lipid produced by several cell types including postmitotic neurons and activated platelets, is thought to be involved in various biological processes, including brain development. Three cognate G protein-coupled receptors encoded by lpa(1)/lp(A1)/Edg-2/Gpcr26, lpa(2)/lp(A2)/Edg-4, and lpa(3)/lp(A3)/Edg-7 mediate the cellular effects of LPA. We have previously shown that deletion of lpa(1) in mice results in craniofacial dysmorphism, semilethality due to defective suckling behavior, and generation of a small fraction of pups with frontal hematoma. To further investigate the role of these receptors and LPA signaling in the organism, we deleted lpa(2) in mice. Homozygous knockout (lpa(2)((-/-))) mice were born at the expected frequency and displayed no obvious phenotypic abnormalities. Intercrosses allowed generation of lpa(1)((-/-)) lpa(2)((-/-)) double knockout mice, which displayed no additional phenotypic abnormalities relative to lpa(1)((-/-)) mice except for an increased incidence of perinatal frontal hematoma. Histological analyses of lpa(1)((-/-)) lpa(2)((-/-)) embryonic cerebral cortices did not reveal obvious differences in the proliferating cell population. However, many LPA-induced responses, including phospholipase C activation, Ca(2+) mobilization, adenylyl cyclase activation, proliferation, JNK activation, Akt activation, and stress fiber formation, were absent or severely reduced in embryonic fibroblasts derived from lpa(1)((-/-)) lpa(2)((-/-)) mice. Except for adenylyl cyclase activation [which was nearly abolished in lpa(1)((-/-)) fibroblasts], these responses were only partially affected in lpa(1)((-/-)) and lpa(2)((-/-)) fibroblasts. Thus, although LPA(2) is not essential for normal mouse development, it does act redundantly with LPA(1) to mediate most LPA responses in fibroblasts.

Lysophosphatidic acid (LPA), a bioactive lipid produced by several cell types including postmitotic neurons and activated platelets, is thought to be involved in various biological processes, including brain development. Three cognate G protein-coupled receptors encoded by lpa1/lpA1/Edg-2/Gpcr26, lpa2/lpA2/Edg-4, and lpa3/lpA3/Edg-7mediate the cellular effects of LPA. We have previously shown that deletion of lpa1in mice results in craniofacial dysmorphism, semilethality due to defective suckling behavior, and generation of a small fraction of pups with frontal hematoma. To further investigate the role of these receptors and LPA signaling in the organism, we deleted lpa2in mice. Homozygous knockout (lpa2(−/−)) mice were born at the expected frequency and displayed no obvious phenotypic abnormalities. Intercrosses allowed generation of lpa1(−/−)lpa2(−/−)double knockout mice, which displayed no additional phenotypic abnormalities relative to lpa1(−/−)mice except for an increased incidence of perinatal frontal hematoma. Histological analyses of lpa1(−/−)lpa2(−/−)embryonic cerebral cortices did not reveal obvious differences in the proliferating cell population. However, many LPA-induced responses, including phospholipase C activation, Ca2+mobilization, adenylyl cyclase activation, proliferation, JNK activation, Akt activation, and stress fiber formation, were absent or severely reduced in embryonic fibroblasts derived from lpa1(−/−)lpa2(−/−)mice. Except for adenylyl cyclase activation [which was nearly abolished in lpa1(−/−)fibroblasts], these responses were only partially affected in lpa1(−/−)and lpa2(−/−)fibroblasts. Thus, although LPA2is not essential for normal mouse development, it does act redundantly with LPA1to mediate most LPA responses in fibroblasts.

ABSTRACTLysophosphatidic acid (LPA), a bioactive lipid produced by several cell types including postmitotic neurons and activated platelets, is thought to be involved in various biological processes, including brain development. Three cognate G protein-coupled receptors encoded by lpa1/lpA1/Edg-2/Gpcr26, lpa2/lpA2/Edg-4, and lpa3/lpA3/Edg-7mediate the cellular effects of LPA. We have previously shown that deletion of lpa1in mice results in craniofacial dysmorphism, semilethality due to defective suckling behavior, and generation of a small fraction of pups with frontal hematoma. To further investigate the role of these receptors and LPA signaling in the organism, we deleted lpa2in mice. Homozygous knockout (lpa2(−/−)) mice were born at the expected frequency and displayed no obvious phenotypic abnormalities. Intercrosses allowed generation of lpa1(−/−)lpa2(−/−)double knockout mice, which displayed no additional phenotypic abnormalities relative to lpa1(−/−)mice except for an increased incidence of perinatal frontal hematoma. Histological analyses of lpa1(−/−)lpa2(−/−)embryonic cerebral cortices did not reveal obvious differences in the proliferating cell population. However, many LPA-induced responses, including phospholipase C activation, Ca2+mobilization, adenylyl cyclase activation, proliferation, JNK activation, Akt activation, and stress fiber formation, were absent or severely reduced in embryonic fibroblasts derived from lpa1(−/−)lpa2(−/−)mice. Except for adenylyl cyclase activation [which was nearly abolished in lpa1(−/−)fibroblasts], these responses were only partially affected in lpa1(−/−)and lpa2(−/−)fibroblasts. Thus, although LPA2is not essential for normal mouse development, it does act redundantly with LPA1to mediate most LPA responses in fibroblasts.

Five cognate G protein-coupled receptors (S1P1–5) have been shown to mediate various cellular effects of sphingosine 1-phosphate (S1P). Here we report the generation of mice null for S1P2and for both S1P2and S1P3. S1P2-null mice were viable and fertile and developed normally. The litter sizes from S1P2S1P3double-null crosses were remarkably reduced compared with controls, and double-null pups often did not survive through infancy, although double-null survivors lacked any obvious phenotype. Mouse embryonic fibroblasts (MEFs) were examined for the effects of receptor deletions on S1P signaling pathways. Wild-type MEFs were responsive to S1P in activation of Rho and phospholipase C (PLC), intracellular calcium mobilization, and inhibition of forskolin-activated adenylyl cyclase. S1P2-null MEFs showed a significant decrease in Rho activation, but no effect on PLC activation, calcium mobilization, or adenylyl cyclase inhibition. Double-null MEFs displayed a complete loss of Rho activation and a significant decrease in PLC activation and calcium mobilization, with no effect on adenylyl cyclase inhibition. These data extend our previous findings on S1P3-null mice and indicate preferential coupling of the S1P2and S1P3receptors to Rho and PLC/Ca2+pathways, respectively. Although either receptor subtype supports embryonic development, deletion of both produces marked perinatal lethality, demonstrating an essential role for combined S1P signaling by these receptors.

Activation of Gq-coupled α1-adrenergic receptors leads to hypertrophic growth of neonatal rat ventricular cardiomyocytes that is associated with increased expression of hypertrophy-related genes, including atrial natriuretic peptide (ANP) and myosin light chain-2 (MLC), as well as increased ribosome synthesis. The role of inositol phosphates in signaling pathways involved in these changes in gene expression was examined by overexpressing inositol phosphate-metabolizing enzymes and determining effects on ANP, MLC, and 45 S ribosomal gene expression following co-transfection of appropriate reporter gene constructs. Overexpression of enzymes that metabolize inositol 1,4,5-trisphosphate did not reduce ANP or MLC responses, but overexpression of the enzyme primarily responsible for metabolism of inositol 4,5-bisphosphate (Ins(1,4)P2), inositol polyphosphate 1-phosphatase (INPP), reduced ANP and MLC responses associated with α1-adrenergic receptor-mediated hypertrophy. Similarly overexpressed INPP reduced ANP and MLC responses associated with contraction-induced hypertrophy. In addition, overexpression of INPP reduced the increase in ribosomal DNA transcription associated with both hypertrophic models. Hypertrophied cells from both cell models as well as ventricular tissue from mouse hearts hypertrophied by pressure overload in vivocontained heightened levels of Ins(1,4)P2, suggesting reduced INPP activity in three different models of hypertrophy. These studies provide evidence for an involvement of Ins(1,4)P2in hypertrophic signaling pathways in ventricular myocytes.

The delta isoform of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) predominates in the heart. To investigate the role of CaMKII in cardiac function, we made transgenic (TG) mice that express the nuclear delta(B) isoform of CaMKII. The expressed CaMKIIdelta(B) transgene was restricted to the myocardium and highly concentrated in the nucleus. Cardiac hypertrophy was evidenced by an increased left ventricle to body weight ratio and up-regulation of embryonic and contractile protein genes including atrial natriuretic factor, beta-myosin heavy chain, and alpha-skeletal actin. Echocardiography revealed ventricular dilation and decreased cardiac function, which was also observed in hemodynamic measurements from CaMKIIdelta(B) TG mice. Surprisingly, phosphorylation of phospholamban at both Thr(17) and Ser(16) was significantly decreased in the basal state as well as upon adrenergic stimulation. This was associated with diminished sarcoplasmic reticulum Ca(2+) uptake in vitro and altered relaxation properties in vivo. The activity and expression of protein phosphatase 2A were both found to be increased in CaMKII TG mice, and immunoprecipitation studies indicated that protein phosphatase 2A directly associates with CaMKII. Our findings are the first to demonstrate that CaMKII can induce hypertrophy and dilation in vivo and indicate that compensatory increases in phosphatase activity contribute to the resultant phenotype.

Sphingosine 1-phosphate (S1P) exerts diverse physiological actions by activating its cognate G protein-coupled receptors. Five S1P receptors have been identified in mammals: LPB1/EDG-1, LPB2/H218/AGR16/EDG-5, LPB3/EDG-3, LPB4/NRG-1/EDG-8, and LPC1/EDG-6. One of these receptors, LPB1, has recently been shown to be essential for mouse embryonic development. Here we disrupted the lpB3gene in mice, resulting in the complete absence of lpB3gene, transcript, and LPB3protein. LPB3-null mice were viable and fertile and developed normally with no obvious phenotypic abnormality. We prepared mouse embryonic fibroblast (MEF) cells to examine effects of LPB3deletion on S1P-induced signal transduction pathways. Wild-type MEF cells expressedlpB1, lpB2, andlpB3but neither lpB4nor lpC1, and they were highly responsive to S1P in phospholipase C (PLC) activation, adenylyl cyclase inhibition, and Rho activation. Identically prepared LPB3-null MEF cells showed significant decreases in PLC activation, slight decreases in adenylyl cyclase inhibition, and no change in Rho activation. Retrovirus-mediated rescue of the LPB3receptor in LPB3-null MEF cells restored S1P-dependent PLC activation and adenylyl cyclase inhibition. These results indicate a nonessential role for LPB3in normal development of mouse but show nonredundant cellular signaling mediated by a single type of S1P receptor.

Recent reports have shown that several heterotrimeric protein-coupled receptors that signal through Gαqcan induce Rho-dependent responses, but the pathways that mediate the interaction between Gαqand Rho have not yet been identified. In this report we present evidence that Gαqexpressed in COS-7 cells coprecipitates with the Rho guanine nucleotide exchange factor (GEF) Lbc. Furthermore, Gαqexpression enhances Rho-dependent responses. Coexpressed Gαqand Lbc have a synergistic effect on the Rho-dependent rounding of 1321N1 astrocytoma cells. In addition, serum response factor-dependent gene expression, as assessed by the SRE.L reporter gene, is synergistically activated by Gαqand Rho GEFs. The synergistic effect of Gαqon this response is inhibited by C3 exoenzyme and requires phospholipase C activation. Surprisingly, expression of Gαq, in contrast to that of Gα12and Gα13, does not increase the amount of activated Rho. We also observe that Gαqenhances SRE.L stimulation by activated Rho, indicating that the effect of Gαqoccurs downstream of Rho activation. Thus, Gαqinteracts physically and/or functionally with Rho GEFs; however this does not appear to lead to or result from increased activation of Rho. We suggest that Gαq-generated signals enhance responses downstream of Rho activation.

Thrombin induces astrocytoma cell rounding through a Rho-dependent pathway (Majumdar, M., Seasholtz, T. M., Goldstein, D., de Lanerolle, P., and Brown, J. H. (1998) J. Biol. Chem.273, 10099–10106). The involvement of the G12family of G proteins and the role of specific Rho exchange factors in transducing signals from the thrombin receptor to Rho-dependent cytoskeletal responses was examined. Microinjection of cDNAs for activated Gα12or Gα13induced cell rounding, and antibodies to Gα12or Gα13blocked the response to thrombin. In contrast, activation or inhibition of Gαqfunction had relatively little effect. The cytoskeletal response to Gα12was inhibited by microinjection of C3 exoenzyme, indicating Rho dependence. Two Rho-specific guanine nucleotide exchange factors (GEFs), oncogenic lbc and p115, increased the percentage of rounded cells 4–5-fold, and this was inhibited by C3. Mutant GEFs lacking the Dbl homology (DH) domain required for exchange factor activity failed to induce cell rounding. However, the DH mutants of lbc and p115 were efficacious inhibitors of rounding induced by thrombin or Gα12. The effects of lbc were dependent on an intact pleckstrin homology domain, which may be required for appropriate targeting of the Rho-GEF. These findings identify the Gα12protein family as transducers of thrombin signaling to the cytoskeleton and provide the first evidence that a Rho-GEF transduces signals between G protein-coupled receptors and Rho-mediated cytoskeletal responses.

ABSTRACTWe evaluated the role of the G alpha-q (Gaq) subunit of heterotrimeric G proteins in the insulin signaling pathway leading to GLUT4 translocation. We inhibited endogenous Gaq function by single cell microinjection of anti-Gaq/11 antibody or RGS2 protein (a GAP protein for Gaq), followed by immunostaining to assess GLUT4 translocation in 3T3-L1 adipocytes. Gaq/11 antibody and RGS2 inhibited insulin-induced GLUT4 translocation by 60 or 75%, respectively, indicating that activated Gaq is important for insulin-induced glucose transport. We then assessed the effect of overexpressing wild-type Gaq (WT-Gaq) or a constitutively active Gaq mutant (Q209L-Gaq) by using an adenovirus expression vector. In the basal state, Q209L-Gaq expression stimulated 2-deoxy-d-glucose uptake and GLUT4 translocation to 70% of the maximal insulin effect. This effect of Q209L-Gaq was inhibited by wortmannin, suggesting that it is phosphatidylinositol 3-kinase (PI3-kinase) dependent. We further show that Q209L-Gaq stimulates PI3-kinase activity in p110a and p110? immunoprecipitates by 3- and 8-fold, respectively, whereas insulin stimulates this activity mostly in p110a by 10-fold. Nevertheless, only microinjection of anti-p110a (and not p110?) antibody inhibited both insulin- and Q209L-Gaq-induced GLUT4 translocation, suggesting that the metabolic effects induced by Q209L-Gaq are dependent on the p110a subunit of PI3-kinase. In summary, (i) Gaq appears to play a necessary role in insulin-stimulated glucose transport, (ii) Gaq action in the insulin signaling pathway is upstream of and dependent upon PI3-kinase, and (iii) Gaq can transmit signals from the insulin receptor to the p110a subunit of PI3-kinase, which leads to GLUT4 translocation.

We evaluated the role of the G alpha-q (Gαq) subunit of heterotrimeric G proteins in the insulin signaling pathway leading to GLUT4 translocation. We inhibited endogenous Gαq function by single cell microinjection of anti-Gαq/11 antibody or RGS2 protein (a GAP protein for Gαq), followed by immunostaining to assess GLUT4 translocation in 3T3-L1 adipocytes. Gαq/11 antibody and RGS2 inhibited insulin-induced GLUT4 translocation by 60 or 75%, respectively, indicating that activated Gαq is important for insulin-induced glucose transport. We then assessed the effect of overexpressing wild-type Gαq (WT-Gαq) or a constitutively active Gαq mutant (Q209L-Gαq) by using an adenovirus expression vector. In the basal state, Q209L-Gαq expression stimulated 2-deoxy-d-glucose uptake and GLUT4 translocation to 70% of the maximal insulin effect. This effect of Q209L-Gαq was inhibited by wortmannin, suggesting that it is phosphatidylinositol 3-kinase (PI3-kinase) dependent. We further show that Q209L-Gαq stimulates PI3-kinase activity in p110α and p110γ immunoprecipitates by 3- and 8-fold, respectively, whereas insulin stimulates this activity mostly in p110α by 10-fold. Nevertheless, only microinjection of anti-p110α (and not p110γ) antibody inhibited both insulin- and Q209L-Gαq-induced GLUT4 translocation, suggesting that the metabolic effects induced by Q209L-Gαq are dependent on the p110α subunit of PI3-kinase. In summary, (i) Gαq appears to play a necessary role in insulin-stimulated glucose transport, (ii) Gαq action in the insulin signaling pathway is upstream of and dependent upon PI3-kinase, and (iii) Gαq can transmit signals from the insulin receptor to the p110α subunit of PI3-kinase, which leads to GLUT4 translocation.

Thrombin treatment causes a dose-dependent rounding of 1321N1 astrocytoma cells. This cytoskeletal response is rapid, peaking 2 h after thrombin stimulation, and reverses by 50% after 24 h. The thrombin receptor peptide SFLLRNP also induces cell rounding, whereas other G protein-linked receptor agonists such as carbachol, lysophosphatidic acid, or bradykinin fail to do so. Results of studies using pharmacological inhibitors do not support a requirement for phosphatidylinositol 3-kinase, mitogen-activated protein kinase, or Ca2+mobilization in this response. Inhibition of protein kinase C or tyrosine kinase produces minimal blockade. Pertussis toxin treatment is also without effect. However, thrombin-induced rounding is fully blocked by the C3 toxin from Clostridium botulinum, which specifically ADP-ribosylates and inactivates the small G protein Rho. Thrombin also leads to a rapid, 2.4-fold increase in 32P incorporation into myosin light chain while carbachol does not. Myosin phosphorylation, like cell rounding is inhibited by inactivation of Rho with C3 exoenzyme, suggesting that myosin phosphorylation is necessary for this cytoskeletal response. This is supported by the observation that thrombin-induced rounding is also blocked by the myosin light chain kinase inhibitor KT5926. However, treatment with KT5926 fails to inhibit mitogenesis. Thus, cell rounding is not prerequisite to thrombin-induced DNA synthesis. We conclude that stimulation of the heterotrimeric G protein-coupled thrombin receptor in 1321N1 cells activates Rho-dependent pathways for both DNA synthesis and cell rounding, the cytoskeletal response being mediated in part through increases in myosin phosphorylation.

G protein-coupled receptor agonists initiate a cascade of signaling events in neonatal rat ventricular myocytes that culminates in changes in gene expression and cell growth characteristic of hypertrophy. These responses have been previously shown to be dependent on Gqand Ras. Rho, a member of the Ras superfamily of GTPases, regulates cytoskeletal rearrangement and transcriptional activation of the c-fosserum response element. Immunofluorescence staining of cardiomyocytes shows that Rho is present and predominantly cytosolic. We used two inhibitors of Rho function, dominant negative N19RhoA and Clostridium botulinumC3 transferase, to examine the possible requirement for Rho in α1-adrenergic receptor-mediated hypertrophy. Both inhibitors markedly attenuated atrial natriuretic factor (ANF) reporter gene expression induced by α1-adrenergic receptor stimulation with phenylephrine, and virtually abolished the increase in ANF reporter gene expression induced by GTPase-deficient Gαq. These effects were reproduced with the myosin light chain-2 reporter gene. Notably, N19RhoA did not block the ability of activated Ras to induce ANF and myosin light chain-2 reporter gene expression. Furthermore, activation of the extracellular signal-regulated kinase by phenylephrine was not blocked by N19RhoA, nor was it stimulated by an activated mutant of RhoA. Since activated RhoA and Ras produce a large synergistic effect on ANF-luciferase gene expression, we conclude that Rho functions in a pathway separate from but complementary to Ras. Our results provide direct evidence that Rho is an effector of Gαqsignaling and suggest for the first time that a low molecular weight GTPase other than Ras is involved in regulating myocardial cell growth and gene expression in response to heterotrimeric G protein-linked receptor activation.

Hormones that interact with seven‐ transmembrane spanning receptors, generally con‐sidered to be involved in acute signaling functions, also induce longer term effects on gene expression and cell growth. These genetic and proliferative effects can be induced by activation of receptors that signal through heterotrimeric GTP‐binding proteins (G‐proteins) of the Gq family, pertussis toxin‐sensitive Gi/Go proteins, Gs, or G12/G13. Numerous growth‐promoting G protein‐coupled receptors activate the low molecular weight G‐protein Ras and stimulate mitogen‐activated protein kinase. Recent data suggest that c‐Jun NH2‐terminal kinase is also activated, possibly through interaction with low molecular weight G‐proteins of the Rho family. Because G protein‐coupled receptors lack intrinsic tyrosine kinase activity, the mechanisms by which heterotrimeric G‐proteins couple to these kinase cascades remain to be elucidated. By analogy to growth factor receptors, G protein‐coupled receptors may access these kinase cascades through binding of adapter proteins or recruitment of cytosolic tyrosine kinases. It is likely that interactions between multiple signaling pathways are required for G protein‐coupled receptors to propagate signals to the nucleus.—Post, G. R., Brown, J. H. G protein‐coupled receptors and signaling pathways regulating growth responses. FASEBJ.10, 741‐749 (1996)

Exogenous purified rabbit skeletal-muscle glycogen synthase was used as a substrate for adipose-tissue phosphoprotein phosphatase from fed and starved rats in order to (1) compare the relationship between phosphate released from, and the kinetic changes imparted to, the substrate and (2) ascertain if decreases in adipose-tissue phosphatase activity account for the apparent decreased activation of endogenous glycogen synthase from starved as compared with fed rats. Muscle glycogen synthase was phosphorylated with [γ-32P]ATP and cyclic AMP-dependent protein kinase alone, or in combination with a cyclic AMP-independent protein kinase, to 1.7 or 3mol of phosphate per subunit. Adipose-tissue phosphatase activity determined with phosphorylated skeletal-muscle glycogen synthase as substrate was decreased by 35–60% as a consequence of starvation. This decrease in phosphatase activity had little effect on the capacity of adipose-tissue extracts to activate exogenous glycogen synthase (i.e. to increase the glucose 6-phosphate-independent enzyme activity), although there were marked differences in the activation profiles for the two exogenous substrates. Glycogen synthase phosphorylated to 1.7mol of phosphate per subunit was activated rapidly by adipose-tissue extracts from either fed or starved rats, and activation paralleled enzyme dephosphorylation. Glycogen synthase phosphorylated to 3mol of phosphate per subunit was activated more slowly and after a lag period, since release of the first mol of phosphate did not increase the glucose 6-phosphate-independent activity of the enzyme. These patterns of enzyme activation were similar to those observed for the endogenous adipose-tissue glycogen synthase(s): the glucose 6-phosphate-independent activity of the endogenous enzyme from fed rats increased rapidly during incubation, whereas that of starved rats, like that of the more highly phosphorylated muscle enzyme, increased only very slowly after a lag period. The observations made here suggest that (1) changes in glucose 6-phosphate-independent glycogen synthase activity are at best only a qualitative measure of phosphoprotein phosphatase activity and (2) the decrease in glycogen synthase phosphatase activity during starvation is not sufficient to explain the differential glycogen synthase activation in adipose tissue from fed and starved rats. However, alterations in the phosphorylation state of glycogen synthase combined with decreased activity of phosphoprotein phosphatase, both as a consequence of starvation, could explain the apparent markedly decreased enzyme activation.

Activation of stress-activated protein kinases, including the p38 and the c-Jun NH2-terminal kinases (JNK), have been associated with the onset of cardiac hypertrophy and cell death in response to hemodynamic overload and ischemia/reperfusion injury. Upon infection of cultured neonatal rat cardiac myocytes with recombinant adenoviral vectors expressing a wild type and a constitutively active mutant of MKK7 (or JNKK2), JNK was specifically activated without affecting other mitogen-activated protein kinases, including extracellular signal-regulated protein kinases and p38. Specific activation of the JNK pathway in cardiac myocytes induced characteristic features of hypertrophy, including an increase in cell size, elevated expression of atrial natriuretic factor, and induction of sarcomere organization. In contrast, co-activation of both JNK (by MKK7) and p38 (by MKK3 or MKK6) in cardiomyocytes led to an induction of cytopathic responses and suppression of hypertrophic responses. These data provide the first direct evidence that activation of JNK alone is sufficient to induce characteristic features of cardiac hypertrophy, thereby supporting an active role for the JNK pathway in the development of cardiac hypertrophy. The cytopathic response, as a result of co-activation of both JNK and p38, may contribute to the loss of contractile function and viability of cardiomyocytes following hemodynamic overload and cardiac ischemia/reperfusion injury.

The pertussis toxin (PTX) insensitive heterotrimeric G protein G12has been implicated in mitogenesis and transformation, but its direct effectors remain unknown. To define potential signaling pathways utilized by G12, we expressed an activated mutant of its α subunit, Gα12(Q229L), in HEK293 cells and examined its effects on Ras and mitogen-activated protein kinases (MAPKs). Transient expression of activated Gα12increased the percentage of Ras in the active, GTP-bound state, stimulated c-Jun NH2-terminal kinase (JNK) activity, and enhanced the transcriptional activity of c-Jun. Dominant negative Ras (N17Ras) inhibited Gα12-mediated JNK activation in NIH3T3 cells but failed to do so in HEK293 cells. In contrast, dominant negative Rac (N17Rac1) inhibited JNK activation by Gα12in HEK293 cells as well as three other cell lines. In 1321N1 cells, where thrombin stimulates G12-dependent mitogenesis, coexpression of N17Rac1 or a dominant negative mutant of MEKK1 (MEKKΔ(K432M)) inhibits c-Jun/AP-1 sensitive reporter gene expression stimulated by thrombin or Gα12. These data demonstrate that the α subunit of the heterotrimeric G protein G12, like tyrosine kinase growth factor receptors, activates Ras and recruits a signal transduction pathway involving the small GTP-binding protein Rac that leads to JNK activation.

A ribosyltransferase from C. botulinumtype D ADP-ribosylated a protein of 22 kDa (p22) in human astrocytoma (1321N1) cells. ADP-ribosylation of membrane-bound p22 was potentiated by 2 mM MgCl 2or guanine nucleotides but was much reduced in the presence of 10 mM Mg 2+plus GTPγS. p22 was immunoprecipitated by a monoclonal antibody 142-24E05 raised against a peptide sequence common to the rasgene family but not by other rasor G-protein antibodies. p22 was also ADP-ribosylated in Drosophilabut was not detected in Diclyostelium. These data suggest that the 22 kDa botulinum toxin substrate is a GTP-binding protein and a member of the rasprotein family.

A ribosyltransferase from C. botulinumtype D ADP‐ribosylated a protein of 22 kDa (p22) in human astrocytoma (1321N1) cells. ADP‐ribosylation of membrane‐bound p22 was potentiated by 2 mM MgCl2or guanine nucleotides but was much reduced in the presence of 10 mM Mg2+plus GTPγS. p22 was immunoprecipitated by a monoclonal antibody 142‐24E05 raised against a peptide sequence common to the rasgene family but not by other rasor G‐protein antibodies. p22 was also ADP‐ribosylated in Drosophilabut was not detected in Diclyostelium. These data suggest that the 22 kDa botulinum toxin substrate is a GTP‐binding protein and a member of the rasprotein family.

Cultured neonatal ventricular myocytes display features of myocardial hypertrophy including increased cell size, myofilament organization, and reexpression of the embryonic gene for atrial natriuretic factor (ANF). KN-93, an inhibitor of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase II), blocked the induction of these responses by the α1-adrenergic receptor agonist phenylephrine, whereas its inactive analog KN-92 did not. To directly determine whether CaM kinase II could regulate ANF gene expression, we transiently expressed each of three isoforms of CaM kinase II (α, δB, and δC) along with an ANF promoter/luciferase reporter gene. The δBisoform markedly increased luciferase gene expression, whereas comparable levels of the δCand α isoforms were ineffective. Expression of δB-CaM kinase II also potentiated phenylephrine-mediated ANF gene expression, and this effect was blocked by KN-93 but not by KN-92. The ability of δB-CaM kinase II to transactivate a truncated ANF promoter, containing a serum response element (SRE) required for phenylephrine-inducible gene expression, was lost when this SRE was mutated. The δBisoform of CaM kinase II has been shown to exhibit nuclear localization. Coexpression of the non-nuclear δCor α isoforms, which can form multimers with the δBisoform, prevented the nuclear localization of δB-CaM kinase II and also blocked its effects on ANF reporter gene and protein expression. In addition, a chimeric α-CaM kinase II which contains the nuclear localization signal of the δBisoform was able to induce ANF reporter gene expression, albeit to a lesser extent than δB-CaM kinase II. These data are the first to assign a function to the δBisoform of CaM kinase II and to link its nuclear localization to subsequent activation of cardiac gene expression.

The relationship between the effects of isoproterenol and prostaglandin E1(PGE1) on contractile state, cyclic AMP accumulation, and the activation states of protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37), phosphorylase kinase, glycogen synthase, and glycogen phosphorylase have been studied in the isolated perfused rat heart. Perfusion of hearts with isoproterenol (10 or 80 nM) caused enhancement of left ventricular dP/dt(P, pressure), increased intracellular cyclic AMP, increased the activation states of protein kinase, phosphorylase kinase, glycogen phosphorylase, and conversion of glycogen synthase to a less active form. PGE1(2 or 30 μM) increased cyclic AMP accumulation and activated protein kinase, but caused no detectable changes in dP/dtor the activation states of the protein kinase substrates involved in glycogen metabolism. Perfusion of hearts with either 10 nM isoproterenol or 30 μM PGE1produced comparable increases in cyclic AMP accumulation and protein kinase activity. Exposure of hearts to a combination of these agents caused additive effects on cyclic AMP content and protein kinase activity. However, values for phosphorylase kinase, glycogen phosphorylase, glycogen synthase, and dP/dtdid not differ from those observed in the presence of 10 nM isoproterenol alone. The failure of PGE1to stimulate phosphorylation of protein kinase substrates was not due to an increase in phosphorylase phosphatase activity. We conclude that an increase in intracellular cyclic AMP and the subsequent activation of protein kinase are insufficient to change either the activities of phosphorylase kinase, glycogen phosphorylase, and glycogen synthase or the inotropic state of heart muscle.

Thrombin stimulation of 1321N1 astrocytoma cells leads to Ras-dependent AP-1-mediated transcriptional activation and to DNA replication. In contrast to what has been observed in most cell systems, in 1321N1 cells these responses are pertussis toxin-insensitive. The pertussis toxin-insensitive G-protein G12has been implicated in cell growth and transformation in different cell systems. We have examined the potential role of this protein in AP-1-mediated transcriptional activation and DNA synthesis in 1321N1 cells. Transient expression of an activated (GTPase-deficient) mutant of Gα12increased AP-1-dependent gene expression. This response was inhibited by co-expression of a dominant negative Ala-15 Ras protein. To determine whether the pertussis toxin-insensitive G12protein is involved in the thrombin-stimulated DNA synthesis, an inhibitory antibody against the C-terminal sequence of Gα12subunit was microinjected into 1321N1 cells. Microinjection of the anti-Gα12resulted in a concentration-dependent inhibition of thrombin-stimulated DNA synthesis. In contrast, microinjection of nonimmune IgG or an antibody directed against the C terminus of Gαodid not reduce the mitogenic response to thrombin. Furthermore, microinjection of the anti-Gα12antibody had no effect on fibroblast growth factor-stimulated DNA synthesis. These results demonstrate a specific role for Gα12in the mitogenic response to thrombin in human astroglial cells.

Stimulation of α1-adrenergic receptors in neonatal ventricular cardiomyocytes induces hypertrophic changes including activation of the atrial natriuretic factor (ANF) gene. This receptor couples to Gqto activate phospholipase C (PLC) and protein kinase C, which have been implicated as mediators of the hypertrophic response. To directly determine whether receptor coupling to Gq/PLC is sufficient to induce ANF expression, we expressed wild-type and chimeric muscarinic cholinergic receptors (mAChRs) with altered G-protein coupling properties in cardiac myocytes and examined their ability to activate an ANF promoter/luciferase reporter gene. The cholinergic agonist carbachol failed to induce transcriptional activation of the ANF reporter gene through endogenous Gi-linked M2mAChRs or in cells transfected with M2mAChRs. In contrast, in cells transfected with M1mAChRs, which effectively couple to Gq/PLC, carbachol increased ANF reporter gene expression 10-fold and also increased ANF protein, as determined by immunofluorescence. Carbachol-mediated ANF gene expression was inhibited by the mAChR antagonist pirenzepine with a Kivalue characteristic of an M1mAChR. Studies using chimeric M1- and M2mAChRs demonstrated that the N-terminal 21 amino acids of the third intracellular loop of the M1mAChR were required for receptor coupling to ANF gene expression. This region, previously shown to specify receptor coupling to Gq/PLC, also conferred partial activity to a chimeric M2receptor. We further demonstrated that M1mAChR coupling to ANF gene expression was Ras-dependent since co-expression of dominant-interfering Ala-15 Ras inhibited M1mAChR-induced ANF expression by 60%. In contrast, ANF expression induced by the chimeric M2receptor was not blocked by dominant-interfering Ras. We suggest that receptor coupling to Gq/PLC is sufficient to induce ANF expression and that a Ras-dependent pathway contributes additional signals required for maximal M1mAChR-mediated ANF gene expression.

Recent studies indicate that antagonism of calcium channels may not be the only mechanism whereby drugs such as verapamil alter myocardial function. We have examined the effect of verapamil on the binding of [3H]prazosin (α1-adrenergic), [3H]quinuclidinyl benzilate (QNB, muscarinic) and [3H]dihydroalprenolol (DHA, β-adrenergic) to membranes prepared from rat heart. Verapamil competed for the binding of these radioligands in the following rank order: [3H]prazosin > [3H]QNB > [3H]DHA (K, for verapamil = 0.6 μM, 7 μM, and 72 μM, respectively). Verapamil (10 μM) competitively inhibited [3H]prazosin binding to rat ventricular membranes: the apparent dissociation constant (KD) of [3H]prazosin increased from 0.13 ± 0.02 to 1.5 ± 0.6 nM(SD) without change in maximal binding capacity (Bmax). The effect of verapamil on the affinity of [3H]prazosin was completely reversed by washing the membranes. The verapamil derivative D-600 also inhibited [3H]prazosin binding (KD= 1.1 μM). Verapamil (30 μM) competitively inhibited [3H]QNB binding in both atria and ventricles and increased the apparent KDof [3H]QNB fivefold (from 0.07 nMto 0.32 nM) without decreasing Bmax. Verapamil was a less potent inhibitor of [3H]DHA binding and its effect was noncompetitive: the KDfor DHA was unaltered by 100 μMverapamil, while the Bmaxdecreased severalfold. We conclude that verapamil, at concentrations clinically achieved in the myocardium (∼1 μM), competitively inhibits binding to α1-adrenergic and muscarinic receptors and that this inhibition may play a role in the effects of verapamil on the heart.

Cardiac myocyte survival is of central importance in the maintenance of the function of heart, as well as in the development of a variety of cardiac diseases. To understand the molecular mechanisms that govern this function, we characterized apoptosis in cardiac muscle cells following serum deprivation. Cardiotrophin 1 (CT-1), a potent cardiac survival factor (Sheng, Z., Pennica, D., Wood, W. I., and Chien, K. R. (1996) Development(Camb.) 122, 419-428), is capable of inhibiting apoptosis in cardiac myocytes. To explore the potential downstream pathways that might be responsible for this effect, we documented that CT-1 activated both signal transducer and activator of transcription 3 (STAT3)- and mitogen-activated protein (MAP) kinase-dependent pathways. The transfection of a MAP kinase kinase 1 (MEK1) dominant negative mutant cDNA into myocardial cells blocked the antiapoptotic effects of CT-1, indicating a requirement of the MAP kinase pathway for the survival effect of CT-1. A MEK-specific inhibitor (PD098059) (Dudley, D. T., Pang, L., Decker, S.-J., Bridges, A. J., and Saltiel, A. R. (1995) Proc. Natl. Acad. Sci. USA92, 7686-7689) is capable of blocking the activation of MAP kinase, as well as the survival effect of CT-1. In contrast, this inhibitor did not block the activation of STAT3, nor did it have any effect on the hypertrophic response elicited following stimulation of CT-1. Therefore, CT-1 promotes cardiac myocyte survival via the activation of an antiapoptotic signaling pathway that requires MAP kinases, whereas the hypertrophy induced by CT-1 may be mediated by alternative pathways, e.g.Janus kinase/STAT or MEK kinase/c-Jun NH2-terminal protein kinase.

In neonatal rat ventricular myocytes, stimulation of the α1-adrenergic receptor (α1-AdrR) activates a program of genetic and morphological changes characterized by transcriptional activation of the atrial natriuretic factor (ANF) gene and enlargement (hypertrophy) of the cells. The low molecular weight GTPase Ras has been established as an important regulator of hypertrophy bothin vitroand in vivo. Ras activates a kinase cascade involving Raf, the mitogen-activated protein kinase kinase (MEK), and the extracellular signal-regulated protein kinase (ERK). However, the extent of involvement of this pathway in regulating hypertrophic responses is controversial. We demonstrate here that both α1-AdrR stimulation and Ras can also activate the c-Jun NH2-terminal kinase (JNK) in cardiomyocytes. The α1-AdrR effect on JNK occurs through a pathway requiring Ras and MEK kinase (MEKK). A constitutively activated mutant of MEKK that preferentially activates JNK, stimulates ANF reporter gene expression, while a dominant negative MEKK mutant inhibits ANF expression induced by PE. Furthermore, JNK activity is increased in the ventricles of mice overexpressing oncogenic Ras, whereas ERK activity is not. These results suggest that the α1-AdrR mediates ANF gene expression through a Ras-MEKK-JNK pathway and that activation of this pathway is associated with in vitroand in vivohypertrophy.

p38 mitogen-activated protein (MAP) kinase activities were significantly increased in mouse hearts after chronic transverse aortic constriction, coincident with the onset of ventricular hypertrophy. Infection of cardiomyocytes with adenoviral vectors expressing upstream activators for the p38 kinases, activated mutants of MAP kinase kinase 3b(E) (MKK3bE) and MAP kinase kinase 6b(E) (MKK6bE), elicited characteristic hypertrophic responses, including an increase in cell size, enhanced sarcomeric organization, and elevated atrial natriuretic factor expression. Overexpression of the activated MKK3bE in cardiomyocytes also led to an increase in apoptosis. The hypertrophic response was enhanced by co-infection of an adenoviral vector expressing wild type p38β, and was suppressed by the p38β dominant negative mutant. In contrast, the MKK3bE-induced cell death was increased by co-infection of an adenovirus expressing wild type p38α, and was suppressed by the dominant negative p38α mutant. This provides the first evidence in any cell system for divergent physiological functions for different members of the p38 MAP kinase family. The direct involvement of p38 pathways in cardiac hypertrophy and apoptosis suggests a significant role for p38 signaling in the pathophysiology of heart failure.

A catecholamine-sensitive adenylate cyclase system is present in homogenates of both calf and rat retinas. Dopamine is a more potent activator of the bovine enzyme than is norepinephrine or epinephrine. Cyclic AMP concentrations in intact bovine retina are increased by dopamine, as well as by other catecholamines, and by depolarizing agents. Studies with adrenergic blocking agents suggest that the stimulation of retinal adenylate cyclase by catecholamines cannot be clearly defined in terms of the characteristics of alpha or beta adrenergic receptors. Bovine retina also contains a protein kinase that is stimulated 20-fold by cyclic AMP. It is proposed that dopamine is the major activator of a retinal adenylate cyclase, and that this activation is related to its role as a neurotransmitter.

Introduction and Hypothesis:Sterile inflammation is associated with cardiac remodeling in response to non-ischemic stress, but how it is initiated in the absence of cell death and how it is propagated are not known. We tested the hypothesis that activation of CaMKII? in cardiomyocytes in response to pressure overload initiates inflammatory responses leading to adverse remodeling.Methods and Results:Cardiomyocyte specific CaMKII? knockout (CKO) mice were subjected to transverse aortic constriction (TAC). CaMKII activity and NFkB activation were significantly increased in control fl/fl (CTL) but not in CKO hearts. Cardiac mRNA levels for pro-inflammatory chemokines and cytokines also increased and peaked at 3 days with 4 to 25 fold increases vs sham in MCP-1, MIP1?, CXCL1, CXCL2 and IL-6. These responses were significantly decreased in the CKO mice. Priming and activation of the NLRP3 inflammasome, assessed by measuring NLRP3 mRNA levels and caspase-1 activity, were increased by 3 days in isolated cardiomyocytes of CTL while attenuated in CKO. Macrophage accumulation by 14 days was attenuated in the CKO and NLRP3 inhibitor MCC950 treated mice. Cardiac fibrosis, increased by 28 days was also significantly attenuated in the CKO and MCP-1 KO mice. Left ventricular ejection fraction at day 42 was less impaired in the CKO, MCP-1 KO and KN-93 treated mice vs CTL (43 ? 6%, 45 ? 4%, 42 ? 2% vs 33 ? 5%). Cardiac-specific CaMKII deletion by AAV9-Cre injection prior to TAC ameliorated inflammasome activity, macrophage infiltration, fibrosis and heart failure. AAV9-Cre injection or pharmacological CaMKII inhibition by KN-93 2 weeks after TAC failed to mitigate inflammatory response and remodeling, suggesting that it is the early CaMKII signals after TAC indispensable for the progression to adverse cardiac remodeling.Conclusions:Our results suggest that activated CaMKII? in response to pressure overload triggers inflammatory gene expression and NLRP3 inflammasome cascade in cardiomyocytes. These responses are then propagated and provide signals for immune cell recruitment, fibrosis and cardiac dysfunction. Early inflammatory responses induced by cardiomyocyte CaMKII? could be a therapeutic target to prevent progression of non-ischemic heart failure.

In this Perspective, former and current editors of Molecular Pharmacology, together with the guest editors for this 50th Anniversary Issue, provide a historical overview of the journal since its founding in 1965. The substantial impact that Molecular Pharmacologyhas had on the field of pharmacology as well as on biomedical science is discussed, as is the broad scope of the journal. The authors conclude that, true to the original goals for the journal, Molecular Pharmacologytoday remains an outstanding venue for work that provides a mechanistic understanding of drugs, molecular probes, and their biologic targets.

Protecting mitochondria from oxidative stress–induced signals protects the heart.