Mitochondrial pyruvate carrier abundance mediates pathological cardiac hypertrophy.

Cardiomyocytes rely on metabolic substrates, not only to fuel cardiac output, but also for growth and remodelling during stress. Here we show that mitochondrial pyruvate carrier (MPC) abundance mediates pathological cardiac hypertrophy. MPC abundance was reduced in failing hypertrophic human hearts, as well as in the myocardium of mice induced to fail by angiotensin II or through transverse aortic constriction. Constitutive knockout of cardiomyocyte MPC1/2 in mice resulted in cardiac hypertrophy and reduced survival, while tamoxifen-induced cardiomyocyte-specific reduction of MPC1/2 to the attenuated levels observed during pressure overload was sufficient to induce hypertrophy with impaired cardiac function. Failing hearts from cardiomyocyte-restricted knockout mice displayed increased abundance of anabolic metabolites, including amino acids and pentose phosphate pathway intermediates and reducing cofactors. These hearts showed a concomitant decrease in carbon flux into mitochondrial tricarboxylic acid cycle intermediates, as corroborated by complementary 1,2-[13C2]glucose tracer studies. In contrast, inducible cardiomyocyte overexpression of MPC1/2 resulted in increased tricarboxylic acid cycle intermediates, and sustained carrier expression during transverse aortic constriction protected against cardiac hypertrophy and failure. Collectively, our findings demonstrate that loss of the MPC1/2 causally mediates adverse cardiac remodelling.

Loss of Protein Kinase Novel 1 (PKN1) is associated with mild systolic and diastolic contractile dysfunction, increased phospholamban Thr17 phosphorylation, and exacerbated ischaemia-reperfusion injury.

Aims: PKN1 is a stress-responsive protein kinase acting downstream of small GTP-binding proteins of the Rho/Rac family. The aim was to determine its role in endogenous cardioprotection. Methods and results: Hearts from PKN1 knockout (KO) or wild type (WT) littermate control mice were perfused in Langendorff mode and subjected to global ischaemia and reperfusion (I/R). Myocardial infarct size was doubled in PKN1 KO hearts compared to WT hearts. PKN1 was basally phosphorylated on the activation loop Thr778 PDK1 target site which was unchanged during I/R. However, phosphorylation of p42/p44-MAPK was decreased in KO hearts at baseline and during I/R. In cultured neonatal rat ventricular cardiomyocytes (NRVM) and NRVM transduced with kinase dead (KD) PKN1 K644R mutant subjected to simulated ischaemia/reperfusion (sI/R), PhosTag® gel analysis showed net dephosphorylation of PKN1 during sI and early R despite Thr778 phosphorylation. siRNA knockdown of PKN1 in NRVM significantly decreased cell survival and increased cell injury by sI/R which was reversed by WT- or KD-PKN1 expression. Confocal immunofluorescence analysis of PKN1 in NRVM showed increased localization to the sarcoplasmic reticulum (SR) during sI. GC-MS/MS and immunoblot analysis of PKN1 immunoprecipitates following sI/R confirmed interaction with CamKIIδ. Co-translocation of PKN1 and CamKIIδ to the SR/membrane fraction during sI correlated with phospholamban (PLB) Thr17 phosphorylation. siRNA knockdown of PKN1 in NRVM resulted in increased basal CamKIIδ activation and increased PLB Thr17 phosphorylation only during sI. In vivo PLB Thr17 phosphorylation, Sarco-Endoplasmic Reticulum Ca2+ ATPase (SERCA2) expression and Junctophilin-2 (Jph2) expression were also basally increased in PKN1 KO hearts. Furthermore, in vivo P-V loop analysis of the beat-to-beat relationship between rate of LV pressure development or relaxation and end diastolic P (EDP) showed mild but significant systolic and diastolic dysfunction with preserved ejection fraction in PKN1 KO hearts. Conclusion: Loss of PKN1 in vivo significantly reduces endogenous cardioprotection and increases myocardial infarct size following I/R injury. Cardioprotection by PKN1 is associated with reduced CamKIIδ-dependent PLB Thr17 phosphorylation at the SR and therefore may stabilize the coupling of SR Ca2+ handling and contractile function, independent of its kinase activity.

ROS-dependent activation of RhoA/Rho-kinase in pulmonary artery: Role of Src-family kinases and ARHGEF1.

The role of reactive oxygen species (ROS) in smooth muscle contraction is poorly understood. We hypothesised that G-protein coupled receptor (GPCR) activation and hypoxia induce Rho-kinase activity and contraction in rat intra-pulmonary artery (IPA) via stimulation of ROS production and subsequent Src-family kinase (SrcFK) activation. The T-type prostanoid receptor agonist U46619 induced ROS production in pulmonary artery smooth muscle cells (PASMC). U46619 also induced c-Src cysteine oxidation, SrcFK auto-phosphorylation, MYPT-1 and MLC20 phosphorylation and contraction in IPA, and all these responses were inhibited by antioxidants (ebselen, Tempol). Contraction and SrcFK/MYPT-1/MLC20 phosphorylations were also inhibited by combined superoxide dismutase and catalase, or by the SrcFK antagonist PP2, while contraction and MYPT-1/MLC20 phosphorylations were inhibited by the Rho guanine nucleotide exchange factor (RhoGEF) inhibitor Y16. H2O2 and the superoxide-generating quinoledione LY83583 both induced c-Src oxidation, SrcFK auto-phosphorylation and contraction in IPA. LY83583 and H2O2-induced contractions were inhibited by PP2, while LY83583-induced contraction was also inhibited by antioxidants and Y16. SrcFK auto-phosphorylation and MYPT-1/MLC20 phosphorylation was also induced by hypoxia in IPA and this was blocked by mitochondrial inhibitors rotenone and myxothiazol. In live PASMC, sub-cellular translocation of RhoA and the RhoGEF ARHGEF1 was triggered by both U46619 and LY83583 and this translocation was blocked by antioxidants and PP2. RhoA translocation was also inhibited by an ARHGEF1 siRNA. U46619 enhanced ROS-dependent co-immunoprecipitation of ARHGEF1 with c-Src. Our results demonstrate a link between GPCR-induced cytosolic ROS or hypoxia-induced mitochondrial ROS and SrcFK activity, Rho-kinase activity and contraction. ROS and SrcFK activate RhoA via ARHGEF1.

Expression and regulation of type 2A protein phosphatases and alpha4 signalling in cardiac health and hypertrophy.

Cardiac physiology and hypertrophy are regulated by the phosphorylation status of many proteins, which is partly controlled by a poorly defined type 2A protein phosphatase-alpha4 intracellular signalling axis. Quantitative PCR analysis revealed that mRNA levels of the type 2A catalytic subunits were differentially expressed in H9c2 cardiomyocytes (PP2ACß > PP2ACα > PP4C > PP6C), NRVM (PP2ACß > PP2ACα = PP4C = PP6C), and adult rat ventricular myocytes (PP2ACα > PP2ACß > PP6C > PP4C). Western analysis confirmed that all type 2A catalytic subunits were expressed in H9c2 cardiomyocytes; however, PP4C protein was absent in adult myocytes and only detectable following 26S proteasome inhibition. Short-term knockdown of alpha4 protein expression attenuated expression of all type 2A catalytic subunits. Pressure overload-induced left ventricular (LV) hypertrophy was associated with an increase in both PP2AC and alpha4 protein expression. Although PP6C expression was unchanged, expression of PP6C regulatory subunits (1) Sit4-associated protein 1 (SAP1) and (2) ankyrin repeat domain (ANKRD) 28 and 44 proteins was elevated, whereas SAP2 expression was reduced in hypertrophied LV tissue. Co-immunoprecipitation studies demonstrated that the interaction between alpha4 and PP2AC or PP6C subunits was either unchanged or reduced in hypertrophied LV tissue, respectively. Phosphorylation status of phospholemman (Ser63 and Ser68) was significantly increased by knockdown of PP2ACα, PP2ACß, or PP4C protein expression. DNA damage assessed by histone H2A.X phosphorylation (γH2A.X) in hypertrophied tissue remained unchanged. However, exposure of cardiomyocytes to H2O2 increased levels of γH2A.X which was unaffected by knockdown of PP6C expression, but was abolished by the short-term knockdown of alpha4 expression. This study illustrates the significance and altered activity of the type 2A protein phosphatase-alpha4 complex in healthy and hypertrophied myocardium.

Protein kinase Cε-calcineurin cosignaling downstream of toll-like receptor 4 downregulates fibrosis and induces wound healing gene expression in cardiac myofibroblasts.

The pathways which regulate resolution of inflammation and contribute to positive remodeling of the myocardium following injury are poorly understood. Here we show that protein kinase C epsilon (PKCε) cooperates with the phosphatase calcineurin (CN) to potentiate induction of cardioprotective gene expression while suppressing expression of fibrosis markers. This was achieved by detailed analysis of the regulation of cyclooxygenase 2 (COX-2) expression as a marker gene and by using gene expression profiling to identify genes regulated by coexpression of CN-Aα/PKCε in adult rat cardiac myofibroblasts (ARVFs) on a larger scale. GeneChip analysis of CN-Aα/PKCε-coexpressing ARVFs showed that COX-2 provides a signature for wound healing and is associated with downregulation of fibrosis markers, including connective tissue growth factor (CTGF), fibronectin, and collagens Col1a1, Col3a1, Col6a3, Col11a1, Col12a1, and Col14a1, with concomitant upregulation of cardioprotection markers, including COX-2 itself, lipocalin 2 (LCN2), tissue inhibitor of metalloproteinase 1 (TIMP-1), interleukin-6 (IL-6), and inducible nitric oxide synthase (iNOS). In primary rat cardiomyocyte cultures Toll-like receptor 4 (TLR4) agonist- or PKCε/CN-dependent COX-2 induction occurred in coresident fibroblasts and was blocked by selective inhibition of CN or PKC α/ε or elimination of fibroblasts. Furthermore, ectopic expression of PKCε and CN in ARVFs showed that the effects on COX-2 expression are mediated by specific NFAT sites within the COX-2 promoter as confirmed by site-directed mutagenesis and chromatin immunoprecipitation (ChIP). Therefore, PKCε may negatively regulate adverse myocardial remodeling by cooperating with CN to downregulate fibrosis and induce transcription of cardioprotective wound healing genes, including COX-2.

Human flavin-containing monooxygenase 2.1 catalyzes oxygenation of the antitubercular drugs thiacetazone and ethionamide.

The second-line antitubercular drugs thiacetazone (TAZ) and ethionamide (ETA) are bioactivated by the mycobacterial enzyme EtaA. We report here that human flavin-containing monooxygenase 2.1 (FMO2.1), which is expressed predominantly in the lung, catalyzes oxygenation of TAZ. The metabolites generated, the sulfenic acid, sulfinic acid, and carbodiimide derivatives, are the same as those produced by EtaA and human FMO1 and FMO3. Two of the metabolites, the sulfenic acid and carbodiimide, are known to be harmful to mammalian cells. FMO2.1 also catalyzes oxygenation of ETA, producing the S-oxide. We have developed a novel spectrophotometric assay for TAZ oxygenation. The assay was used to determine kinetic parameters for TAZ oxygenation catalyzed by human FMO1, FMO2.1, and FMO3 and by EtaA. Although the K(M) values for the four enzyme-catalyzed reactions are similar, k(cat) and, consequently, k(cat)/K(M) (the specificity constant) for FMO2.1-catalyzed TAZ oxygenation are much higher than those of FMO1, FMO3, or EtaA. This indicates that FMO2.1 is more effective in catalyzing TAZ oxygenation than are the other three enzymes and thus is likely to contribute substantially to the metabolism of TAZ, decreasing the availability of the prodrug to mycobacteria and producing toxic metabolites. Because of a genetic polymorphism, Europeans and Asians lack FMO2.1. However, in sub-Saharan Africa, a region in which tuberculosis is a major health problem, a substantial proportion of individuals express FMO2.1. Thus, our results may explain some of the observed interindividual differences in response to TAZ and ETA and have implications for the treatment of tuberculosis in sub-Saharan Africa.