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1.
Cardiovasc Res ; 114(1): 138-157, 2018 01 01.
Article in English | MEDLINE | ID: mdl-29045568

ABSTRACT

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.


Subject(s)
Calcium-Binding Proteins/metabolism , Myocardial Contraction , Myocardial Infarction/enzymology , Myocardial Reperfusion Injury/enzymology , Myocardium/metabolism , Protein Kinase C/deficiency , Ventricular Dysfunction, Left/enzymology , Ventricular Function, Left , Animals , Calcium Signaling , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Cells, Cultured , Diastole , Disease Models, Animal , Humans , Membrane Proteins/metabolism , Mice, Knockout , Myocardial Infarction/genetics , Myocardial Infarction/pathology , Myocardial Infarction/physiopathology , Myocardial Reperfusion Injury/genetics , Myocardial Reperfusion Injury/pathology , Myocardial Reperfusion Injury/physiopathology , Myocardium/pathology , Phosphorylation , Protein Kinase C/genetics , Rats, Sprague-Dawley , Sarcoplasmic Reticulum/enzymology , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Severity of Illness Index , Stroke Volume , Systole , Ventricular Dysfunction, Left/genetics , Ventricular Dysfunction, Left/physiopathology , Ventricular Pressure
2.
Cardiovasc Res ; 71(4): 672-83, 2006 Sep 01.
Article in English | MEDLINE | ID: mdl-16828070

ABSTRACT

OBJECTIVE: To determine the role of calcineurin and Src tyrosine kinase in the regulation of inducible nitric oxide synthase (iNOS) expression and protection in cardiomyocytes. METHODS: iNOS expression was studied in isolated neonatal rat ventricular myocyte cultures in response to bacterial lipopolysaccharide (LPS) or following transfection with constitutively active calcineurin or Src and in hearts isolated from wild-type or calcineruin Abeta knockout mice. Cell injury in response to simulated ischemia-reperfusion was studied following overexpression of active calcineurin. Regulation of the iNOS gene promoter by calcineurin was studied using promoter-luciferase reporter and chromatin immunoprecipitation assays. RESULTS: Overexpression of constitutively active Src co-operated with [Ca2+]c elevation to induce iNOS expression, and LPS-induced iNOS expression was abrogated by pharmacological inhibition of calcineurin or tyrosine kinase. LPS also induced tyrosine kinase-dependent but calcineurin-independent phosphorylation of Src Tyr418. LPS induced myocardial iNOS expression in wild-type but not calcineurin Abeta knockout mice. Overexpression of constitutively active calcinuerin in isolated cardiomyocytes caused dephosphorylation and nuclear accumulation of the c1 isoform of nuclear factor of activated T-cells (NFATc1), induced strong iNOS expression, and induced NOS-dependent protection against simulated ischemia-reperfusion prior to cardiomyocyte hypertrophy. Co-transfection of a mouse iNOS promoter-luciferase reporter in combination with active calcineurin and wild-type or dominant negative Src confirmed that constitutive activation of calcineurin was sufficient for transactivation. Chromatin immunoprecipitation confirmed calcineurin-dependent in vivo binding of NFATc1 to consensus sites within the iNOS promoter. CONCLUSIONS: These results support a cardioprotective role for calcineurin mediated by NFAT-dependent induction of iNOS expression and co-operativity between calcineurin and Src.


Subject(s)
Calcineurin/metabolism , Myocardial Ischemia/metabolism , Myocytes, Cardiac/metabolism , NFATC Transcription Factors/metabolism , Nitric Oxide Synthase Type II/metabolism , Up-Regulation , Animals , Calcineurin/genetics , Calcium/metabolism , Cells, Cultured , Immunoprecipitation , Ionomycin/pharmacology , Ionophores/pharmacology , Male , Mice , Mice, Knockout , Microscopy, Fluorescence , Rats , Signal Transduction , Transfection/methods , src-Family Kinases/metabolism
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