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P42 Kinase independent cardioprotection by protein kinase novel 1 (PKN1) and SR remodelling
  1. AA Francois1,
  2. JE Clark1,
  3. PJ Parker2,
  4. MS Marber1,
  5. RJ Heads1
  1. 1Cardiovascular Division, Faculty of Life Sciences and Medicine, King’s College London, UK
  2. 2Division of Cancer Studies, Faculty of Life Sciences and Medicine, King’s College London and Protein Phosphorylation Laboratory, Francis Crick Institute, London, UK

Abstract

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. Hearts from PKN1 knockout (KO) or wild type (WT) littermate control mice were perfused in Langendorff mode and subjected to global ischemia and reperfusion (I/R). Myocardial infarct size was doubled in PKN1 KO hearts compared to WT. siRNA knockdown of PKN1 in cultured neonatal rat ventricular cardiomyocytes (NRVM) significantly decreased cell survival and increased cell injury induced by simulated ischaemia/reperfusion (sI/R) which was reversed in NRVM transduced with a wild type (WT) or kinase dead (KD) PKN1 K644R mutant. PKN1 was basally phosphorylated on the activation loop Thr774 PDK1 target site and PhosTag® gel analysis showed net dephosphorylation of PKN1 during sI and early R despite Thr774 phosphorylation. Immunoprecipitation of PKN1 and GC-MS/MS following sI/R confirmed interaction with CamKIIδ. Confocal immunofluorescence analysis showed co-localisation of PKN1 and CamKIIδ at the SR/membrane during sI which correlated with phospholamban (PLB) Thr17 phosphorylation. siRNA knockdown of PKN1 resulted in increased CamKIIδ activation and increased PLB Thr17 phosphorylation during sI. PLB Thr17 phosphorylation and Sarco-Endoplasmic Reticulum Ca2+ ATPase (SERCA2) expression were also increased in PKN1 KO hearts. Therefore, loss of PKN1 in vivo significantly reduces endogenous cardioprotection and increases myocardial infarct size associated with remodelling of SR calcium handling proteins. PKN1-dependent cardioprotection is independent of its kinase activity.

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