These results support a cardioprotective role for calcineurin mediated by NFAT-dependent induction of iNOS expression and co-operativity between calcineurin and Src.
AimsPKN1 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 resultsHearts 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.ConclusionLoss 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.
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