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

Abstract

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.

PKC and PKN in heart disease

“…Published results have demonstrated that PKN1 and/or PKN2 protein levels and/or activity are deregulated in different cohorts of human cardiac pathologies such as atrial fibrosis [91], hibernating myocardium [92] or dilated cardiomyopathy [93], act as cardiomyocyte survival factors after I/R injury [94][95][96] and can regulate ANF expression in vitro [96]. Moreover, patients suffering from 19p13.12 microdeletion syndrome, which results in the loss of the PKN1 gene, are characterized by congenital heart abnormalities and arrhythmias [97,98].…”

“…Loss of PKN1 in 19p13.12 microdeletion syndrome supports the importance of PKN1 in cardiovascular development. Global knockouts for PKN1 and the more homologous PKN3, as well as PKN1/3 double knockouts displayed no overt cardiovascular phenotype [94,100]. However, PKN1 knockouts did exhibit larger infarcts in response to MI, suggesting that endogenous PKN1 provides cardioprotection in vivo [94].…”

“…Global knockouts for PKN1 and the more homologous PKN3, as well as PKN1/3 double knockouts displayed no overt cardiovascular phenotype [94,100]. However, PKN1 knockouts did exhibit larger infarcts in response to MI, suggesting that endogenous PKN1 provides cardioprotection in vivo [94]. In addition, evidence indicates that PKN1 is not only involved in cardioprotection upon I/R, but it is also involved in mediating pro-hypertrophic signaling.…”

“…There have been less than 20 protein substrates identified for PKNs (Table 1), and for the majority of them physiological relevance of their phosphorylation by PKNs has not been yet confirmed [104,105]. However, it was shown that PKN can be involved in the regulation of important cardiac molecules and structures including atrial natriuretic factor (ANF) [106], αB-crystallin [107], calcium/calmodulin-dependent protein kinase (CamKIIδ) and phospholamban (PLN) [94]. Moreover, it is striking to note that among the PKN substrates are several protein kinases with known cardiac roles, including mammalian sterile 20-like kinase 1 and 2 (MST1, MST2) or PKCδ, as well as sarcomeric αactinin [104,105].…”

IGF-1C domain–modified hydrogel enhanced the efficacy of stem cells in the treatment of AMI

“…On the one hand, co-culture was incubated with DMEM supplemented with 10% fetal bovine serum at 37°C and 5% CO 2 for 12 h. Then, culture medium was changed to complete medium which contained 500 μM H 2 O 2 , to simulate the oxidative damage caused by myocardial infarction in vivo. On the other hand, NMVC ischemia model was performed as previously described [25]. Briefly, NRVCs were cultured in a serum-free DMEM in an anaerobic chamber with 95% N 2 /5% CO 2 for 8 h and reperfusion at 37°C for 1 h. Then, co-culture was maintained with DMEM supplemented with 10% fetal bovine serum at 37°C and 5% CO 2 for 12 h. Next, Calcein-AM/PI double staining was performed.…”