Protein Kinase Novel 2 (PKN2) in heart

PROJECT SUMMARY The Protein Kinase Novel (PKNs) family of kinases, also known as Protein Kinase C-related kinases, belong to the PKC superfamily. A single nucleotide polymorphism at the Pkn2 locus is associated with greater risk for coronary artery disease/myocardial infarction, and elevated levels of PKN2 protein are associated with heart

disease, highlighting the importance of PKN2 in heart. Global deletion of Pkn2 in mouse results in lethality at embryonic day (E) 10 with cardiac defects. Conditional deletion of Pkn2, utilizing SM22α-Cre mice, results in partial lethality between E13.5 and weaning, with surviving mutants displaying abnormal cardiac phenotypes.

These observations strongly suggest that PKN2 plays a critical role in the developing heart, however, because SM22α-Cre is expressed not only in early developing cardiomyocytes, but also in smooth muscle, skeletal muscle, and myeloid and lymphoid immune cells, this previous study does not address the cardiomyocyte-

specific requirement. Intriguingly, a recent study showed that deletion of Pkn1 and Pkn2 in adult cardiomyocytes, utilizing αMHC-MerCreMer mice, did not affect basal cardiac function, but protected mice from pressure overload- and angiotensin II-induced cardiac hypertrophy and heart failure, suggesting that PKN

inactivation could be a unique therapeutic target for heart failure. The contradiction between the partial embryonic lethality of SM22α-Cre:Pkn2 knockout mice and protective effects observed in adult αMHC- MerCreMer:Pkn1/2 double knockout mice highlights a critical need to define potential roles of PKN2 in

cardiomyocytes at different developmental stages. To address this contradiction, we have generated novel Pkn2 cardiomyocyte-specific constitutive knockout (cKO) and Pkn2 tamoxifen-inducible cardiomyocyte-specific knockout (icKO) mouse models utilizing Xmlc2-Cre and Tnnt2-MerCreMer mouse lines, respectively. Upon

preliminary characterization, Pkn2 cKO mice displayed partial postnatal lethality and cardiac morphological defects as early as E12.5. Echocardiographic studies of surviving mutants revealed a dilated cardiomyopathy phenotype in Pkn2 cKO mutants at both 1 and 3 months of age. In contrast to published reports that loss of

Pkn2 in adult cardiomyocytes does not affect basal cardiac function, our preliminary observations suggest that Pkn2 deficiency in developing cardiomyocytes is detrimental. Taken together, the above evidence leads us to the hypothesis that PKN2 plays distinct roles at different stages of cardiomyocyte development through the

phosphoregulation of specific substrates. Accordingly, our specific aims are: 1. To elucidate the role of PKN2 in cardiomyocytes by analysis of cardiac and cardiomyocyte structure and function in Pkn2 cKO mice, and to identify endogenous substrates of PKN2 in cardiomyocytes by utilizing unbiased phosphoproteomics and a

chemical-genetics approach with an analog-sensitive PKN2 mutant, and 2. To determine the cardiomyocyte- specific requirement for PKN2 in postnatal development by analysis of Pkn2 icKO mice.