Mitochondrial Dysfunction in Embryonic and Neonatal Mouse Cardiomyocytes Suggests Diverse Roles of Cyclin Dependent Kinase Inhibitors in Myocardial Stress Response

Objectives: Embryonic and neonatal cardiomyocytes (CM) show higher stress resistance and proliferative capacity following injury compared with adult CM. We have previously shown these abilities in a mouse model in which 50% of CM exhibit mitochondrial dysfunction at midgestation (referred to as mosaic hearts). The latter is compensated until birth by hyperproliferation of healthy CM, while the surviving defective CM activated several stress responses. Along with signs of inflammatory signaling this resembles characteristics of cellular senescence (CS), a stress response with regenerative potential marked by cell cycle inhibition and an inflammatory secretory phenotype (referred to as SASP). The aim of this study was to investigate CS and SASP in mosaic hearts to explore their role in CM stress resistance and regeneration.

Methods: Markers of CS and inflammatory pathways were molecularly and histologically analyzed in embryonic and neonatal mosaic hearts.

Result: Neonatal mosaic hearts showed induction of &γ-H2A.X (fold change [FC] 2.23 ± 0.59, p < 0.01), a histone variant indicating activated DNA damage response (DDR). DDR can induce CS via p53, which was unaltered, however. Conversely p21CIP, a cyclin dependent kinase inhibitor (CDKI) characteristic for CS, exhibited increased protein expression in embryonic (fc: 2.87 ± 0.41, p < 0.01) and neonatal (fc: 3.71 ± 0.67, p < 0.01) mosaic hearts. Strikingly, this induction was not restricted to defective cells. p21CIP rather exhibited increased cytoplasmic staining in healthy compared with defective CM. By contrast, p16INK4a, another CS-marking CDKI, showed increased cytoplasmic staining in embryonic and neonatal defective CM. Notably, cell cycle inhibition by CDKIs is usually associated with nuclear localization. &β-galactosidase activity, which accumulates in senescent cells, was not detectable in neonatal mosaic hearts. A protein array of 111 cytokines, including typical SASP components like IL-1 and IL-6, and the SASP upstream regulators NF&κB and p38MAPK revealed no induction of inflammatory signaling. Accordingly, mRNA levels of IL-1&β, IL-6, and TNF&α showed no significant upregulation.

Conclusion: Whereas mitochondrial dysfunction can induce CS in adult cardiomyocytes, our data do not support such scenario in embryonic and neonatal mosaic hearts. In contrast, this study suggests a role of CDKIs beyond CS and cell cycle regulation, which might involve cytoprotective effects in stressed embryonic and neonatal cardiomyocytes.