Abstract 13122: STK25 Regulates the Cardiac PKA Pathway in Human Cardiomyocytes

Abstract

Hypothesis: STK25 regulates cell survival, metabolism and cardiac function through phosphorylating PKA R1α and modulating PKA signaling pathway in human cardiomyocytes. Background: Dysregulation of cardiac kinases are related with many cardiovascular diseases including hypertrophy, remodeling and ischemia injury. STK25 is a stress response kinase that regulates cell survival through modulating several signaling pathways and is involved in the metabolism regulation. However, the direct substrates and mechanism of STK25 remain undefined. Methods: CRISPR-Cas9 was used to generate a homozygous knockout (KO) of STK25 in induced pluripotent stem cell (iPSC) which were then differentiated into cardiomyocytes (iPSC-CMs) and used for engineered human cardiac tissue generation. Role of STK25 in gene expression, cell proliferation/apoptosis, metabolism and cardiac function were characterized. STK25 was quantified in human left ventricular tissue from heart failure (HF) patients. Results: In iPSC-CMs, phospho-proteomic analysis identified PKA signaling as the most significantly upregulated pathway by STK25 deletion. PKA regulatory subunit I α (R1α) was identified as a downstream substrate of STK25 and validated by IP. Loss of STK25 resulted in elevated PKA kinase activity and decreased phosphorylation of PKA targets implicated in cardiac contractility and Ca2+ handling, such as cardiac myosin-binding protein C (cMyBPC), cardiac troponin (cTnT), ryanodine receptor-2 (RyR2) and L-type calcium channel (CaV1.2). Further mechanistic studies revealed that STK25 mediates PKA activity through phosphorylation of the R1α regulatory subunit at S77/83 sites, leading to inhibition of PKA kinase activity. In functional assays, STK25 modulated cell cycle progression, proliferation, hypoxia induced apoptosis, beta adrenergic response, and mitochondrial respiration in iPSC-CMs. We also detected significant increase in STK25 expression and PKA R1α phosphorylation in HF patients versus normal control, demonstrating STK25 altered regulation in heart diseases. Conclusions: STK25 kinase is a critical regulator of the PKA pathway in human cardiomyocytes modulating cell growth, metabolism and cardiac function and is aberrantly expressed in heart failure.