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GRK5‐Dependent p53 Activity Controls Basal Cardiac Function and Survival
Author(s) -
Marzano Federica,
Rapacciuolo Antonio,
Koch Walter,
Cannavo Alessandro
Publication year - 2021
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2021.35.s1.02884
Subject(s) - pressure overload , medicine , cardiac function curve , context (archaeology) , g protein coupled receptor kinase , heart failure , biology , basal (medicine) , endocrinology , receptor , g protein coupled receptor , cardiac hypertrophy , paleontology , insulin
Background G protein‐coupled receptor (GPCR) kinase 5 (GRK5) is a multifunctional protein. When anchored to the plasma membrane, GRK5 exerts “canonical” effects regulating several GPCRs, including beta‐adrenergic receptors. Conversely, when in the cytosol or in the nucleus, GRK5 interacts with non‐GPCR proteins. In this context, our group has previously demonstrated that GRK5 activity into the nucleus can trigger cardiac maladaptive hypertrophy, leading to left ventricular (LV) dysfunction and early heart failure (HF). However, despite the plethora of effects attributed to GRK5, the role played by its catalytic activity in governing basal cardiac function and response to stress has not been investigated. Objective The aim of this study is to determine whether and how cardiac GRK5 catalytic activity impacts basal cardiac function and the heart's ability to respond to stress. Methods In vivo , we generated a new knock‐in mouse line with lysine (K) 215 within the catalytic domain replaced by arginine (R) in GRK5 (GRK5‐K215R mice), thus virtually abolishing GRK5's kinase activity. GRK5‐K215R mice were studied under normal basal conditions and after chronic pressure overload via transverse aortic constriction (TAC). In vitro, we used 3T3 fibroblasts and H9c2 cardiomyocytes to study signaling. Results Compared to age‐matched wild‐type (WT) littermates, 9‐week old, unstressed GRK5‐K215R mice exhibited larger LV chamber dimensions and increased heart rate. These mutant mice also had higher rates of myocardial apoptosis, but no signs of maladaptive hypertrophy or fibrosis. However, TAC induced a faster progression towards HF in GRK5‐K215R mice, along with manifestly worsened LV functional deterioration and chamber remodeling compared to WT control mice. Molecularly, GRK5‐K215R mice showed increased p53 expression levels with a consequent augmented apoptotic cell death rate. Thus, in order to better investigate the effects of GRK5 on apoptosis, we induced the K215R mutation in GRK5 within 3T3 cells. Of note, this GRK5 mutated protein resulted in a robust increase in p53 levels with consequent augmented apoptotic rate. Subsequently, we transduced H9c2 cells with adenoviruses (Ad), encoding for WT GRK5 (Ad‐GRK5) or a mutant GRK5 lacking its nuclear localization signal (Ad‐NLS). Importantly, when GRK5 is overexpressed outside the nucleus, there is significant protection from apoptosis, in terms of reduced p53 expression levels and cell death. Conversely, when we overexpressed GRK5‐∆NES, a mutant GRK5 without nuclear export signal, we found a significant increase of the apoptotic response, with increased p53 protein and mRNA levels. Conclusions Alterations in basal GRK5 catalytic activity impairs basal cardiac function and accelerates HF progression in mice with pressure overload due to repressed pro‐survival signaling pathways within the nucleus of myocytes.