Introduction:
G protein-coupled receptor (GPCR) kinase 5 (GRK5) is a multifunctional protein and depending on its localization within the cell, it has been shown to elicit either protective or deleterious effects. For instance in the heart, when anchored to the plasma membrane, this kinase can regulate specific GPCRs via canonical phosphorylation that can confer cardioprotection. However, when it accumulates in the nucleus its non-canonical activity can drive pathological hypertrophic gene transcription. Interestingly, the latter effects may not be kinase-dependent.
Hypothesis:
The role played by GRK5’s catalytic activity in the heart has not been fully elucidated and for that reason we sought to assess the in vivo consequences of inactivating the catalytic site of GRK5 with an initial focus at examining the basal cardiac phenotype and response to stress.
Methods:
We used CRISPR/Cas9 technology to generate a novel knock-in mouse model, with the ATP binding lysine (K) 215 in the catalytic cleft replaced by arginine (R) (GRK5-K215R) resulting in mice devoid of any GRK5 catalytic activity. We studies baseline cardiac function in these mutant mice compared to wild-type (WT) littermates and then stressed them via transverse aortic constriction (TAC). In vitro, we used H9c2 cardiomyocytes and various GRK5 mutants for mechanistic studies.
Results:
Compared to age-matched WT littermates, GRK5-K215R mice revealed marked and early (9 weeks) deterioration of cardiac function, with augmented apoptosis and fibrosis basally. Importantly, mutant knock-in mice displayed increased p53 gene expression (both at mRNA and protein levels). Moreover, TAC induced increased dysfunction and fibrosis in GRK5-K215R mice compared to WT. Mechanistically, we transduced H9c2 cells with adenoviruses (Ad), encoding for WT GRK5 (Ad-GRK5) or a mutant GRK5 lacking its nuclear localization signal (Ad-NLS) and when GRK5 was localized only outside the nucleus, there was a significant protection against apoptosis, with reduced p53 protein and mRNA levels. Conversely, when we overexpressed a mutant GRK5 without nuclear export signal (GRK5-ΔNES) to trap GRK5 within the nucleus, we found a significant increase in apoptosis, with high p53 protein expression levels.
Conclusions:
Inactivating GRK5’s catalytic activity impairs its nuclear regulation of p53. This can result in higher levels of p53 mRNA and protein resulting in higher rates of apoptosis in the heart leading to significant cardiac dysfunction and an intolerance to stress.
A novel LIM protein Cal promotes cardiac differentiation by association with CSX/NKX2-5