Abstract Body: Introduction: A major pathomechanism triggered by GRK2 in failing hearts is the phosphorylation and desensitization of beta-adrenoceptors. But non-receptor substrates of GRK2 could also contribute to cardiac dysfunction caused by GRK2.
Research Question: This study explored whether phosphorylation of non-receptor substrates by GRK2 could contribute to GRK2-mediated cardiac degeneration.
Aims: We focused on SRSF1 (serine/arginine-rich splicing factor 1) which we identified as a non-receptor substrate of GRK2.
Methods: As a model of heart failure, we used non-transgenic B6 mice with symptoms of heart failure induced by chronic pressure overload imposed by abdominal aortic constriction (AAC). GRK2-interacting proteins in failing hearts were identified by immunoaffinity-enrichment followed by nano-LC-ESI-MS/MS analysis. In vitro phosphorylation assays were performed with purified recombinant proteins. Cardiac effects of SRSF1 and GRK2 were analyzed in transgenic mice with myocardium-specific expression of SRSF1, GRK2, or the GRK2-inhibitory mutant, GRK2-K220R. Srsf1 was down-regulated in Tg-GRK2 mice by lentiviral transduction of miSrsf1.
Results: Immunoaffinity enrichment and nano-LC-ESI-MS/MS analysis identified SRSF1 as a GRK2-interacting protein in hearts from mice with AAC-induced heart failure. Increased SRSF1 caused cardiac dysfunction in vivo as documented by a significantly reduced left ventricular ejection fraction (LVEF) of 28.7 ± 2.3 % in Tg-SRSF1 mice compared to the LVEF of 51.0 ± 1.8 % in non-transgenic B6 controls (mean ± s.d.; n = 6; p < 0.0001; unpaired, two-tailed t-test). In vitro phosphorylation studies found that GRK2 promoted the activating phosphorylation of SRSF1 in the arginine-serine-rich RS domain of SRSF1. In vivo, phospho-SRSF1 contents were also increased in Tg-GRK2 mice. Endogenous Srsf1 contributed to GRK2-induced cardiac dysfunction because down-regulation of Srsf1 by miSrsf1 improved the cardiac function of Tg-GRK2 mice. In addition, mice with AAC-induced heart failure had increased cardiac contents of SRSF1, which were lowered by transgenic expression of GRK2-inhibitory GRK2-K220R.
Conclusions: SRSF1 is a non-receptor substrate of GRK2 and aggravates GRK2-induced cardiac damage.