Cardiac cverexpression of RKIP – an endogenous inhibitor of GRK-2 – causes long-term hypercontractility and is protective in heart failure

Kristina Lorenz, Sabrina Denzinger, Katrin Kahlert, Evelyn Schmid, Katharina Deiss, Joachim Schmitt, Martin J. Lohse. Institute of Pharmacology and Toxicology, Versbacher Str.9, 97078 Würzburg, Germany

Background. G-protein coupled receptor kinases (GRKs) phosphorylate and thereby desensitize activated G-protein coupled receptors (GPCRs) and are thus important negative feedback regulators of GPCR signalling. Since GRK-2 expression is strongly up-regulated in human heart failure, it was proposed that inhibition of GRK-2 attenuates receptor desensitisation and may thereby normalise β-adrenergic receptor function in heart failure patients. Previously, we have characterised the Raf-1 kinase inhibitor protein (RKIP) as an endogenous GRK-2 inhibitor (Lorenz et al., Nature 2003). It was shown that RKIP mediated GRK-2 inhibition results in increased receptor signalling and in increased contractility of cardiomyocytes in vitro.

Objective. The aim of this study was to investigate the physiological role of RKIP in the heart and to evaluate whether chronic GRK-2 inhibition via RKIP may improve cardiac contractility.

Methods and Results. We generated mice with cardiac overexpression of wild-type RKIP (TgRKIP). GRK-2 activity in heart lysates of these transgenic mice was significantly reduced as assessed by an receptor phosphorylation assay in vitro. In line with these results, downstream targets of β-adrenergic receptors such as phospholamban (PLN) and troponin I (TnI), important players in the cardiomyocyte contraction-relaxation cycle, showed increased phosphorylation in TgRKIP mice compared to wild-type mice indicating increased receptor activity. Cardiac function and morphology of TgRKIP mice were characterised by echocardiography, left ventricular hemodynamics with and without dobutamine stress, histological analyses and apoptosis assays. These studies revealed significantly increased fractional shortening (P < 0.01) as well as increased speeds of contraction (P < 0.01) and relaxation (P < 0.01) of left ventricles in 8-week-old TgRKIP mice compared to control mice. Interestingly, this hypercontractile phenotype of the TgRKIP mice was still present at the age of 12-14 month. While heart to body weight ratios were indistinguishable from wild-type mice, TgRKIP mice developed much less interstitual fibrosis and apoptosis than wild-type controls until the age of 12-14 month. To assess the effects of RKIP overexpression on heart failure, we analysed heart function and structure of RKIP transgenic mice (backcrossed into the C57BL/6 background). While left ventricular hypertrophy was increased to similar extents in wild-type and RKIP transgenic mice, RKIP mice did neither develop dilatation of the left ventricle nor a decrease in fractional shortening. In contrast to wild-type mice, the expression of the heart failure markers BNP and ANP was not up-regulated in banded TgRKIP mice and also fibrosis and apoptosis was not increased after 3 weeks of TAC.

Summary. In summary, we found that RKIP triggers cardiac hypercontractility without any harmful long-term effects on the aging or pressure overloaded heart. Cardiac overexpression of RKIP prevented left ventricular dilatation and loss of contractile function in a mouse model of pressure overload induced heart failure. RKIP may represent a new, promising therapeutic principle to improve cardiac performance in heart failure patients not only for short-term but also for long-term treatment.