Abstract 12243: Inhibition of the Plasma Membrane Calcium Pump 4 by a Novel Substance from a Chemical Screen Reduces Pressure-Overload Induced Hypertrophy
In contrast to the classical premise that the sarcolemmal calcium pump (PMCA=Plasma Membrane Ca2+/Calmodulin dependent ATPase) has a minor role in diastolic Ca2+ removal and hence little function, we have recently shown that PMCA4 is major player in cardiac signaling through its interaction and regulation of neuronal nitric oxide synthase (nNOS) and calcineurin. Further studies revealed that PMCA4 is involved in regulating cardiac hypertrophy, prompting us to hypothesise that specific pharmacological inhibition of PMCA4 will modify the development of cardiac hypertrophy. Using an in vitro assay for PMCA4 ATPase activity, we screened a medically optimised chemical library and successfully identified a novel specific PMCA4 inhibitor (AP4) with an IC50 of 150 nM. In isolated rat cardiomyocytes, treatment with AP4 at 90% inhibitory concentration (1uM) significantly reduced phenylephrine-induced hypertrophy by ∼40% (cell size) as well as leucine incorporation and BNP expression. Injection of AP4 in mice (5 mg/kg body weight/day ip.) significantly reduced cardiac hypertrophy following transverse aortic constriction (TAC) for 2 weeks (heart weight/tibia length (mg/mm): sham, 5.5±0.3, TAC+vehicle, 8.7±0.2, TAC+AP4, 7.1±0.5, n=10 in each group, p<0.01). AP4 treated mice displayed lower cardiac ANP and BNP expression and reduced cardiomyocyte cross-sectional area following TAC. Moreover, AP4 reversed pre-established TAC-induced hypertrophy (16% decrease of left ventricular mass/body weight in AP4 treated mice vs 11% increase in controls, P<0.05). To detect PMCA4 activity in situ we cloned a novel fluorescent Ca2+ sensor (GCaMP2) attached to the N-terminus of PMCA4. This sensor showed that AP4 decreased PMCA4 activity in the cellular context; a control sensor for cytoplasmic calcium transients showed no change with AP4. Furthermore, AP4 inhibited the NFAT pathway as shown by a. NFAT-luciferase assay; b. expression of the bona fide calcineurin target RCAN 1.4; and c. NFAT phosphorylation levels. Overall, our results show that specific inhibition of PMCA4 prevents and reverses pressure-overload hypertrophy, likely through interference with calcineurin signaling making it a potential target for the treatment of cardiac hypertrophy.
- © 2010 by American Heart Association, Inc.