Regulation of the Inositol 1,4,5-Trisphosphate growth signaling pathway in cardiomyocytes

Hypertrophy is an adaptive response of the heart to increased hemodynamic load with both pressure and volume overload resulting in increased cardiomyocyte size in the absence of DNA replication. Activation of Gq/11 proteins via stimulation of a1-adrenergic receptors is a well known initiator of cardiac hypertrophy. Upon receptor activation of Gq, phospholipase Cβ hydrolyzes the membrane phospholipid phophatidylinositol 4,5-bisphosphate to yield inositol 1,4,5-triphosphate (Ins(1,4,5)P3) and the PKC activator sn-1,2-diacylglycerol (DAG). Though the role of PKC activation in cardiac hypertrophy has been well studied, not all hypertrophic response can be explained by PKC signalling events, thus implicating Ins(1,4,5)P3 as playing a role in hypertrophic growth. The aim of this study was to elucidate the role and requirement of PLCβ and Ins(1,4,5)P3 in mediating the hypertrophic phenotype. RNA interference was used to reduce PLCβ1 or PLCβ3 protein expression levels in cultured neonatal rat ventricular myocytes (NRVM). Following testing of over 30 unique siRNA delivered as both dsRNA and shRNA via adenoviral transduction, it was found that though PLCβ mRNA is targeted by siRNA for degradation, no reduction in PLCβ protein levels was observed due to the short lived nature of the NRVM culture and the stability of the PLCβ protein. An alternative approach utilising an Ins(1,4,5)P3-specific 5’-phosphatase to deplete Ins(1,4,5)P3 levels revealed a tight regulation of intracellular Ins(1,4,5)P3 in NRVM. Though initial reduction of Ins(1,4,5)P3 levels was achieved by viral delivery of a 5’-phosphatase, a compensatory mechanism was observed where Ins(1,4,5)P3 returned to resting levels within 20-minutes of α1-adrenergic receptor stimulation coupled with an increase in all other InsPs measured. Taken together, the data described here show that the PLCβ/Ins(1,4,5)P3 hypertrophic signalling pathway in NRVM is tightly regulated and any attempts at manipulation of its constituents is compensated for in NRVM.