ABSTRACT
Voltage-gated T-type Ca(2+) channels (T-channels) are normally expressed during embryonic development in ventricular myocytes but are undetectable in adult ventricular myocytes. Interestingly, T-channels are reexpressed in hypertrophied or failing hearts. It is unclear whether T-channels play a role in the pathogenesis of cardiomyopathy and what the mechanism might be. Here we show that the alpha(1H) voltage-gated T-type Ca(2+) channel (Ca(v)3.2) is involved in the pathogenesis of cardiac hypertrophy via the activation of calcineurin/nuclear factor of activated T cells (NFAT) pathway. Specifically, pressure overload-induced hypertrophy was severely suppressed in mice deficient for Ca(v)3.2 (Ca(v)3.2(-/-)) but not in mice deficient for Ca(v)3.1 (Ca(v)3.1(-/-)). Angiotensin II-induced cardiac hypertrophy was also suppressed in Ca(v)3.2(-/-) mice. Consistent with these findings, cultured neonatal myocytes isolated from Ca(v)3.2(-/-) mice fail to respond hypertrophic stimulation by treatment with angiotensin II. Together, these results demonstrate the importance of Ca(v)3.2 in the development of cardiac hypertrophy both in vitro and in vivo. To test whether Ca(v)3.2 mediates the hypertrophic response through the calcineurin/NFAT pathway, we generated Ca(v)3.2(-/-), NFAT-luciferase reporter mice and showed that NFAT-luciferase reporter activity failed to increase after pressure overload in the Ca(v)3.2(-/-)/NFAT-Luc mice. Our results provide strong genetic evidence that Ca(v)3.2 indeed plays a pivotal role in the induction of calcineurin/NFAT hypertrophic signaling and is crucial for the activation of pathological cardiac hypertrophy.
Subject(s)
Blood Pressure , Calcium Channels, T-Type/metabolism , Calcium Signaling , Cardiomegaly/metabolism , Hypertension/complications , Myocardium/metabolism , Angiotensin II , Animals , Animals, Newborn , Aorta/surgery , Calcineurin/metabolism , Calcium Channel Blockers/pharmacology , Calcium Channels, T-Type/deficiency , Calcium Channels, T-Type/drug effects , Calcium Channels, T-Type/genetics , Calcium Signaling/drug effects , Cardiomegaly/etiology , Cardiomegaly/physiopathology , Cardiomegaly/prevention & control , Cells, Cultured , Constriction , Disease Models, Animal , Ethosuximide/pharmacology , Genes, Reporter , Hypertension/etiology , Hypertension/metabolism , Hypertension/physiopathology , Male , Membrane Potentials , Mice , Mice, Inbred C57BL , Mice, Knockout , Myocardium/pathology , NFATC Transcription Factors/genetics , NFATC Transcription Factors/metabolism , Time FactorsABSTRACT
It was shown previously that a bacterially expressed mungbean defensin VrCRP exhibited both antifungal and insecticidal activities. To isolate this protein in a large quantity for its characterization, the defensin cDNA was expressed in Pichia pastoris and the recombinant defensin (rVrD1) was purified. The recombinant VrD1 was shown to inhibit the growth of fungi such as Fusarium oxysporum, Pyricularia oryza, Rhizoctonia solani, and Trichophyton rubrum and development of bruchid larva. The protein also inhibits in vitro protein synthesis. These biological activities are similar to that of the bacterially expressed defensin. Functional expression of VrD1 in Pichia pastoris provides a highly feasible system to study the structure-function relationship of VrD1 using the mutagenesis approach.
