Exploring Protein Kinase G Inhibition of Canonical Transient Receptor Potential 6 Channels in the Heart

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Date
2015-03-24
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Johns Hopkins University
Abstract
Maladaptive ventricular remodeling often involves pathologic cardiac hypertrophy and at the molecular level the activation of multiple pathways, including G-protein coupled receptor (GPCR) activation. Stimulation of GPCRs is implicated in the activation of calcium entry through the plasma membrane via canonical transient receptor potential (TRPC) channels. TRPC6, a subtype of TRPC channels, is linked to hypertrophic signaling through the dephosphorylation of the transcription factor nuclear factor of activated T-cells (NFAT) by a phosphatase, calcineurin. Channel activity is regulated in part by protein kinase G (PKG) phosphorylation at two residues (T70 and S322) to suppress channel conductance and associated signaling. The impact of this regulation on pathologic processes in vivo and how phosphorylation results in channel inhibition remain unknown. In this thesis, I hypothesized that TRPC6 phosphorylation by PKG could play an important role in hypertrophic remodeling of the in vivo heart. To test this, we developed a mouse model with cardiac-restricted expression of a phosphomimetic TRPC6 (T70E, S322E). At baseline, these animals are similar to littermate controls, and when subjected to pressure-overload, these mice developed similar responses at the chamber level, though there were some differences in molecular signaling. Thus, while capable of suppressing cellular and molecular hypertrophic signaling in vitro, expression of phosphomimetic TRPC6 was not sufficient to block hypertrophic responses in vivo. Another proposed regulatory mechanism was the interaction of TRPC channels with stromal interacting molecule 1 (Stim1), a resident ER-protein that is involved in store-operated calcium entry. We tested the hypothesis that Stim1 inhibition would blunt the development of pathologic hypertrophy by generating a cardiac-restricted knockdown of Stim1 in a mouse model of heart failure, but it was not protective against the development of hypertrophy. Lastly, we conducted studies in the glomerular podocyte focusing on the ability of PKG to modulate TRPC6 activation and NFAT signaling, a pathological signaling cascade in focal and segmental glomerusclerosis (FSGS). We determined channel conductance and podocyte behavior in the face of PKG signaling. Our results demonstrated the potency of PKG to inhibit pathophysiology (podocyte dysfunction, ie. motility) and blunt TRPC6 hyperactivity linked to FSGS.
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Keywords
Heart Failure, TRPC6, Protein Kinase G
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