Abstract 186: Identification of Novel Protein Kinase G I Alpha Antiremodeling Substrates in the Myocardium
Protein kinase G I α (PKGIα) inhibits cardiac remodeling, and this effect requires the PKGIα leucine zipper (LZ) binding domain. However, PKGIα LZ-dependent cardiac substrates remain poorly understood. Clinical trials of PKGI activating drugs have been limited to date by hypotension arising from vascular PKGI activation. Therefore, we explored downstream PKGIα substrates in the heart which may inhibit remodeling, yet circumvent the hypotensive effects of systemic PKGI activation. A screen for PKGIα LZ-interacting proteins identified: 1)cardiac myosin binding protein-C (cMyBP-C) and 2) mixed lineage kinase 3 (MLK3).
cMyBP-C is a cardiac myocyte protein known to inhibit remodeling when phosphorylated. Co-precipitations with cGMP-conjugated beads confirmed the PKGIα-cMyBP-C interaction. Purified PKGIα phosphorylated cMyBP-C in vitro at Ser-273, Ser-282, and Ser-302. cGMP induced cMyBP-C phosphorylation at these sites in COS cells transfected with WT PKGIα, but not in cells transfected with either LZ mutant PKGIα or kinase-inactive PKGIα. In hearts of 9 month old PKGIα Leucine Zipper mutant mice, which have LV hypertrophy (LVH) and diastolic dysfunction, we observed decreased phosphorylated cMyBP-C as well as decreased total cMyBP-C, compared with WT littermate hearts.
We next tested the effect of MLK3, which interacts with PKGIα in the heart, on remodeling in vivo. We performed 7 day Transaortic Constriction (TAC) on MLK3 KO mice and WT littermates (n=5 shams, 8 TAC per genotype). MLK3 KO TAC mice had increased LVH (LV mass/tibia length 71.1 ± 2.7 g/cm KO TAC vs 62.1 ± 2.7 WT TAC; p<0.05). Further, MLK3 KO mice developed overt CHF compared with WT littermates (LV end diastolic pressure 14.8 ± 1.9 mmHg KO TAC vs 7.7 ± 2.1 WT TAC, p <0.05), as well as accelerated decrements in LV preload recruitable stroke work (36.6 ± 11.9 mmHg/ul KO TAC vs 94.6 ± 12.9 WT TAC, p<0.05) and min dP/dt (-6292 ± 519 mmHg/s KO TAC vs −8157 ± 554 WT TAC , p <0.05). We observed no differences in LV structure or function between sham genotypes.
These studies reveal 2 novel PKGIα anti-remodeling substrates, and they support that exploring PKGIα substrates in the heart may identify novel therapeutic targets to inhibit cardiac remodeling but avoid excessive PKGI induced vasodilation.
Author Disclosures: R.M. Blanton: None R.A.U. Baumgartner: None R. Thoonen: None S. Giovanni: None S. Govindan: None M. Aronovitz: None S. Sadayappan: None D.A. Kass: None R.H. Karas: None.
This research has received full or partial funding support from the American Heart Association, National Center.
- © 2014 by American Heart Association, Inc.