Decreasing tropomyosin phosphorylation rescues tropomyosin-induced familial hypertrophic cardiomyopathy

Abstract

Studies indicate that tropomyosin (Tm) phosphorylation status varies in different mouse models of cardiac disease. Investigation of basal and acute cardiac function utilizing a mouse model expressing an α-Tm protein that cannot be phosphorylated (S283A) shows a compensated hypertrophic phenotype with significant increases in SERCA2a expression and phosphorylation of phospholamban Ser-16 (Schulz, E. M., Correll, R. N., Sheikh, H. N., Lofrano-Alves, M. S., Engel, P. L., Newman, G., Schultz Jel, J., Molkentin, J. D., Wolska, B. M., Solaro, R. J., and Wieczorek, D. F. (2012) J. Biol. Chem. 287, 44478-44489). With these results, we hypothesized that decreasing α-Tm phosphorylation may be beneficial in the context of a chronic, intrinsic stressor. To test this hypothesis, we utilized the familial hypertrophic cardiomyopathy (FHC) α-Tm E180G model (Prabhakar, R., Boivin, G. P., Grupp, I. L., Hoit, B., Arteaga, G., Solaro, R. J., and Wieczorek, D. F. (2001) J. Mol. Cell. Cardiol. 33, 1815-1828). These FHC hearts are characterized by increased heart:body weight ratios, fibrosis, increased myofilament Ca(2+) sensitivity, and contractile defects. The FHC mice die by 6-8 months of age. We generated mice expressing both the E180G and S283A mutations and found that the hypertrophic phenotype was rescued in the α-Tm E180G/S283A double mutant transgenic animals; these mice exhibited no signs of cardiac hypertrophy and displayed improved cardiac function. These double mutant transgenic hearts showed increased phosphorylation of phospholamban Ser-16 and Thr-17 compared with the α-Tm E180G mice. This is the first study to demonstrate that decreasing phosphorylation of tropomyosin can rescue a hypertrophic cardiomyopathic phenotype.

Keywords: Calcium; Cardiac Hypertrophy; Muscle; Phosphorylation; Transgenic Mice; Tropomyosin.

FIGURE 1.

A, α-Tm E180G/S283A DMTG construct. The α-MHC promoter drives cardiac-specific expression of the striated muscle α-Tm with encoded substitutions at amino acids (aa) 180 (E180G) and 283 (S283A) on the same molecule. B, myofibrillar preparations resolved by SDS-PAGE and stained with Coomassie Blue. Note the small shift in migration between the α-Tm E180G and DMTG Tm samples. Arrows indicate DMTG endogenous and TG protein. C, quantification of endogenous Tm protein remaining in NTG, α-Tm E180G, α-Tm S283A, and DMTG samples. Error bars represent S.E.

FIGURE 2.

A, immunoblot of phosphorylation status of α-Tm E180G hearts at 3 months of age. B, immunoblot of phosphorylation status of α-Tm S283A hearts at 3 months of age. C, immunoblot of phosphorylation status of DMTG hearts at 3 months of age. D, quantification of α-Tm phosphorylation in hearts from 3-month-old NTG, α-Tm E180G, α-Tm S283A, and DMTG mice. *, versus NTG, p < 0.05. E, two-dimensional SDS-PAGE of Tm from α-Tm E180G TG myofibers. Error bars represent S.E. pTm, phosphorylated Tm; α-Tm-p, phosphorylated α-Tm.

FIGURE 3.

A, histological studies of NTG, α-Tm E180G, and DMTG hearts at 3 months of age stained with H&E, Masson's trichrome, and wheat germ agglutinin (WGA). Images were taken at 40×, and scale bars indicate 50 μm. B, cardiomyocyte cross-sectional area measurements (n = 6). *, versus NTG, p < 0.05; #, versus α-Tm E180G, p < 0.05. C, heart weight to body weight (HW:BW) ratios of 3-month-old mice (n = 6). Error bars represent S.E.

FIGURE 4.

A, Ca²⁺-tension relations in skinned fiber bundles from NTG, α-Tm E180G, and DMTG hearts. B, normalized force relations in skinned fiber bundles from NTG, α-Tm E180G, and DMTG hearts. Error bars represent S.E. mN, millinewtons.

FIGURE 5.

A–C, quantitative RT-PCR analysis of expression of the cardiomyopathy marker genes in 3-month-old NTG, α-Tm E180G, α-Tm S283A, and DMTG hearts. For NTG, n = 12; for E180G, n = 8; for S283A, n = 8; and for DMTG, n = 10. *, versus NTG, p < 0.05; #, versus α-Tm E180G, p < 0.05; @, versus α-Tm S283A, p < 0.05. Error bars represent S.E.

FIGURE 6.

A, Western blots of sarcoplasmic Ca²⁺ flux proteins in NTG, α-Tm E180G, α-Tm S283A, and DMTG hearts. B, quantification of phosphorylation levels of PLN Ser-16 and PLN Thr-17. C, quantification of expression of PLN and SERCA2a. D, Western blots of TnI and phosphorylated TnI (pTnI) from NTG, α-Tm E180G, α-Tm S283A, and DMTG hearts. E, quantification of data from D. *, versus NTG, p < 0.05; #, versus α-Tm E180G, p < 0.05; @, versus α-Tm S283A, p < 0.05; n = 8. Error bars represent S.E.