Long-term rescue of a familial hypertrophic cardiomyopathy caused by a mutation in the thin filament protein, tropomyosin, via modulation of a calcium cycling protein

Abstract

We have recently shown that a temporary increase in sarcoplasmic reticulum (SR) cycling via adenovirus-mediated overexpression of sarcoplasmic reticulum ATPase (SERCA2) transiently improves relaxation and delays hypertrophic remodeling in a familial hypertrophic cardiomyopathy (FHC) caused by a mutation in the thin filament protein, tropomyosin (i.e., α-TmE180G or Tm180). In this study, we sought to permanently alter calcium fluxes via phospholamban (PLN) gene deletion in Tm180 mice in order to sustain long-term improvements in cardiac function and adverse cardiac remodeling/hypertrophy. While similar work has been done in FHCs resulting from mutations in thick myofilament proteins, no one has studied these effects in an FHC resulting from a thin filament protein mutation. Tm180 transgenic (TG) mice were crossbred with PLN knockout (KO) mice and four groups were studied in parallel: 1) non-TG (NTG), 2) Tm180, 3) PLNKO/NTG and 4) PLNKO/Tm180. Tm180 mice exhibit increased heart weight/body weight and hypertrophic gene markers compared to NTG mice, but levels in PLNKO/Tm180 mice were similar to NTG. Tm180 mice also displayed altered function as assessed via in situ pressure-volume analysis and echocardiography at 3-6 months and one year; however, altered function in Tm180 mice was rescued back to NTG levels in PLNKO/Tm180 mice. Collagen deposition, as assessed by Picrosirius Red staining, was increased in Tm180 mice but was similar in NTG and in PLNKO/Tm180 mice. Extracellular signal-regulated kinase (ERK1/2) phosphorylation increased in Tm180 mice while levels in PLNKO/Tm180 mice were similar to NTGs. The present study shows that by modulating SR calcium cycling, we were able to rescue many of the deleterious aspects of FHC caused by a mutation in the thin filament protein, Tm.

Figure 1. Ablation of PLN reduces hypertrophy and expression of hypertrophic marker genes in Tm180 mice

Heart weight/body weight ratios were increased in Tm180 mice but returned to NTG levels in PLNKO/Tm180 mice at both 3 - 6 months (A) and one year of age (B). Atrial weights were also increased in Tm180 hearts at both 3 – 6 months (C) and one year (D) but returned to NTG levels in PLNKO/Tm180 hearts. n=10-25 for all heart weight values. The hypertrophic gene makers, β-MHC (E, F) and ANF (G, H), were also increased in Tm180 mice but decreased in PLNKO/Tm180 mice (n=4-10 for hypertrophic gene marker data). * denotes P ≤ 0.05 vs. NTG, + denotes P ≤ 0.05 vs. Tm180, # denotes P ≤ 0.05 vs. PLNKO/NTG.

Figure 2. Ablation of PLN improves systolic and diastolic hemodynamic parameters in Tm180 mice at 3 – 6 months of age (in situ pressure-volume recordings)

(A) dP/dt_max, maximum rate of LV pressure development (an index of contractility), increases in PLNKO/Tm180 mice compared to Tm180 mice at baseline. Following intravenous infusion of the β-adrenergic agonist (ISO, 0.08 ng/g*min), dP/dt_max decreases in Tm180 mice compared to NTGs, yet PLNKO/Tm180 mice are similar to NTG values. (B) E_max, peak value of time-varying elastance (a load-independent parameter of contractility) during inferior vena cava occlusion. E_max decreases in Tm180 mice but is restored to NTG values in PLNKO/Tm180 mice. (C) dP/dt_min, maximum rate of LV pressure decline (an index of relaxation), decreases in Tm180 mice compared to NTGs but is restored to NTG values in PLNKO/Tm180 mice both at baseline and following ISO. (D) Tau, an index of relaxation time, is prolonged in Tm180 mice compared to NTGs, but this is restored to NTG values in PLNKO/Tm180 mice (baseline only). * denotes P ≤ 0.05 vs. NTG, + denotes P ≤ 0.05 vs. Tm180, # denotes P ≤ 0.05 vs. PLNKO/NTG, ◆ denotes P ≤ 0.05 vs. same group at baseline (NTG, Tm180, PLNKO/NTG or PLNKO/Tm180), n=8-12 animals for each group.

Figure 3. Echocardiography parameters from 4 month and one-year-old mice

A - C, morphology parameters: LVIDd = LV internal dimensions during diastole (B), SWT = septal wall thickness (C). Note that PLNKO/Tm180 mice have a decreased left atrial diameter and septal wall thickness compared to Tm180 mice. D – F, diastolic parameters: E/A ratio (D), Em = peak of myocardial tissue velocity during the early phase of diastole (E) and E/Em = ratio of peak velocity of mitral blood flow in early diastole (E) to Em, an indicator of LV filling pressure (F). Note that the significant changes in Tm180 mice return to NTG levels in PLNKO/Tm180 mice. G – I, systolic parameters: ejection fraction (G), cardiac output (H) and Sm = peak systolic tissue velocity (I). Decreased Sm in Tm180 returns to NTG levels in PLNKO/Tm180 mice. * denotes P ≤ 0.05 vs. NTG, + denotes P ≤ 0.05 vs. Tm180, # denotes P ≤ 0.05 vs. PLNKO/NTG. n=4-6 animals for each group.

Figure 4. Ablation of PLN reduces myocyte disarray and fibrosis in Tm180 mice

Histological sections (40x amplification) from one year-old mice stained with H&E (left panels) and Picrosirius Red (right panels). Note the myocyte disarray (H&E) and fibrosis (Picrosirius Red) in Tm180 compared to NTG and its reduction in PLNKO/Tm180.

Figure 5. Ablation of PLN reduces phosphorylation of ERK1/2

Left. Densitometry of Phospho-ERK1/2 T202,Y204/ ERK1/2 in 3-4 month-old mice. Right. Western blots of Phospho-ERK1/2 T202,Y204 and ERK1/2. * denotes P ≤ 0.05 vs. NTG, + denotes P ≤ 0.05 vs. Tm180, n=6 per group.