Malignant familial hypertrophic cardiomyopathy D166V mutation in the ventricular myosin regulatory light chain causes profound effects in skinned and intact papillary muscle fibers from transgenic mice

Abstract

Transgenic (Tg) mice expressing approximately 95% of the D166V (aspartic acid to valine) mutation in the ventricular myosin regulatory light chain (RLC) shown to cause a malignant familial hypertrophic cardiomyopathy (FHC) phenotype were generated, and the skinned and intact papillary muscle fibers from the Tg-D166V mice were examined using a Guth muscle research system. A large increase in the Ca(2+) sensitivity of force and ATPase (Delta pCa(50)>0.25) and a significant decrease in maximal force and ATPase were observed in skinned muscle fibers from Tg-D166V mice compared with control mice. The cross-bridge dissociation rate g was dramatically decreased, whereas the energy cost (ATPase/force) was slightly increased in Tg-D166V fibers compared with controls. The calculated average force per D166V cross-bridge was also reduced. Intact papillary muscle data demonstrated prolonged force transients with no change in calcium transients in Tg-D166V fibers compared with control fibers. Histopathological examination revealed fibrotic lesions in the hearts of the older D166V mice. Our results suggest that a charge effect of the D166V mutation and/or a mutation-dependent decrease in RLC phosphorylation could initiate the slower kinetics of the D166V cross-bridges and ultimately affect the regulation of cardiac muscle contraction. Profound cellular changes observed in Tg-D166V myocardium when placed in vivo could trigger a series of pathological responses and result in poor prognosis for D166V-positive patients.

Figure 1.

Schematic representation of the D166V mutation (labeled in red) in the myosin RLC (National Center for Biotechnology Information Accession Number 2MYS). The heavy chain of myosin is labeled in yellow, the ELC in dark blue, and the RLC in green.

Figure 2.

Transgenic protein expression in Tg-D166V and Tg-WT mouse hearts. A) Representative Coomassie-stained gel (top panel) and Western blot (bottom panel) of atrial muscle samples from WT and mutant mice. Lanes 1 and 2, NTg; lanes 3 and 8, Tg-WT; lanes 4–7, Tg-D166V; lane 9, D166V standard protein. B) Percent protein expression in Tg-WT and Tg-D166V mice. ELC_endog, endogenous atrial essential light chain; RLC_endog, endogenous atrial regulatory light chain; RLC_transg, transgenic ventricular regulatory light chain.

Figure 3.

The effect of the D166V mutation on the phosphorylation status of RLC and TnI in transgenic mouse ventricular extracts blotted with CT-1 antibody recognizing total RLC protein and 6F9 antibody recognizing total TnI protein (A) and Mab14 MMS-418R antibody recognizing +P-TnI (top panel) and +P-human RLC antibody recognizing +P-RLC human (Tg) (bottom panel) (B). hWT_st, human WT standard protein; +P-hWT_st, phosphorylated human WT standard protein; +P-hTnI_st, phosphorylated human TnI standard protein.

Figure 4.

Representative ventricular heart sections from Tg-D166V, Tg-WT, and NTg mice. A) Microscopic views of ventricular cross-sections stained with H&E and Masson’s trichrome of representative ∼11-month-old mice. B) Cross-sections of ventricles stained with Masson’s trichrome from ∼7- and ∼17-month-old mutant and control mice. Note severe fibrotic lesions found in the tissue of ∼17-month-old Tg-D166V mice.

Figure 5.

ATPase-pCa (left panel) and force-pCa (right panel) relationships in Tg-D166V skinned muscle fibers compared with Tg-WT and NTg littermates. A statistically significant difference in the Ca²⁺ sensitivity of ATPase activity and force was observed between Tg-D166V muscle fibers and all other groups of fibers. No difference in either the Ca²⁺ sensitivity of ATPase or force between Tg-WT and NTg fibers was observed. Data are expressed as means ± se of n experiments (n individual fibers).

Figure 6.

Effect of the D166V mutation in myosin RLC on maximal ATPase activity (s⁻¹ per myosin head) and maximal force (in kN/m²) determined in Tg skinned papillary muscle fibers. Data are expressed as means ± se of n experiments (n fibers isolated from n mouse hearts).

Figure 7.

Fraction of myosin cross-bridges attached at maximal calcium activation in Tg-D166V fibers compared with Tg-WT and NTg fibers. Data are expressed as means ± se of n experiments (n fibers isolated from n mouse hearts).

Figure 8.

Effect of the D166V mutation in myosin RLC on the rate of cross-bridge dissociation g (s⁻¹) in skinned papillary muscle fibers. Data are expressed as means ± se of n = 9 (Tg-D166V), n = 6 (Tg-WT), and n = 5 (NTg) experiments.

Figure 9.

Energy cost of contraction expressed as the ratio of fiber ATPase/fiber force (×10⁻² s⁻¹ kN⁻¹ m²) plotted as a function of muscle activation. Data are expressed as means ± se of n experiments (as in Fig. 8).

Figure 10.

Normalized force (top panel) and [Ca²⁺] (bottom panel) transients in electrically stimulated intact papillary muscle fibers from Tg-D166V compared with Tg-WT and NTg mice. Note significant differences in force transients between Tg-D166V and control papillary muscle fibers (P<0.05). Data are expressed as means ± se of n = 4–6 experiments.