Diastolic dysfunction in familial hypertrophic cardiomyopathy transgenic model mice

Abstract

Aims: Several mutations in the ventricular myosin regulatory light chain (RLC) were identified to cause familial hypertrophic cardiomyopathy (FHC). Based on our previous cellular findings showing delayed calcium transients in electrically stimulated intact papillary muscle fibres from transgenic Tg-R58Q and Tg-N47K mice and, in addition, prolonged force transients in Tg-R58Q fibres, we hypothesized that the malignant FHC phenotype associated with the R58Q mutation is most likely related to diastolic dysfunction.

Methods and results: Cardiac morphology and in vivo haemodynamics by echocardiography as well as cardiac function in isolated perfused working hearts were assessed in transgenic (Tg) mutant mice. The ATPase-pCa relationship was determined in myofibrils isolated from Tg mouse hearts. In addition, the effect of both mutations on RLC phosphorylation was examined in rapidly frozen ventricular samples from Tg mice. Significantly, decreased cardiac function was observed in isolated perfused working hearts from both Tg-R58Q and Tg-N47K mice. However, echocardiographic examination showed significant alterations in diastolic transmitral velocities and deceleration time only in Tg-R58Q myocardium. Likewise, changes in Ca(2+) sensitivity, cooperativity, and an elevated level of ATPase activity at low [Ca(2+)] were only observed in myofibrils from Tg-R58Q mice. In addition, the R58Q mutation and not the N47K led to reduced RLC phosphorylation in Tg ventricles.

Conclusion: Our results suggest that the N47K and R58Q mutations may act through similar mechanisms, leading to compensatory hypertrophy of the functionally compromised myocardium, but the malignant R58Q phenotype is most likely associated with more severe alterations in cardiac performance manifested as impaired relaxation and global diastolic dysfunction. At the molecular level, we suggest that by reducing the phosphorylation of RLC, the R58Q mutation decreases the kinetics of myosin cross-bridges, leading to an increased myofilament calcium sensitivity and to overall changes in intracellular Ca(2+) homeostasis.

Figure 1

Linear representation of the N-terminal myosin regulatory light chain (RLC) sequence containing the Ca²⁺–CaM-activated myosin light chain kinase phosphorylation site at Ser 15 and the EF-hand (helix–loop–helix) Ca²⁺–Mg²⁺-binding site of RLC. Familial hypertrophic cardiomyopathy mutations are labelled in boxes: alanine to threonine (A13T), phenylalanine to leucine (F18L), glutamate to lysine (E22K), asparagine to lysine (N47K), and arginine to glutamine (R58Q). X, first Ca²⁺ ligand; Y, second Ca²⁺ ligand; Z, third Ca²⁺ ligand; G, glycine; -Y, fourth Ca²⁺ ligand, provided by a backbone carbonyl; I, isoleucine (although other aliphatic residues are also found at this position); -X, fifth Ca²⁺ ligand; -Z, sixth and seventh Ca²⁺ ligands, provided by a bidentate glutamate or aspartate residue.

Figure 2

The effect of the N47K and R58Q mutations on the phosphorylation status of myosin regulatory light chain (RLC) and troponin I (TnI) in transgenic ventricular extracts blotted with Mab14 MMS-418R antibody recognizing phosphorylated TnI (+P-TnI) (upper panel), and human specific phospho-RLC antibody recognizing transgenic +P-RLC_human (middle panel). Total TnI (TnI_total) and RLC (RLC_total) proteins were blotted with 6F9 and CT-1 antibodies, respectively (bottom panel). Lane 1, R58Q protein standard (RLC_pr.std.); lane 2, Tg-N47K extract; lane 3, Tg-R58Q extract; lanes 4 and 5, NTg extract; lane 6, Tg-WT extract; lane 7, phosphorylated human cardiac RLC-WT protein standard (+P-RLC_pr.std.); lane 8, phosphorylated human cardiac TnI-WT protein standard (+P-HCTnI_pr.std.).

Figure 3

Transcript expression pattern of Ca²⁺-handling proteins, SERCA 2 and PLB, in transgenic mice. Left ventricular total RNA extracts from Tg-WT, Tg-R58Q, and Tg-N47K mice expressing ≈100% transgene were used. Mouse cardiac α-actin and GAPDH were used as internal loading controls. Lanes 1 and 9, 100 bp and 1 kb plus DNA ladders, respectively; lanes 2 and 3, NTg; lanes 4 and 5, Tg-WT; lane 6, Tg-R58Q; lanes 7 and 8, Tg-N47K.

Figure 4

(A) ATPase–pCa relationships for NTg, Tg-WT, Tg-N47K, and Tg-R58Q mouse cardiac myofibrils. (B) The pCa₅₀ values for the ATPase–pCa dependences of NTg, Tg-WT, Tg-N47K, and Tg-R58Q mouse muscle myofibrils. The differences between the Tg-R58Q myofibrils and myofibrils from other transgenic lines were statistically significant (P < 0.05). Data in (A) and (B) are expressed as mean of n experiments ± SEM.

Figure 5

Representative high-resolution echocardiography B-mode images from control (A) and Tg-R58Q mice (B) show no significant difference in chamber dimensions or wall thickness. Representative pulsed Doppler tracings of the mitral valve in controls (C) and Tg-R58Q mice (D) demonstrating reduced E-wave velocity and longer deceleration times in the latter group.

Figure 6

Assessment of in vivo cardiac function. (A) Early transmitral diastolic velocity ‘E-wave’. (B) Late transmitral diastolic velocity ‘A-wave’. (C) E/A ratio, and (D) deceleration time. Data are expressed as mean of n experiments ± SEM.