Faster cross-bridge detachment and increased tension cost in human hypertrophic cardiomyopathy with the R403Q MYH7 mutation

Abstract

The first mutation associated with hypertrophic cardiomyopathy (HCM) is the R403Q mutation in the gene encoding β-myosin heavy chain (β-MyHC). R403Q locates in the globular head of myosin (S1), responsible for interaction with actin, and thus motor function of myosin. Increased cross-bridge relaxation kinetics caused by the R403Q mutation might underlie increased energetic cost of tension generation; however, direct evidence is absent. Here we studied to what extent cross-bridge kinetics and energetics are related in single cardiac myofibrils and multicellular cardiac muscle strips of three HCM patients with the R403Q mutation and nine sarcomere mutation-negative HCM patients (HCMsmn). Expression of R403Q was on average 41 ± 4% of total MYH7 mRNA. Cross-bridge slow relaxation kinetics in single R403Q myofibrils was significantly higher (P < 0.0001) than in HCMsmn myofibrils (0.47 ± 0.02 and 0.30 ± 0.02 s(-1), respectively). Moreover, compared to HCMsmn, tension cost was significantly higher in the muscle strips of the three R403Q patients (2.93 ± 0.25 and 1.78 ± 0.10 μmol l(-1) s(-1) kN(-1) m(-2), respectively) which showed a positive linear correlation with relaxation kinetics in the corresponding myofibril preparations. This correlation suggests that faster cross-bridge relaxation kinetics results in an increase in energetic cost of tension generation in human HCM with the R403Q mutation compared to HCMsmn. Therefore, increased tension cost might contribute to HCM disease in patients carrying the R403Q mutation.

Figure 1

Original registrations of a multicellular muscle strip and myofibril preparation A and B, the generation of force normalized to cross-sectional area (CSA) (tension) in an HCM_smn and R403Q muscle strip. The muscle strip was activated in a saturating [Ca²⁺] solution (pCa 4.5) until force reached a steady state and subsequently relaxed in a low [Ca²⁺] solution (pCa 9). C and D, corresponding NADH absorbance recorded during the activations shown in A and B. E and F, contraction–relaxation cycles of myofibril preparations from an HCM_smn (black curve) and R403Q (red curve) patient. G, the time course of tension activation following sudden [Ca²⁺] increase of the HCM_smn and R403Q(1) myofibril preparations shown in E and F superimposed on a faster time base after normalization for maximal tension. H, tension relaxation kinetics following sudden Ca²⁺ removal of the HCM_smn and R403Q(1) myofibril preparations superimposed on a faster time base after normalization for maximal tension. k_act is the rate constant of tension generation and k_rel represents the rate constant of relaxation.

Figure 2

Maximal tension, ATPase activity and tension cost in multicellular muscle strips A, maximal tension on average was significantly lower in R403Q muscle strips compared to HCM_smn (*P < 0.0001). B, the lower maximal tension was evident in each individual R403Q patient compared to HCM_smn (*P < 0.0001). In addition, muscle strips of R403Q(1) revealed a lower maximal tension compared to R403Q(2) (^#P = 0.025) and R403Q(3)(⁺P = 0.026). C, maximal ATPase activity was significantly lower in R403Q muscle strips compared to HCM_smn (*P = 0.004). D, R403Q(1) and R403(3) muscle strips showed significantly lower ATPase activity compared to HCM_smn (*P = 0.002 and *P = 0.017, respectively). ATPase activity in R403Q(1) was significantly lower than R403Q(2) (^#P = 0.04). E, tension cost was calculated by dividing maximal ATPase activity by tension. Maximal tension cost was significantly higher in R403Q compared to HCM_smn (*P < 0.0001). F, the increase in tension cost was significant in R403Q(1) and R403Q(2) compared to HCM_smn (*P < 0.0001 and *P = 0.008, respectively). In addition, tension cost in R403Q(1) muscle strips was higher compared to R403Q(2) (^#P = 0.020) and R403Q(3)(⁺P = 0.001). Average data are shown ± SEM, N = number of patients, n = number of individual muscle strips; data points represent individual muscle strips ± SEM.

Figure 3

Tension cost at maximal and submaximal [Ca⁺] A, tension and ATPase activity in muscle strips were measured at submaximal [Ca²⁺] yielding the following relations for the R403Q patients: y = 2.82x + 4.47 and HCM_smn patients: y = 1.65x + 4.47. The average slope (i.e. tension cost) of the R403Q muscle strips was significantly higher compared to HCM_smn (*P = 0.018). B, ATPase activity–tension relations of muscle strips of individual R403Q patients are as follows: R403Q(1): y = 3.66x + 7.26, R403Q(2): y = 2.59x + 5.40 and R403Q(3): y = 2.22x + 0.75. The R403Q(1) slope reached a significant difference compared to both HCM_smn (*P = 0.001) and R403Q(3) (⁺P = 0.026). Average data are shown ± SEM, N = number of individual muscle strips; data points represent individual muscle strips ± SEM.

Figure 4

Cross-bridge kinetics in myofibril preparations A, maximal tension on average did not differ between R403Q and HCM_smn myofibrils. B, myofibrils from R403Q(1) showed a significant decrease in maximal tension compared to the other R403Q patients and HCM_smn patients (*P = 0.02 vs. HCM_smn, ^#P = 0.019 vs. R403Q(2) and ⁺P = 0.003 vs. R403Q(3)). C, slow k_rel (∼g_app∼tension cost) was significantly higher in R403Q myofibrils compared to HCM_smn (*P < 0.0001). D, this increase was visible in myofibrils of all R403Q patients compared to HCM_smn (R403Q(1)(2) *P < 0.0001 and R403Q(3) *P = 0.001). In addition, slow k_rel was significantly higher in myofibrils of R403Q(1) compared to R403Q(2)(^#P = 0.006) and R403Q(3)(⁺P = 0.001). Data are represented as individual myofibril preparations ± SEM and N = number of patients, n = number of individual muscle strips. ^aData from Belus et al. (2008).

Figure 5

Relation between slow relaxation kinetics and sarcomere energetics and function The correlation between slow k_rel of the myofibril preparations and tension cost of the muscle strips can be described by the positive linear equation y = 0.13x + 0.1 with R² = 0.97. The slow k_rel measured in myofibrils correlated very well with the energetic cost of isometric tension generation (tension cost) measured in multicellular muscle strips from the same samples. Data are represented as mean ± SEM of HCM_smn (N = 9) and each individual R403Q patient.

Figure 6

Estimated myofibril force and f_app A, estimated myofibril force of R403Q(1) myofibrils was lower compared to HCM_smn myofibrils. B, estimated f_app was significantly higher in R403Q myofibrils *P < 0.0001 and for myofibrils of each R403Q patient individually compared to HCM_smn (*P < 0.001, *P < 0.0001 and *P < 0.005, respectively).

Figure 7

Histological and mRNA analysis A, representative iamges of WGA-stained cryosections. B, CSA was significantly higher (*P < 0.0001) in the R403Q cardiomyocytes, which was mostly caused (C) by the R403Q(1) and R403Q(2) cardiomyocytes (*P < 0.0001 and P < 0.01, respectively). D, representative images of Picrosirius Red-stained cryosections. E, fibrosis tended to be higher for R403Q compared to HCM_smn (P = 0.06). F, fibrosis was highest for R403Q(3) compared to HCM_smn (P < 0.05). G, RNA was extracted three times from a tissue sample of each patient. The relative expression of the R403Q and wildtype alleles was quantified at least in duplicate. Analysis of mutant R403Q mRNA revealed a deviation from the 50:50 ratio (allelic imbalance) in R403Q(1) with a fraction of mutated mRNA being significantly lower than in R403Q(2) (^#P < 0.0001) and R403Q(3) (⁺P < 0.0002), both showing no allelic imbalance. N = number of individual PCR reactions analysed.

Figure 8

Correlation with age A, there was a significant correlation between tension cost and age (P = 0.03; R² = 0.51), which was not due to HCM_smn. B, there was no significant correlation for slow k_rel and age taking both HCM_smn and R403Q into account (P = 0.23; R² = 0.33).