The familial hypertrophic cardiomyopathy-associated myosin mutation R403Q accelerates tension generation and relaxation of human cardiac myofibrils

Abstract

The R403Q mutation in beta-myosin heavy chain was the first mutation to be identified as responsible for familial hypertrophic cardiomyopathy (FHC). In spite of extensive work on the functional sequelae of this mutation, the mechanism by which the mutant protein causes the disease has not been definitely identified. Here we directly compare contraction and relaxation mechanics of single myofibrils from left ventricular samples of one patient carrying the R403Q mutation to those from a healthy control heart. Tension generation and relaxation following sudden increase and decrease in [Ca(2+)] were much faster in the R403Q myofibrils with relaxation rates being the most affected parameters. The results show that the R403Q mutation leads to an apparent gain of protein function but a greater energetic cost of tension generation. Increased energy cost of tension generation may be central to the FHC disease process, help explain some unresolved clinical observations, and carry significant therapeutic implications.

Figure 1. Tension generation and relaxation in control donor and R403Q heart myofibrils

A, representative tension responses (top traces) of donor and R403Q myofibrils maximally activated and fully relaxed by fast solution switching (pCa changes at arrows as indicated). Temperature 15°C. Fast length changes (bottom traces) are applied to the myofibrils under conditions of steady tension generation. k_ACT is the rate constant of tension generation following fast Ca²⁺ activation; k_TR is the rate constant of tension redevelopment following the release–restretch. B, the time course of tension activation following sudden [Ca²⁺] increase of the donor and R403Q myofibrils shown in A are superimposed on a faster time base after normalization for maximal tension. C, tension relaxation kinetics following sudden Ca²⁺ removal; same traces as in A superimposed on a faster time base after normalization for maximal tension. The rate constant of the early slow force decline (slow k_REL) is estimated from the slope of the regression line fitted to the tension trace normalized to the entire amplitude of the tension relaxation transient. The rate constant for the final fast phase of tension decline (fast k_REL) is estimated from mono-exponential fit. D, mean values of slow k_REL for the donor (open columns) and R403Q (filled columns) myofibrils. Bars above columns are s.e.m.; the number of myofibrils is given above s.e.m. bars.

Figure 2. Reaction pathway for acto-myosin ATPase and energy transduction cycle

A, in this generally accepted scheme (Gordon et al. 2000), nucleotide occupancy of the myosin (M) active site modifies myosin s binding affinity for actin (A), defining two general categories of crossbridges: ‘strong binding states’ (AM, AM.ADP) and ‘weak binding states’ (AM.ATP, AM.ADP.P_i); crossbridges in strong states may generate force; crossbridges in weak states do not and are mostly detached forms (M.ATP, M.ATP.P_i). The release of inorganic phosphate (P_i) is thought to power the working stroke. It is well established that crossbridge transitions involved in P_i release and force generation are reversible and, in the presence of P_i, both forward (ADP release and ATP binding) and backward (P_i rebinding and reversal of the power stroke) transitions of crossbridges from force-generating to non-force-generating states occur. However, myofibril slow k_REL, measured in the absence of P_i, only probes forward detachment transitions that lead to ATP hydrolysis steps. B, 2-state crossbridge scheme. AM_{no force} represents all weak binding states (mostly detached) and AM_force all strong binding states. The transition from the non-force-generating states to the force-generating states has an apparent rate constant f_app whereas g_app describes the return to the non-force-generating states by means of ADP release and ATP binding. The apparent rate constant for the reverse transition f′_app that depends on [P_i] can be neglected under the conditions of the present study.