Optical Trapping Shows a 3-Fold Increase in Myosin Head Stiffness by the FHC-Mutation R723G in the β-Cardiac Myosin Heavy Chain

Missense mutations in the β-cardiac myosin heavy chain (β-MyHC) are the most frequent cause of Familial Hypertrophic Cardiomyopathy (FHC). For these mutations mutant and wildtype β-MyHC are co-incorporated in the contractile apparatus, and direct functional effects of such mutations presumably trigger development of the FHC-phenotype. In M. soleus biopsies of affected patients we previously found increased isometric force generation for the converter mutation R723G, most likely due to increased stiffness of the myosin head domain. Quantification of the increase in head stiffness from fiber studies, however, was complicated by filament compliance, as well as non-equal and variable co-expression of mutant and wildtype protein in the tissue samples.In the present work we directly determined stiffness of individual myosin heads isolated from M. soleus tissue samples of affected patients using the three-bead optical trapping assay. Since each assay contained mutant and wildtype head domains, stiffness of individual heads with and without the R723G mutation could directly be compared. We determined head stiffness by imposing triangular stage displacements and recording bead displacements during binding events, taking into account compliance of the actin-bead link determined by the variance-Hidden-Markov method (Smith et al., BJ 2001). We found two populations of head domains, one with a head stiffness of 0.39 ± 0.24 pN/nm the other with an about 3-fold higher stiffness. The low value is close to our previous optical trapping with β-MyHC (0.38 ± 0.06 pN/nm; Brenner et al., JMRCM 2012), the about 3-fold increase in head stiffness agrees well with our previous estimate from rigor stiffness in fibers with the same mutation (Seebohm et al., BJ 2009) while the absolute value of head stiffness from fibers was about 25% lower than determined here.