We had focussed our earlier work on the functional effects of missense mutations in the converter domain of the β-MyHC, mutations R719W, R723G, and I736T (cf. Figure 1, Rayment et al., 1993). Our goal was to identify direct functional effects of these mutations on muscle function that may be common to all three mutations and common to other FHC-related mutations, including mutations in other proteins.

Tissue samples of M. soleus were obtained by open biopsy. The samples were immediately separated in small bundles and fiber membranes were dissolved by incubation in skinning solution, an ATP-containing solution mimicking the intracellular ionic milieu to which 0.5% Triton X100 was added. Fiber bundles were then equilibrated with solutions containing increasing concentrations of sucrose (maximum 2 M) as a cryoprotectant. Fiber bundles were then rapidly frozen in liquid propane and stored in liquid nitrogen until use. For details see Kraft et al. (1995). For experiments fiber bundles were thawed, individual fibers were isolated and mounted between a strain gage force transducer and a motor to control muscle length or load. As a direct measure of length and length changes of the muscle fibers we measured sarcomere length by laser light diffraction. For further details see Seebohm et al. (2009). The mounted individual fibers were bathed in different experimental solutions that mimicked the ionic composition of the intracellular medium. Different free Ca⁺⁺-ion concentrations were obtained by adding the calcium chelator EGTA and CaEGTA in appropriate proportions. Free Ca⁺⁺-ion concentrations were calculated according to Föhr et al. (1993). For measurements under rigor conditions, fibers were incubated in solutions without MgATP that contained 3 mM EDTA to quickly remove free Mg⁺⁺-ions.

We first measured stiffness of single M. soleus muscle fibers in rigor (Figures 2A,B) and during active contraction (Figures 2C,D, Seebohm et al., 2009). Stiffness was measured by applying small ramp-shaped length changes, stretches (Figure 2A) or releases (Figure 2C) for stiffness in rigor or during active contraction, respectively. During these length changes force and change in sarcomere length were recorded. Fiber stiffness was measured as the slope when force was plotted vs. change in sarcomere length (cf. Figures 2A,C). For stiffness during active contraction the slope over the initial 2–3 nm of length change was determined (red line in Figure 2C) for further details of stiffness measurements see Brenner (1998). Under rigor conditions and during active contraction we observed an increased fiber stiffness for mutations R723G and R719W, while stiffness of fibers with mutation I736T was unchanged (Figures 2B,C). The increased stiffness in rigor, i.e., when all myosin heads are attached to actin, immediately suggested that mutations R723G and R719W both increase the stiffness of the myosin head domain.

For a quantitative estimate of the increase in stiffness per myosin head we had to determine the elastic distortion of the myosin head vs. elastic extension of myofilaments while active force was generated or while fibers were in rigor. This was possible from measurements of active force and active stiffness at different degrees of Ca⁺⁺-activation (cf. Linari et al., 2007). Figure 2E shows these data and illustrates the separation between elastic head distortion and elastic filament extension at different levels of active force. The slope of the solid line in Figure 2E is the compliance of the myofilaments. The intercept of this line with the ordinate at about 5.5 nm/HS represents the elastic extension of the force-generating cross bridges. This is assumed to be the same for all levels of Ca⁺⁺-activation. The y₀-value is the amplitude of fiber release per half-sarcomere that is necessary to drop active force to zero if no fast redistribution of cross-bridges among their different states had occurred (intercept of red solid line in Figure 2C with abscissa at about 8.6 nm/HS). In this concept the y₀ equals the elastic extension/distortion of the force generating cross-bridges, about 5.6 nm, plus the elastic extension of the myofilaments that increases with the forces (isometric force) that act on the myofilaments (cf. Linari et al., 2007).

For a quantitative estimate of the changes in stiffness and in the contribution to force generation by the individual myosin head, caused by the converter mutations, we also had to know how many myosin molecules actually carry the FHC-mutation. From the autosomal dominant inheritance one may have assumed that 50% of the myosin molecules carry the mutation while the other 50% have the wildtype sequence. Analysis by mass spectrometry, however, revealed that the abundance of mutant protein is not equal to 50% but instead is characteristic for each particular mutation (allelic imbalance; Figure 3, black bars). The same allelic imbalance is found for different members of the same family (cf. I734T, R723G) and for different generations (cf. R723G), but also in unrelated families with the same mutation (cf. V606M, R723G). In addition, the same abundance is found in M. soleus fibers and in left ventricular myocardium (cf. R723G). Analysis of expression of the mutant allele at the mRNA-level revealed very similar fractions of mutant mRNA (allelic imbalance) as was found for the mutant protein (Figure 3, gray bars vs. black bars).

The known fraction of mutant myosin in our samples allowed us to estimate the change in head stiffness (change in resistance to elastic distortion) from the observed increase in fiber stiffness, i.e., of the mixture of mutant and wildtype myosin molecules, and the known compliance of the myofilaments (Figure 2C, Seebohm et al., 2009). This estimate yielded an about 2.6-fold increase in head stiffness for mutation R719W and about 2.9-fold increase for mutation R723G while mutation I736T had no such effect (Seebohm et al., 2009).

Based on the fundamental mechanism of force generation, i.e., that active forces result from elastic distortion of actin attached myosin heads caused by structural changes in the myosin head during its working stroke (Huxley, 1957), an increased head stiffness that corresponds to an increased resistance to elastic distortion is expected to result in increased generation of active force. In fact the estimated increase in stiffness of the individual head domain indicated by our stiffness measurements predicted an increase in force generation that was very close to that observed experimentally, about 2-fold for the head domains with mutation R723G or R719W (Seebohm et al., 2009).

The increased head stiffness by converter mutations R719W and R723G implies that the converter domain itself is a major determinant of head stiffness since a point mutation in a particular domain can result in an increase in head stiffness only if the affected domain is a compliant part of the molecule. Stiffening of an already rigid component would not affect overall “stiffness” of a molecule since overall stiffness is limited by the most compliant element(s) of the molecule. But why should increased head stiffness and increased contribution to force generation by an individual myosin head cause disease?

Since mutations R719W and R723G increased myosin head stiffness while mutation I736T did not, we wondered whether the first two mutations were located at particular points in the converter sequence. We therefore compared the amino acid sequence of the β-cardiac myosin heavy chain (which is also the heavy chain of slow skeletal muscle) with the sequences of fast skeletal myosin heavy chains. This revealed that the FHC-mutations R719W and R723G are located at positions where the β-cardiac/slow MHC differs from fast MyHC isoforms in the otherwise highly conserved converter region (cf. Table 1). This raised the question, whether fast and slow skeletal myosins may actually differ in the head stiffness and thus the force generated by a cross-bridge in a force-generating state. We tested this by both stiffness measurements on skinned fibers of human soleus (β-cardiac/slow skeletal MyHC) vs. rabbit psoas (fast skeletal MyHC-2D) and by optical trapping on β-cardiac/slow skeletal myosin subfragment 1 prepared from human and rabbit slow skeletal muscle fibers vs. fast myosin subfragment 1 from the rabbit psoas (Seebohm et al., 2009; Brenner et al., 2012). This revealed that the fast MyHC has ≥3-fold higher head stiffness than the slow/β-cardiac MyHC (cf. Table 1). This was supported by data in the literature (cf. Table 1; Capitanio et al., 2006; Lewalle et al., 2008). So increased head stiffness with increased contribution to force generation appears to be physiological in fast skeletal muscle fibers. This made it even more puzzling as to why converter mutations R719W and R723G cause disease. In addition, increased head stiffness with increased contribution to force generation cannot act as a common trigger for FHC-development, not even for myosin mutations since mutation I736T showed no such effect.

Converter mutations R719W and R723G, both located in the core of the converter at positions where slow and fast myosins differ in amino acid sequence, increase head stiffness. This implies that the converter is a main determinant of head stiffness and thus of the contribution of a myosin head to force generation. Mutation I736T, located at the surface of the converter has no such effect. The other surprising effect is that both R719W and R723G, opposite to the expectation for FHC-related mutations (Marston, 2011) reduce Ca⁺⁺-sensitivity while mutation I736T has no effect on Ca⁺⁺-sensitivity but causes an incomplete relaxation. In addition, mutation R719W showed some changes in cross-bridge cycling kinetics. Altogether, up to this point, the three converter mutations did not show a common direct functional effect. Thus, whilst increased head stiffness is a direct functional effect of the FHC-mutations R723G and R719W, the FHC-phenotype cannot simply be the result of increased head stiffness. Interestingly, higher head stiffness is in fact normal for fast skeletal muscle myosin when compared with the slow skeletal isoform (Table 1). So how does increased head stiffness in the β-myosin heavy chain (β-MyHC) cause disease in myocardium?

A possible insight into the puzzle of how FHC-related mutations in the myosin head domain with different direct functional effects may cause development of an FHC-phenotype was revealed when we compared force-Ca⁺⁺-relationships of individual M. soleus fibers of FHC patients. To our surprise, in some fibers the force-pCa relations were just like those in unaffected controls while other fibers showed substantially different force-pCa relations (cf. Figure 8, Kirschner et al., 2005).

For mutation R719W force-pCa relations of some fibers were shifted substantially further to the right than expected from the “average” fiber (Figure 8 vs. Figure 7A). Similarly, some fibers with mutation R723G were also shifted much further to lower Ca⁺⁺-sensitivity than the average fiber (Figure 8 vs. Figure 7B). Some fibers with mutation I736T showed substantially increased forces at partial activation and had still substantial active forces even at relaxing Ca⁺⁺-concentrations, i.e., showed incomplete relaxation while other fibers of the same tissue sample had force-pCa relations indistinguishable from control fibers. The common feature of all three FHC-mutations, however, is the spectrum of force-pCa relations ranging from normal (control) to substantially shifted to “abnormal” Ca⁺⁺-sensitivity or enhanced partial activation with incomplete relaxation.

The relevance of such spectrum of different Ca⁺⁺-sensitivities for fibers of the same tissue sample of an individual patient becomes most obvious when considering force generation at partial activation. At Ca⁺⁺-concentrations resulting in control fibers in about 60% of full activation, e.g., pCa 6.1, the observed active force generated by individual control fibers varies between about 50% and 70% of full activation. Thus, in control fibers the highest observed force levels observed at pCa 6.1 (70% of full activation; Figure 8) are about 1.5 times higher than the lowest (50% of full activation). At the same pCa value, about 6.1, for mutations R719W and R723G the highest forces of individual fibers in Figure 8 are at least 4- to 5-fold larger than the lowest. For mutation I736T the difference between fibers with highest forces vs. fibers with lowest forces at pCa 6.1 are again in this range (Figure 8). In addition, for mutation I736T some fibers still generated substantial forces at relaxing Ca⁺⁺-concentrations where control fibers were fully relaxed (pCa 8.0).

Thus, the common feature for all three FHC-related mutations in the converter is the much larger spectrum of forces generated by individual fibers of one and the same patient at partial activation levels. In skeletal muscle such different force generation among individual fibers at partial activation means that individual fibers contribute differently to the force generated by a whole muscle. Yet, “stronger” or “weaker” fibers are not expected to interfere with each others function. This is because skeletal muscle fibers do not form cellular networks but contribute to total force of a muscle independently.

We had previously observed that in soleus muscle samples of FHC-patients mutant and wildtype β-cardiac/slow skeletal MyHC were not expressed equally, different to what one may have expected for heterozygous patients with an autosomal dominant disease. Instead each mutation has a characteristic fraction of mutant β-cardiac/slow skeletal MyHC both at the mRNA and protein level (Figure 3, Tripathi et al., 2011). We therefore wondered whether the fraction of β-cardiac/slow skeletal MyHC may also vary from fiber to fiber. Because of the very small amount of material obtainable from individual soleus fiber segments we could only determine the fraction of mutant β-cardiac/slow skeletal MyHC at the mRNA level. Individual M. soleus fibers of a patient with mutation R723G showed quite different fractions of mutant mRNA, ranging from about 10% to essentially 100% (Figure 9). The high number of fibers with almost 100% mutant mRNA is consistent with the observation that in soleus samples (tissue level) the average fraction of mutated mRNA is ≥2/3 of total β-cardiac/slow skeletal MyHC-mRNA (cf. Figure 3).

In tissue samples of FHC-patients we always found a close correlation between fraction of mutant mRNA and fraction of mutant protein, regardless of the FHC-mutation we studied (Figure 3, Tripathi et al., 2011). Thus, the observed large variation in the fraction of mutant mRNA among individual fibers of soleus muscle of affected patients very likely correlates with a similar variation in the fraction of mutant protein. The large variation in mutant mRNA and thus in mutant protein among individual soleus fibers of affected patients could well account for the large functional variation among individual fibers, as judged by the force-pCa relations and the large variation in active force at partial activation (cf. Figure 8, Kirschner et al., 2005).

Assuming that the fibers with the largest shift in force-pCa relations have the highest abundance of mutant β-cardiac/slow skeletal MyHC, e.g., 100% as the maximum, forces generated by these fibers would at most be 2-fold larger than force of control fibers. This is because force generation per head with mutations R719W or R723G was estimated to be about 2-fold higher than force generation by wildtype myosin heads; see above (Kirschner et al., 2005). As a consequence, the forces at partial activation (pCa ≥ 6.3), as low as 1/5 of control fibers in the normalized plots (Figure 8), would still be less than half of control in absolute terms. At pCa around 5.8–5.9 fibers with mutant myosin are expected to generate about the same amount of absolute force as controls, at higher activation levels fibers with mutations R719W or R723G would generate higher forces than controls, reaching up to 2-fold higher forces at maximum activation for fibers with 100% mutant myosin. Most importantly, however, even if absolute forces of individual fibers are considered, the large variation in Ca⁺⁺-sensitivity among individual fibers remains unaffected.

Characterization of functional effects of FHC-related mutations in myocardium for comparison with the effects seen in M. soleus fibers became possible by samples from explanted hearts of patients with the myosin missense mutations, R723G, one of the mutations that we had studied on soleus muscle samples.

Cardiomyocytes were mechanically isolated from the flash-frozen tissue samples (van der Velden et al., 2003; Kraft et al., 2013). After chemical permeabilization the isolated cardiomyocytes were attached to a force transducer and motor such that active and passive forces and kinetics of cross-bridge cycling (k_redev) could be measured at different concentrations of free Ca⁺⁺-ions. To our surprise, force-pCa relations of cardiomyocytes isolated from the flash-frozen samples of explanted myocardium of two patients with mutation R723G showed (i) no difference in Ca⁺⁺-sensitivity and (ii) reduced maximum active force compared to myocytes from donor hearts (Figure 10A). This was in total contrast to the reduced Ca⁺⁺-sensitivity and increased maximum force seen in soleus samples with the same mutation.

Analysis of phosphorylation of contractile proteins revealed substantially lower phosphorylation of troponin I in these myocardial samples with mutations compared to the controls. When the phosphorylation pattern was matched with controls by treatment with protein kinase A (PKA) or by a combination of dephosphorylation by protein phosphatase-1α (PP-1α) followed by phosphorylation with PKA, the mutant samples showed a similarly reduced Ca⁺⁺-sensitivity as the soleus fibers of patients with the R723G-mutation (Figure 10A vs. Figure 10B).

Maximum force, however, was still substantially reduced after matching phosphorylation of patient samples with controls (Figure 10B, left panel). An explanation for the reduced force generation in myocardium in contrast to the increased force generation in soleus fibers with the same mutation was revealed by electron microscopy of tissue samples of affected patients. In myocardium of affected patients prominent intracellular myofibrillar disarray and reduced packing of myofibrils within cardiomyocytes was observed with non-myofibrillar and non-mitochondrial material taking up substantial intracellular volume (Kraft et al., 2013). Quantitatively, myofibrillar density of cardiomyocytes of affected patients with mutation R723G was reduced by about 26%. Without such reduced myofibrillar density within cardiomyocytes, force generation per cross-sectional area had been essentially the same as in control samples (Kraft et al., 2013). The additional myofibrillar disarray with myofibrils deviating from the longitudinal axis of a cardiomyocyte will add to reduced force generation in axial direction (Friedrich et al., 2010) such that increased force generation is not observed even when accounting for reduced myofibrillar density.

Altogether, the opportunity to compare effects of the R723G missense mutation in both soleus fibers and myocardium of affected patients revealed that at least at stages of disease development when myectomies were taken or when transplantation was necessary, direct functional effects of FHC-related mutations can be masked or even reversed by effects most likely subsequent to the direct functional effects. This suggests that e.g., FHC-typical cardiomyocyte and myofibrillar disarray are not direct results of FHC-related mutations but develop subsequently as a result of functional alterations. Functional effects of different mutations apparently converge to a common path of changes resulting in the FHC-phenotype. Cardiomyocyte and myofibrillar disarray may represent the start of this common path leading to e.g., interstitial fibrosis and hypertrophy.

So what may be a feature common to different FHC-related mutations that could trigger cardiomyocyte and myofibrillar disarray as the start on a common path to an FHC-phenotype?

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.