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Background: Mutations in NEB, ACTA1, TPM2, TPM3, KBTBD13, KLHL40 and −41 lead to thin filament myopathies, such as nemaline myopathy, congenital fiber type disproportion and cap disease. A hallmark feature of these myopathies is muscle weakness. Here, we aimed to elucidate whether mutations in NEB, ACTA1, TPM2, TPM3, KBTBD13, KLHL40 and KLHL41 affect the maximal force generating capacity of muscle fibers. Subsequently, by determining the sarcomere length-dependence of force, we investigated whether changes in thin filament length contributed to muscle weakness.Methods: Biopsies from NM, CFTD, and CAP patients (n=39) with mutations in NEB, ACTA1, TPM2, TPM3, KBTBD13, KLHL40 or KLHL41 were compared to biopsies from healthy controls (n=9). Using permeabilized muscle fibers, maximal active tension was determined at incremental sarcomere lengths (range 2.0-3.5 µm) to obtain the force-sarcomere length relationship.Results: The maximal active tension (Fmax (in mN/mm2, mean±SEM)) was significantly lower in biopsies from NEB (46±5), ACTA1 (48±4), TPM2 (71±8), TPM3 (85±10), KBTBD13 (78±3), KLHL40 (2.8±0.2) and KLHL41 (63±4) patients compared to biopsies of controls (129±7).No shift in the force-sarcomere length relationship was observed in TPM3, TPM2, KBTBD 13, KLHL 40 and KLHL41 patients. In contrast, several patients with ACTA1 and NEB mutations showed a leftward shift of the force-sarcomere length relationship indicating shorter thin filaments. Furthermore, the slope of the descending limb of the force-sarcomere length relationship in these patients is less steep than the slope of the CTRL curve, suggesting heterogeneity of thin filaments.Conclusion: Our data suggest that mutations in NEB and ACTA1 result in changes in thin filament length. Insights in the mechanisms underlying weakness in patients with thin filament mutations are necessary to improve specific treatment strategies.

Two mutations, skeletal α-actin K326N and β-tropomyosin ΔK7 were reported in patients with ‘stiff’ muscles and spontaneous contractures, suggesting a hypercontractile phenotype. K326N actin was isolated from a patient biopsy and ΔK7 β-tropomyosin was expressed in baculovirus/sf9 cells. Ca2+ regulation of reconstituted thin filaments was studied by quantitative in vitro motility assay. Both mutations increased Ca2+-sensitivity (EC50 mut/WT =0.37±0.05 and 0.45±0.25 respectively). In recent models of actin-α-tropomyosin in the OFF state, both actin K326 and tropomyosin K7 are located in the actin-tropomyosin interface. We suppose that the charge change due to the mutation would destabilise the OFF state and favour the equilibrium towards the ON state, thus accounting for the higher Ca2+-sensitivity.We also examined a pair of mutations that cause loss of function. Skeletal α-actin D292V (from a patient biopsy) and β-tropomyosin E117K (expressed in baculovirus/sf9 cells ) cause congenital fibre type disproportion ( i.e weak contractility without nemaline bodies). In reconstituted thin filaments β-tropomyosin E117K caused a decrease in Ca2+-sensitivity (EC50 mut/WT = 2.44±0.55). Addition of tropomyosin to actin D292V filaments caused a complete switch-OFF of motility that could not be reversed by troponin at high Ca2+ or even NEM-S-1. Thus congenitally weak muscles correlate with loss of function at the molecular level. These mutations are not at the interface of the OFF state but have an opposite charge change to the gain-of-function mutations and are in a location that could destabilise the ON state, which may account for the loss of function.