Changes in cross-bridge cycling underlie muscle weakness in patients with tropomyosin 3-based myopathy

Abstract

Nemaline myopathy, the most common non-dystrophic congenital myopathy, is caused by mutations in six genes, all of which encode thin-filament proteins, including NEB (nebulin) and TPM3 (α tropomyosin). In contrast to the mechanisms underlying weakness in NEB-based myopathy, which are related to loss of thin-filament functions normally exerted by nebulin, the pathogenesis of muscle weakness in patients with TPM3 mutations remains largely unknown. Here, we tested the hypothesis that the contractile phenotype of TPM3-based myopathy is different from that of NEB-based myopathy and that this phenotype is a direct consequence of the loss of the specific functions normally exerted by tropomyosin. To test this hypothesis, we used a multidisciplinary approach, including muscle fiber mechanics and confocal and electron microscopy to characterize the structural and functional phenotype of muscle fibers from five patients with TPM3-based myopathy and compared this with that of unaffected control subjects. Our findings demonstrate that patients with TPM3-based myopathy display a contractile phenotype that is very distinct from that of patients with NEB-based myopathy. Whereas both show severe myofilament-based muscle weakness, the contractile dysfunction in TPM3-based myopathy is largely explained by changes in cross-bridge cycling kinetics, but not by the dysregulation of sarcomeric thin-filament length that plays a prominent role in NEB-based myopathy. Interestingly, the loss of force-generating capacity in TPM3-based myopathy appears to be compensated by enhanced thin-filament activation. These findings provide a scientific basis for differential therapeutics aimed at restoring contractile performance in patients with TPM3-based versus NEB-based myopathy.

Figures

Figure 1.

(A) The ratio of β-Tm over α-Tm was comparable between the muscle specimens from controls and those from patients with TPM3-based myopathy. (B) Total Tm, normalized to actin, was comparable between controls and patients. Bottom: typical Tm western blot result; note the additional band with higher molecular weight in two patients (T7 and T261) who carry the c.857A > C mutation that eliminates the stop codon.

Figure 2.

(A) Typical force response to saturating Ca2+ levels of a control fiber and a fiber from a patient with TPM3-based myopathy (T856 is shown) with similar fiber CSAs. Note the lower maximal force in the fiber from the patient. (B) The maximal force-generating capacity was significantly decreased in muscle fiber bundles from patients with TPM3-based myopathy. Values shown are the mean ± SEM. *P < 0.05 compared with both type 1 and 2A fibers from controls.

Figure 3.

(A) Representative images and analysis of myofibrils from controls and patients with TPM3-based myopathy stained for α-actinin and tropomodulin (Tmod). Left: note the tropomodulin doublet in the middle of the sarcomere in the control and in the patients' myofibrils (biopsy T239 is shown). Middle: overlay of line scan intensity profile of α-actinin and tropomodulin. Right: the distance between tropomodulin staining (measured across the Z-disk and indicated as Tmod-α-actinin-Tmod) is comparable between control and patient myofibrils. (B) Left: actin staining with phalloidin shows broad and homogenous staining in both control myofibrils and myofibrils from patients with TPM3-based myopathy (note that the fainter phalloidin staining of myofibrils from the TPM3 patient depicted here was not a consistent finding). Middle and right: analysis of phalloidin line scan intensities revealed comparable average thin filament lengths in control and patient myofibrils. (C) Top: typical force response of a patient's muscle fiber preparation that was activated at various SLs. Bottom: the force–SL relation of fiber preparations from patients with TPM3-based myopathy overlap with that from control fibers: both display a characteristic force plateau followed by a comparable descending limb. NS, not significant. Values shown are the mean ± SEM.

Figure 4.

(A) Muscle histology and ultrastructure in patients with TPM3 mutations. Toluidine blue staining (a–c) of Epon-embedded tissue from biopsy (a and d) T863, (b and e) T261 and (c and f) T856 reveals increased variation in fiber size, which corresponds to the selective hypotrophy of type 1 fibers on oxidative stains (a). A subsarcolemmal aggregate of nemaline rods is seen in one fiber from biopsy T856 (c). Ultrastructural examination of muscle from these patients reveals an appropriate organization of the contractile apparatus (d and e), with the exception of areas containing nemaline rods in biopsy T856 (f). Note that the “out of register” appearance of the sarcomeres in (e) is a normal finding that is often due to inadequate stretching of the muscle during fixation or the oblique orientation of the section. Bar = 50 µm for a–c and 500 nm for d–f. (B) Table showing the fiber-type proportions and the relative fiber CSAs for controls and patients. Note the variable degrees of type 1 predominance, but consistent type 1 fiber hypotrophy in patients. (C) Typical SDS–PAGE result showing the relative contributions of the various myosin heavy chain (MHC) isoforms in the different biopsies (indicated below). (D) In line with the histology data, quantification of the myosin heavy chain isoforms obtained from the SDS–PAGE results revealed a decreased proportion of type 1 isoforms in muscle biopsies from patients (with the exception of T7), reflecting the type 1 fiber hypotrophy.

Figure 5.

(A) ktr measurements of fibers from controls and patients with TPM3-based myopathy. Left: example of typical ktr measurements at pCa 4.5 with superimposed results of a patient fiber bundle (biopsy T239 is shown) and a control fiber. Right: ktr is significantly lower in fibers from patients compared with control fibers. (B) Tension cost of patient and control myofibers. Left: example of a maximally activated patient fiber bundle (pCa 4.5) with developed force at the bottom and [ATP] at the top. The slope of the [ATP] versus time trace was divided by fiber volume (in mm3) to determine the ATP consumption rate. Right: the ATP consumption rate was normalized to tension to determine the tension cost. The tension cost is significantly higher in fibers from patients with TPM3-based myopathy. Values shown are the mean ± SEM. *P < 0.05 compared with both type 1 and 2A fibers from controls.

Figure 6.

Force–Ca2+ characteristics of skinned muscle from patients with TPM3-based myopathy and from control muscle. (A) Typical chart recording showing the force response to incremental Ca2+ concentrations in a patient's fiber preparation (biopsy T856 is shown). (B) Left: the relative force generated in response to incubation with incremental increase of [Ca2+]; note the leftward shift of the force–Ca2+ relationship in patients' versus control muscle fibers. Right: same as left panel, but with force normalized to the fiber CSA. The red dotted lines indicate in vivo cytosolic Ca2+ concentrations during activation. Note that T856 produces higher force at these Ca2+ levels than control fibers. (C) The Ca2+ concentration needed for 50% of maximal force generation was significantly lower (i.e. higher pCa50) in patient versus control muscle. Values shown are the mean ± SEM. *P < 0.05 compared with both type 1 and 2A fibers from controls.