A nemaline myopathy mutation in alpha-tropomyosin causes defective regulation of striated muscle force production

Abstract

Nemaline myopathy (NM) is a rare autosomal dominant skeletal muscle myopathy characterized by severe muscle weakness and the subsequent appearance of nemaline rods within the muscle fibers. Recently, a missense mutation inTPM3, which encodes the slow skeletal alpha-tropomyosin (alphaTm), was linked to NM in a large kindred with an autosomal-dominant, childhood-onset form of the disease. We used adenoviral gene transfer to fully differentiated rat adult myocytes in vitro to determine the effects of NM mutant human alphaTm expression on striated muscle sarcomeric structure and contractile function. The mutant alphaTm was expressed and incorporated correctly into sarcomeres of adult muscle cells. The primary defect caused by expression of the mutant alphaTm was a decrease in the sensitivity of contraction to activating Ca(2+), which could help explain the hypotonia seen in NM. Interestingly, NM mutant alphaTm expression did not directly result in nemaline rod formation, which suggests that rod formation is secondary to contractile dysfunction and that load-dependent processes are likely involved in nemaline rod formation in vivo.

Figure 1

(a) Alignment of αTm isoforms. Genebank accession numbers are: TPM3, X04201; TPM1, M19713; rat, SEG_RATTMA; C. elegans., D38540. (b) Alpha-helical wheel diagram showing the predicted position (22) of the M9R mutation within the hydrophobic core “a” position of the coiled coil motif.

Figure 2

(a) Expression of thin filament proteins in adult myocytes expressing normal and NM mutant αTm at days 5–6 after gene transfer. Two different viral titers (300 and 150 moi, respectively) for AdαTmFLAG M9R giving similar levels of expression were used in functional studies shown in Figure 4. Antibodies used in the panels from top to bottom were: Tm311 for Tm, 5C5 for sarcomeric actin, MAB1691 for troponin I, JLT-12 for troponin T. Soleus and superficial vastus lateralis were included to show migration of alternate myofilament protein isoforms. (b) Anti-FLAG immunofluorescence localization of the expressed epitope-tagged normal Tm (αTmFLAG) in a representative adult myocyte 5 days after gene transfer. (c) Anti-FLAG immunofluorescence localization of the expressed epitope-tagged NM mutant Tm (αTmFLAG M9R) in a representative adult myocyte 5 days after gene transfer. Insets in b and c show enlarged views of the striated anti-FLAG immunofluorescence staining in adult myocytes with arrowheads delineating the edges of the sarcomere (at each Z-line). Resting sarcomere length, ∼1.8–1.9 μm. At this sarcomere length, the thin filaments are overlapping, so segregated I-bands are not expected to be seen. Scale bars are 20 μm for the panels and 4 μm for the insets in b and c.

Figure 3

Electron micrographs of day 5 quiescent adult myocytes (a–c) or day 5 adult myocytes contracting at 0.5 Hz (d–f). The representative myocytes shown were treated with no vector (a, d), AdαTmFLAG (b, e), or AdαTmFLAG M9R (c, f). Scale bar is 1 μm.

Figure 4

Steady-state isometric force production in adult myocytes expressing normal and NM mutant αTm. (a) Original fast time–based force recordings from single adult myocytes expressing normal αTm (i–iii) or NM mutant αTm (iv–vi) using maximal (pCa 4.0, i, iii, iv, vi) or submaximal (pCa 5.7, ii, v) Ca²⁺-activating solutions. Arrows depict the slack step of the myocyte for calculation of active tension production. (b) Summary of tension-pCa relationship of a representative control myocyte (triangles) and a myocyte expressing normal αTm (closed circles) or NM mutant Tm (open circles). (c) Summary of the activating [Ca²⁺] required for half-maximal activation (pCa₅₀) in control adult myocytes (n = 10) and expressing normal (n = 9) and NM mutant αTm (n = 23). *Significantly different from control (P < 0.05; ANOVA). +Significantly different from αTmFLAG (P < 0.05; ANOVA).