Early mechanical dysfunction of the diaphragm in the muscular dystrophy with myositis (Ttnmdm) model

Abstract

A complex rearrangement mutation in the mouse titin gene leads to an in-frame 83-amino acid deletion in the N2A region of titin. Autosomal recessive inheritance of the titin muscular dystrophy with myositis (Ttn(mdm/mdm)) mutation leads to a severe early-onset muscular dystrophy and premature death. We hypothesized that the N2A deletion would negatively impact the force-generating capacity and passive mechanical properties of the mdm diaphragm. We measured in vitro active isometric contractile and passive length-tension properties to assess muscle function at 2 and 6 wk of age. Micro-CT, myosin heavy chain Western blotting, and histology were used to assess diaphragm structure. Marked chest wall distortions began at 2 wk and progressively worsened until 5 wk. The percentage of myofibers with centrally located nuclei in mdm mice was significantly (P < 0.01) increased at 2 and 6 wk by 4% and 17%, respectively, compared with controls. At 6 wk, mdm diaphragm twitch stress was significantly (P < 0.01) reduced by 71%, time to peak twitch was significantly (P < 0.05) reduced by 52%, and half-relaxation time was significantly (P < 0.05) reduced by 57%. Isometric tetanic stress was significantly (P < 0.05) depressed in 2- and 6-wk mdm diaphragms by as much as 64%. Length-tension relationships of the 2- and 6-wk mdm diaphragms showed significantly (P < 0.05) decreased extensibility and increased stiffness. Slow myosin heavy chain expression was aberrantly favored in the mdm diaphragm at 6 wk. Our data strongly support early contractile and passive mechanical aberrations of the respiratory pump in mdm mice.

Fig. 1.

Two- and 6-wk-old control mice (A and D) and age-matched mice with muscular dystrophy with myositis (mdm; B and C). At 2 wk, mdm mouse was phenotypically indistinguishable from its wild-type littermate (A and B). Progression of disease was marked at 6 wk, manifesting in severe wasting of hindlimb muscles, dorsal kyphosis, decreased ambulation, and decreased body mass (C).

Fig. 2.

Three-dimensional surface reconstructions of thoracic skeleton and lungs from high-resolution micro-CT. Images are representative of control and mdm mice at 2 and 6 wk of age (n = 3 for each genotype and age group). A, E, I, and M (column 1) represent wild-type mice for comparison with images of mdm mice (columns 2, 3 and 4). Sagittal views are shown for 2-wk-old (A–D) and 6-wk-old (E–H) mice. Corresponding axial views are also shown for the same 2-wk-old (I–L) and 6-wk-old (M–P) mice. Note normal spinal curvature, symmetric thoracic rib cage configuration, and normal lung shape in 2-wk-old mdm compared with wild-type mice. Note severe scoliosis and dorsal kyphosis, in combination with asymmetric distortion of the thoracic rib cage, namely, lateral constriction and anterior-posterior lengthening, in addition to decreased skeletal and lung mass in 6-wk-old mdm mice. Lung shapes in 6-wk-old mdm mice and appeared to be distorted concomitantly with rib cage changes and appeared smaller than controls.

Fig. 3.

Whole body mass, costal diaphragm mass, and total protein content. A and B: mean whole body mass and left costal diaphragm mass of control and mdm mice at 2 and 6 wk (n > 20 for all groups). Decreases in total body mass (A) and left costal diaphragm mass (B) were statistically significant at 6 wk compared with controls. Reduction in diaphragm mass was slightly (10%) greater than whole body weight loss at 2 and 6 wk. C: total protein content per unit mass of muscle was significantly reduced (31%) in mdm compared with control mice at 2 wk (n = 3 per genotype). *P < 0.05 vs. age-matched controls.

Fig. 4.

Thickness calculated from surface area and mass of excised left costal diaphragms (A) and from histological tissue sections (B). A: calculated thickness for control (n = 6) and mdm (n = 6) mice at 2 and 6 wk. Although mean values were ∼20% less for mdm than wild-type mice, these differences were not statistically significant. B: mean histological thickness of mdm (n = 6, 0.0317 and 0.0290 cm at 2 and 6 wk, respectively) and wild-type (n = 6, 0.0407 and 0.0362 cm at 2 and 6 wk, respectively) diaphragm. Differences in mean calculated thickness between genotype groups were not statistically different in either age group.

Fig. 5.

Histological staining of 2- and 6-wk diaphragms. A: hematoxylin-eosin staining of wild-type and mdm diaphragms (n = 3 for each age × genotype group) at 2 and 6 wk. Rows 1 and 2 are images of wild type diaphragms. Rows 3 and 4 are images of mdm diaphragms. Columns 1 and 2 show examples of the same age × genotype groups. At 2 wk, histopathological signs were largely absent in mdm diaphragm. At 6 wk, mdm diaphragms showed marked foci of necrosis, scar tissue, and variation in myofiber size. B: small foci of increased perimysial connective tissue deposition in 2-wk mdm diaphragm stained with Masson's trichrome. At 6-wk, large endomysial and perimysial connective tissue deposition was noted in localized foci of diaphragm necrosis. Scale bar, 60 μm.

Fig. 6.

Myofiber morphometry of 2- and 6-wk mdm diaphragms. A: percentage of myofibers with centrally located nuclei was significantly greater at 6 wk. Total number of individual myofibers for 2-wk control (553) and mdm (596) and 6-wk control (1,842) and mdm (651) diaphragms was ≥500 per group (n = 3 for each age × genotype group; *P < 0.05). B and C: myofiber Feret's diameter frequency plots in 2-wk control and mdm and 6-wk control and mdm diaphragms. Feret's diameter values are as follows: 31.52 ± 0.266, 33.00 ± 0.35, 34.91 ± 0.20, and 36.79 ± 0.38 for 2-wk control, 2-wk mdm, 6-wk control, and 6-wk mdm, respectively. Corresponding variance coefficients were 179.24, 166.75, 143.93, and 178.07, respectively. Mean Feret's diameters of 2- and 6-wk mdm diaphragms were significantly increased compared with age-matched controls (P < 0.01). Total number of individual myofibers for 2-wk control (431) and mdm (249) and 6-wk control (608) and mdm (212) diaphragms was ≥200 per group. For frequency histograms, 8 control (4 each at 2 and 6 wk of age) and 6 mdm (2 at 2 wk of age and 4 at 6 wk of age) diaphragms were analyzed.

Fig. 7.

In vitro isometric twitch characteristics of left costal diaphragm at 2 and 6 wk. Maximum twitch stress (P_max), time to peak (TTP) twitch, and half-relaxation time (1/2RT) were significantly reduced at 6 wk (n = 10 control and 3 mdm), but not at 2 wk (n = 7 per genotype). P_max, TTP, and 1/2RT in mdm diaphragms were reduced by 71%, 57%, and 52%, respectively. Rate of force development (dP/dt) was not different for either age group. *Significantly different from age-matched controls (P < 0.05).

Fig. 8.

Mean costal diaphragm force-frequency response curves. At 2 wk, mdm diaphragm (n = 5) generated less tetanic stress than control (n = 9), with significance reached at 60, 100, and 150 Hz. At 6 wk, maximum tetanic stress was reduced 64% in mdm diaphragms (n = 5) relative to controls, with significance at all stimulation frequencies (#P < 0.05). All force-frequency response curves were nonlinear over the range of stimulation frequencies. %Statistically different from 2-wk and 6-wk mdm (P < 0.05).

Fig. 9.

Western blot of myosin heavy chain (MHC) isoforms. Anti-fast and anti-slow MHC primary antibodies were used to detect changes in extracted myosin myofilaments from wild-type (control) and mdm diaphragms at 2 and 6 wk (n = 3 for all age × genotype groups). At 2 wk, anti-fast MHC appeared similar in control and mdm diaphragms. Anti-slow MHC appeared to be slightly decreased in mdm diaphragms. At 6 wk, relative proportions of anti-fast MHC and anti-slow MHC detection were reversed in mdm diaphragm. Anti-slow MHC signal was much stronger in mdm diaphragm.

Fig. 10.

Passive length-tension relationship plotted as tension vs. extension ratio at 2 wk. Representative length-tension relationships compare control and mdm diaphragm extensibilities in response to applied mechanical loading and unloading. Passive length-tension relationships were shifted leftward for mdm diaphragm vs. age-matched control.

Fig. 11.

Extension ratios vs. passive lengthening at 4 tension levels for control (n = 9) and mdm (n = 10) diaphragms. Significant decreases in extensibility were detected at 3 levels of tension at 2 wk (A) and 2 levels of tension at 6 wk (B). P < 0.05. Extensibility was measured from 3 successive stretch cycles in each diaphragm and averaged for each age group. #,%Statistically different from control (P < 0.05).