Nebulin protein levels in NEM2 patients and controls
We studied skeletal muscle of four NM patients with nebulin mutations and four control subjects with no history of skeletal muscle disease (for control subject and patient characteristics see ). Three of the patients had homozygous deletions of exon 55 of the nebulin gene and the fourth patient had a heterozygous single base pair deletion resulting in a premature stop codon (p.Ser1908AlafxX8) and a second unidentified mutation.
To study protein levels of nebulin, as well as the levels of other sarcomeric proteins, we used SDS-PAGE and Western blotting techniques on skeletal muscle fibers from NEM2 patients and control subjects. As shown in , control muscle shows a clear nebulin band at ~800 kDa, with an expression level of 0.070 ± 0.001 (nebulin/MHC ratio). SDS-PAGE showed that nebulin in NEM2 patients has a mobility that is indistinguishable from that of controls. Importantly, gel analysis indicated significantly reduced nebulin levels relative to MHC in NM muscle (NM: 21 ± 13% of controls, , middle panel). The actin/MHC ratio was significantly reduced as well, although to a lesser extent when compared to the nebulin/MHC ratio (NM: 78 ± 3% of controls, , right panel).
Figure 2 A) SDS-PAGE (for typical example gel result of a control and NM patient, see left panel; NM patient ID: 174-1) revealed that nebulin protein levels (normalized to MHC) in nebulin-based NM muscle are reduced to ~20% of the nebulin levels found in control (more ...)
To test for stoichiometric changes in other components of the thin filament we studied the thin filament regulatory proteins TnI, TnT, TnC and tropomyosin (Tm) and included in the analysis slow and fast skeletal isoforms and both α- and β-Tm. indicates that the total expression level of TnI, TnT, and TnC, normalized to actin, were on average reduced in NM compared to control muscle, although this reduction was not found to be significant. Total tropomyosin was higher in NM than in control muscle (), with no difference in the relative expression of α- and β-Tm (0.41 ± 0.03 vs. 0.51 ± 0.04, NM and control respectively). Analysis of the slow and fast isoforms of the troponins revealed that the NM patients express mainly slow skeletal (ss) isoforms, whereas controls express both slow and fast skeletal (fs) isoforms (ss/fs, TnC: 4.5 ± 1.2 vs. 0.5 ± 0.2; TnI: 4.6 ± 1.7 vs. 0.09 ± 0.01; TnT: 5.3 ± 2.0 vs. 0.4 ± 0.1, NM vs. control respectively, for typical WB result see ).
As the contractile performance of skeletal muscle fibers might depend on the fibers’ MHC isoform composition, we used specialized SDS-PAGE to study MHC isoform expression in NM and control muscle. As shown in , control muscle expressed a mixture of MHC I, 2A and 2X isoforms (49 ± 3%, 33 ± 3%, and 18 ± 0.4%, respectively). In contrast, muscle from the four NM patients contained only MHC type I, with one patient expressing a minor band (~30% of total MHC) corresponding to MHC 2A, see .
In summary, muscle of NM patients had greatly reduced nebulin protein levels relative to MHC, whereas the expression level of actin was only slightly reduced. Nebulin protein levels, relative to actin, were reduced to a larger extent than the relative levels of the other thin-filament based proteins troponin and tropomyosin, which showed no or only slightly reduced expression. Furthermore, consistent with pathological data on the biopsies demonstrating a characteristic fiber type 1 predominance (), the muscle biopsies of the NM patients mainly contained MHC type slow as well as slow Tn isoforms.
Muscle structure analysis by confocal microscopy
To study the effect of reduced nebulin levels on muscle structure and thin filament length we used immunofluorescence confocal scanning laser microscopy with fluorescently labeled phalloidin (which binds filamentous actin with high affinity) on NM and control muscle. Both control and NM myofibrils showed the expected striated labelling patterns, although the labelling appeared more variable and more diffuse in NM myofibrils. We also found dense actin accumulations in the NM muscle fiber (), which most likely indicate nemaline rods. Importantly, controls showed broad actin labeling with uniform density (except for the Z-disk area where actin filaments overlap), whereas in NM myofibrils the labelling was narrower with decreasing intensity from the Z-disk towards the middle of the sarcomere (). Densitometric analysis revealed that the width at half-maximal intensity was significantly reduced in NM compared to control myofibrils (). Thus, these studies suggest shorter and non-uniform thin filament lengths in NM myofibrils.
Figure 3 Myofibrils from controls and nebulin-based NM patients stained for actin. A) Actin staining with phalloidin shows broad and homogenous staining in control myofibrils, whereas actin staining intensity in NM myofibrils gradually decreases from Z-disk towards (more ...)
Muscle fiber mechanics
As NM muscle expressed mainly MHC type I (), and considering that a muscle fiber’s contractile properties can depend on MHC isoform composition, the contractile data from NM muscle preparations were compared to those from control fibers expressing solely slow MHC. We found that the maximal Ca2+-activated active tension (tension at pCa 4.5) was significantly reduced from 71 ± 8 mN/mm2 in control to 5 ± 1 mN/mm2 in the NM muscle fibers. We also measured active force at a range of calcium levels and expressed force relative to the maximal force. An example of active force development of a NM muscle preparation in response to incremental calcium is shown in . The obtained force-pCa relations were shifted to the right in NM muscle fibers () with a pCa50 value of 5.67 ± 0.08 for control muscle fibers and 5.28 ± 0.07 for NM muscle fibers. NM muscle fibers also had on average a reduced Hill coefficient (nH), a measure of the cooperativity of myofilament activation, but this difference was not statistically significant ().
Figure 4 Force–Ca2+ characteristics of skinned muscle from NM and control muscle (note that the results from NM muscle fibers were compared to those from control muscle fibers expressing solely MHC slow). A) Typical chart recording showing the force response (more ...)
The tension cost was determined simultaneously by measurement of the breakdown of NADH and force during contraction, with NADH levels enzymatically coupled to ATP utilization (see Methods). An example of a maximally-activated NM muscle preparation with [NADH] falling linearly during the tension plateau is shown in . The slope of the [NADH] vs. time curve was normalized by the fiber volume to obtain ATP consumption rates that can be compared for differently sized muscle preparations. By normalizing ATP consumption rates to the tension generated and fiber volume, the tension cost can be determined. As shown in , the tension cost was significantly higher in NM muscle compared to control muscle (8.5 ± 0.8 vs. 3.0 ± 0.2 pmol/mN/mm/s, NM vs. control respectively).
We also measured the rate of tension redevelopment (Ktr). Muscle preparations were first isometrically activated at pCa 4.5 and when a steady tension was reached, crossbridges were disengaged by performing a quick release, a brief period of unloaded shortening, and then a rapid restretch to the original length. Following restretch, tension rebuilds with a time course that can be fit to a monoexponential with rate constant Ktr. illustrates a Ktr measurement on an NM and control muscle preparation, revealing that tension recovers slower in the NM than in the control muscle preparation. The averaged results from different fibers are shown in . Ktr is significantly lower in NM fibers compared to control fibers (1.5 ± 0.2 vs. 2.2 ± 0.2 s−1).
Ktr measurements of NM and control fibers. A) Example of ktr measurement at pCa 4.5 with superimposed the results of a NM (174-1) and a control fiber. B) Ktr is significantly lower in NM compared to control fibers.