SEARCH

SEARCH BY CITATION

Keywords:

  • contraction velocity;
  • MLC3f;
  • isometric muscle contraction;
  • myofibrillar proteins;
  • sarcopenia;
  • gene delivery

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgment
  8. References

Aging is characterized by a progressive loss of muscle mass and impaired contractility (e.g., decline in force, velocity, and power). Although the slowing of contraction speed in aging muscle is well described, the underlying molecular mechanisms responsible for the decrement in speed are unknown. Myosin heavy chain (MHC) isoforms are the primary molecules determining contractile velocity; however, the contraction speed of single fibers within a given MHC isoform type is variable. Recent evidence proposes that the decline in shortening velocity (Vo) with aging is associated with a decrease in the relative content of essential myosin light chain 3f (MLC3f) isoform. In the current study, we first evaluated the relative content of MLC3f isoform and Vo in adult and old rats. We then used recombinant adenovirus (rAd) gene transfer technology to increase MLC3f protein content in the MHC type II semimembranosus muscle (SM). We hypothesized that (i) aging would decrease the relative MLC3f content and Vo in type II fibers, and (ii) increasing the MLC3f content would restore the age-induced decline in Vo. We found that there was an age-related decrement in relative MLC3f content and Vo in MHC type II fibers. Increasing MLC3f content, as indicated by greater % MLC3f and MLC3f/MLC2f ratio, provided significant protection against age-induced decline in Vo without influencing fiber diameter, force generation, MHC isoform distribution, or causing cellular damage. To the best of our knowledge, these are the first data to demonstrate positive effects of MLC3f against slowing of contractile function in aged skeletal muscle.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgment
  8. References

Aging is associated with a progressive loss of muscle mass and impairment of muscle contractile functions (i.e., strength, velocity, and power generation). The relevance of those phenomena with age is widely recognized as the life expectancy greatly increases. To date, the underlying mechanisms responsible for muscle weakness and slowing of contractile speed are not clearly identified. Single muscle fiber contractile studies and in vitro motility experiments support the idea that the age-related decline in strength is, in part, because of modifications of myosin (Lowe et al., 2001). Although several studies point to myosin as one of the key players, the reported age-induced slowing of velocity cannot be fully explained by myosin modification or myosin isoform expression. Myosin is composed of two myosin heavy chains (MHCs) and four myosin light chains (MLCs). In rodents, four different types (I, IIA, IIX, IIB) of MHC isoforms are identified. Each MHC consists of two types of MLCs: a pair of essential light chains (ELC) and a pair of regulatory light chains (RLC). In adult skeletal muscle, there are three isoforms of ELC (MLC1f, MLC1s, and MLC3f) and two isoforms of RLC (MLC2f, MLC2s). MHC isoforms are primarily responsible for determining contractile velocity, whereas the ELC isoforms have a modulatory, or fine-tuning, role in the regulation of muscle speed. The contraction speed, as demonstrated by unloaded shortening velocity (Vo), in skeletal muscle increases three to ninefold in the order of MHC type I [RIGHTWARDS ARROW] IIA [RIGHTWARDS ARROW] IIX [RIGHTWARDS ARROW] IIB (Bottinelli et al., 1991, 1994). However, the large variability of Vo within a given MHC fiber type supports the idea that other factors, in addition to MHC, contribute to Vo. Evidence suggests that the variability is, in part, related to the modulatory influence of the ELC isoforms (Sweeney et al., 1988; Lowey et al., 1993a,b; Bottinelli et al., 1994). Using the in vitro motility assay (isolated proteins), the removal of the ELCs from myosin results in a 10-fold decrease in sliding velocity of actin filaments compared with native myosin (Lowey et al., 1993a,b). Likewise, myosin containing only the MLC1f isoform move actin at a significantly slower velocity than myosin containing the MLC3f isoform. Using the permeabilized fiber preparation, MHC type II fibers with a relatively larger amount of MLC3f shorten faster than fibers with a greater amount of MLC1f and Vo is proportional to the relative content of MLC3f (Sweeney et al., 1988; Lowey et al., 1993b; Bottinelli et al., 1994). These results suggest that the relative content of the essential myosin light chains, MLC1f and MLC3f, is an important determinant in the regulation of Vo in MHC type II single fibers.

To date, the aging effect on the relative MLC1f and MLC3f isoform content in skeletal muscle fibers, mainly MHC type II fibers, is unknown. We predict that a decrease in relative MLC3f (concomitant increase in MLC1f content) content with age underlies the slowing of contraction in MHC type II fibers. Therefore, the primary purpose of this study was to use single, permeabilized fibers to determine whether aging decreases the relative MLC3f content (increases the relative MLC1f content) and Vo. The second goal was to restore the loss in Vo with age by increasing MLC3f content via recombinant adenovirus (rAd) gene transfer technology in MHC type II fibers from the semimembranosus (SM) muscle (a muscle composed predominantly of MHC type II fibers). We hypothesize that (i) aging decreases the relative MLC3f content (increases the relative MLC1f content) and Vo in MHC type II SM fibers, and (ii) increasing the MLC3f content by rAd-MLC3f in the SM fibers would restore Vo of individual fibers. We further postulate that rAd-empty vector and MLC3f injection in SM muscle will not result in cellular damage, protein damage, or changes in single fiber diameter and force generation in aged rats.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgment
  8. References

Adenoviral vector transduction in SM muscle

We investigated the fluorescent properties of the SM muscles between nontreated controls and treated (rAd injection) experimental groups. As shown in Fig. 1, there was a clear difference in adenovirus DNA delivery between nontreated muscle, which was nonfluorescent (Fig. 1A,B) and treated muscle with red fluorescence because both empty vector (rAd-empty) and MLC3f vector (rAd- MLC3f) carry DsRed gene (Fig. 1C,D). Both empty vector and MLC3f vector injections showed stripe and speckle fluorescent patterns. We also noticed that the fluorescence covered 20-30% of the total muscle area. The 20-30% transduction levels of rAd in postmitotic muscle may be partly due to inefficient binding of rAd particles to the cell surface (Nalbantoglu et al., 2001).

image

Figure 1.  Representative examples of recombinant adenovirus (rAd) transduction in SM muscle. The transduction in tissues was screened using a fluorescent microscope. (A) Nontreated adult control (AC), (B) nontreated old control (OC), (C) old rAd-empty vector (OE), and (D) old rAd-MLC3f-treated (OM) groups. Bar = 1 mm.

Download figure to PowerPoint

No changes in fiber and protein damage with rAd injection

To determine whether the adenoviral vector injection causes muscle damage and subsequent repair, macrophage markers ED1+ and ED2+ were evaluated by immunohistochemistry. We observed an increase in % area of ED1+ with age and no further increase in ED1+ following injection with the vectors (Fig. 2A). In contrast to ED1+, the percentage of nonphagocytic ED2+ macrophages was greater with both rAd-empty vector and rAd-MLC3f injection compared to adult and old groups (Fig. 2B). To determine whether the adenovirus injection causes an increase in protein damage, we determined protein carbonylation in both the cytosolic (Fig. 2C) and the myofibrillar fractions (Fig. 2D). Overall, the adenovirus injection did not appear to induce an increase in protein damage.

image

Figure 2.  Fiber damage and protein damage of SM muscle. (A) Average % area of phagocytic macrophages (ED1+) and (B) nonphagocytic macrophages (ED2+) per total area in SM muscle was determined using NIH image J program. Protein carbonylation, a marker of protein damage, in (C) cytosolic and (D) myofibrillar fractions was compared among the three experimental groups within the left and right hindlimbs. ‘a’ indicates significantly different than AC. ‘†’ indicates significantly different than OM. Values represent mean ± SEM. Significance was set at < 0.05.

Download figure to PowerPoint

No changes in MHC type II fiber diameter, force, and composition with rAd injection

A total of 500 single fibers were evaluated for diameter and force generation. We observed a significant decrease in the diameter of MHC type II single fibers with age (Fig. 3A; P = 0.015). More importantly, the treatment of either rAd-empty vector or rAd-MLC3f into the muscles of aged rats did not affect the diameter of the single fibers. Single fibers in the OC group showed lower absolute force (Po) than those fibers from the AC group (Fig. 3B; P < 0.001). Specific force (Po/CSA) of single fibers from the OC rats was significantly lower than fibers from the AC rats (Fig. 3C; P = 0.015). However, there was no difference in Po and Po/CSA between the OC and rAd-treated groups. These data provide evidence to support that contractile parameters, fiber size, Po, and Po/CSA are not affected following rAd injection.

image

Figure 3.  Fiber diameter, absolute force, specific force, and MHC isoform composition in type II single fibers. (A) Diameter, (B) absolute force (Po), (C) specific force (Po/CSA), and (D) MHC isoform distribution (%) of type II fibers were compared among experimental groups. ‘a’ indicates significantly different than AC. ‘†’ indicates significantly different than OM. ‘NS’ indicates no significant difference. Values represent mean ± SEM. Significance was set at < 0.05.

Download figure to PowerPoint

We evaluated the MHC isoform composition of 587 single fibers. The percentage of type IIB fibers was significantly higher in the AC group compared with the OC group (Fig. 3D; P < 0.001). The expression of MHC type IIX/IIB fibers was significantly greater in the OC group compared with the AC group (P = 0.03). The proportion of MHC type IIX fibers was not different between the AC and OC groups. Noticeably, the rAd injection into the SM muscle did not affect the MHC isoform composition of type II fibers.

Single fiber MLC isoform identification

To identify the essential and regulatory myosin light chain proteins in our one-dimensional silver-stain gels, we used Mass spectrometry (MS) technology. As listed in Fig. 4, MS identified the 21 and 19 kD bands (Spots 1 and 2) as the fast myosin alkali light chain1f and the phosphorylatable fast myosin light chain with 46% and 41% sequence coverage, respectively. These results indicate that the 21 and 19 kD bands in our silver-stain gels are the MLC1f and MLC2f proteins. The 17 kD band/Spot 3 was matched with a fast myosin alkali light chain1f with 19% sequence coverage. Evidence suggests that MLC1f (190aa) and MLC3f (149aa) have complete sequence homology for the last 141aa at the C-terminus and are different in length and sequence (MLC1f = 49aa, MLC3f = 8aa) in N-terminus (Periasamy et al., 1984). Therefore, the extension in amino termini of MLC1f results in a greater molecular mass and, hence, lowers electrophoretic mobility (21 kD). The results of other papers identify the MLC3f protein band almost at the bottom on the 12% SDS-PAGE silver-stain gels (below MLC2f protein band), which also support the idea that 17 kD band is the MLC3f protein (Bottinelli et al., 1994; Larsson et al., 1997).

image

Figure 4.  Myosin light chain (MLC) isoform identification in SM muscle by Mass Spectrometry (MS). The specified protein’s accession number was used from the National Center for Biotechnology Information database. The amino acid peptides with identified sequence were highlighted in yellow, and modified amino acid peptides were highlighted in green.

Download figure to PowerPoint

The rAd-MLC3f attenuates age-induced decline in MLC3f content and Vo in MHC type II fibers

To determine whether there are age and adenovirus-associated differences in MLC isoform compositions, we examined the relative contents of fast MLCs in each MHC type II single fiber with/without physiology testing. There was a shift in fast MLC isoforms of type II fibers with age. The relative content of MLC1f was significantly greater in the OC group than in the AC group (Fig. 5A; P < 0.001). In contrast, the relative content of MLC2f (Fig. 5B; P < 0.001) and MLC3f (Fig. 5C; P < 0.001) was significantly greater in the AC group than in the OC group. We also analyzed the ratio of MLC3f/MLC2f as an indicator for the fraction of MLC3f per myosin head because the amount of regulatory MLC isoforms (MLC2f or MLC2f + MLC2s) reflects the content or amount of myosin present (Lowey & Risby, 1971). The ratio of MLC3f/MLC2f was lower in the OC group compared to the AC group (Fig. 5D; P < 0.001). Noticeably, rAd-MLC3f injection significantly increased the % MLC3f content to 8% and the MLC3f/MLC2f ratio to 0.20 in type II fibers.

image

Figure 5.  Myosin light chain (MLC) isoform composition and maximal unloaded shortening velocity (Vo) in MHC type II single fibers. (A-D) MLC isoform composition and (E) Vo in MHC type II single fibers were compared among experimental groups. The relative content of the individual MLC isoforms (MLC1f, MLC2f, and MLC3f) was determined densitometrically from 12% SDS-PAGE and silver staining. Single fiber Vo was determined by the slack test. ‘a’ indicates significantly different than AC. ‘†’ indicates significantly different than OM. Values represent mean ± SEM. Significance was set at < 0.05.

Download figure to PowerPoint

The MHC type II single fibers had a significant decrement in Vo with age by 30% (Fig. 5E; P < 0.001). Importantly, the Vo of the rAd-MLC3f-treated group was significantly faster than that of the OC group (P = 0.013) and no different than the individual fibers from the AC group. We also found that the rAd-MLC3f injection showed a faster Vo compared to the empty vector treated counterpart (P < 0.001). These results are consistent with the hypothesis that rAd-MLC3f injection may provide significant protection against age-induced decline in contraction speed in type II fibers.

The rAd-MLC3f rescues age-associated decline in Vo in MHC type IIB and type IIX/IIB fibers

To investigate whether age and adenovirus injection affect Vo of single fibers within a specific combination of MHC isoform expression, we separated the MHC type II fibers based on MHC isoforms. Table 1 illustrates the change of Vo depending on the presence of the different MHC isoforms within the single fibers. The fibers with the same MHC isoform content from the AC group showed a significantly higher Vo than those from the OC group (3.3 ± 0.2 vs. 2.5 ± 0.2 FL s−1, P = 0.02 in MHCIIB and 3.2 ± 0.5 vs. 2.3 ± 0.2 FL s−1, P = 0.04 in MHCIIX/IIB). The Vo in MHCIIB and MHCIIB/IIX fibers was not different between the OC and OE groups. Noticeably, the Vo of MHCIIB fibers in the OM group was significantly higher than in the OC and OE groups. However, the Vo in MHCIIX/IIB fibers from the OM was not different than OC and OE groups.

Table 1.   Vo and MLC isoforms of SM MHC type II fibers (MHC type IIB, IIX/IIB, IIX, IIAX)
GroupACOCOEOM
 MHCs
Vo MLCsIIBIIX/IIBIIXIIAXIIBIIX/IIBIIXIIAXIIBIIX/IIBIIXIIAXIIBIIX/IIBIIXIIAX
  1. Vo and MLC isoforms in semimembranosus muscle (SM) myosin heavy chain (MHC) type II fibers were compared among groups: adult control (AC), old control (OC), old empty vector (OE), and old MLC3f (OM). The % MLC1f, % MLC2f, % MLC3f, and MLC3f/MLC2f ratio were determined from densitometric analysis of individual fibers. ‘a’ indicates significantly different than AC. ‘†’ indicates significantly different than OM. Values represent mean ± SEM. Significance was set at < 0.05.

Vo (FL s−1)3.3 ± 0.23.2 ± 0.52.7 ± 0.0/2.5 ± 0.2a†2.3 ± 0.2a1.6 ± 0.32.4 ± 0.32.0 ± 0.2a†1.9 ± 0.2a1.7 ± 0.30.7 ± 0.33.6 ± 0.42.5 ± 0.32.9 ± 0.42.4 ± 0.0
(40)(6)(1)(14)(15)(5)(6)(8)(22)(11)(2)(14)(17)(9)(1)
MLClf (%)29.4 ± 1.526.4 ± 4.226.0 ± 0.0/51.6 ± 2.7a56.4 ± 2.4a50.0 ± 4.246.1 ± 5.148.8 ± 1.5a50.4 ± 3.1a42.3 ± 6.745.9 ± 10.847.7 ± 2.1a50.9 ± 2.4a46.1 ± 1.856.8 ± 0.0
(48)(4)(1)(28)(49)(6)(13)(8)(22)(11)(2)(14)(17)(9)(1)
MLC2f (%)56.6 ± 1.762.0 ± 3.464.9 ± 0.0/44.5 ± 2.6a40.1 ± 2.3a45.4 ± 3.648.6 ± 4.645.2 ± 1.3a45.5 ± 2.8a52.1 ± 6.153.7 ± 10.843.7 ± 2.2a41.1 ± 2.4a46.8 ± 1.342.3 ± 0.0
(48)(4)(1)(28)(49)(6)(13)(8)(22)(11)(2)(14)(17)(9)(1)
MLC3f (%)14.1 ± 0.911.6 ± 1.39.2 ± 0.0/3.9 ± 0.6a†3.5 ± 0.4a†4.6 ± 1.15.4 ± 0.96.0 ± 0.9a4.1 ± 0.5a†5.6 ± 0.90.4 ± 0.18.6 ± 1.1a8.0 ± 1.07.1 ± 1.61.0 ± 0.0
(48)(4)(1)(28)(49)(6)(13)(8)(22)(11)(2)(14)(17)(9)(1)
MLC3F/2f (%)0.28 ± 0.030.19 ± 0.020.14 ± 0.00/0.09 ± 0.02a†0.09 ± 0.0 la†0.10 ± 0.020.12 ± 0.020.13 ± 0.02a0.09 ± 0.0 la†0.14 ± 0.030.01 ± 0.000.21 ± 0.030.21 ± 0.030.15 ± 0.040.02 ± 0.00
(48)(4)(1)(28)(49)(6)(13)(8)(22)(11)(2)(14)(17)(9)(1)

The MLC isoform composition of the MHCIIB fibers in the AC group was 29.4% MLC1f, 56.6% MLC2f, and 14.1% MLC3f, which was different than the OC group (51.6% MLC1f, 44.5% MLC2f, and 3.9% MLC3f). Similarly, MHCIIX/IIB fibers showed a higher amount of MLC1f and a lower amount of MLC2f and MLC3f with age. The MLC3f/MLC2f ratio overlapped considerably among the different fiber types, but the AC group showed a higher MLC3f/MLC2f ratio in MHCIIB and MHCIIX/IIB fibers compared with the OC group. There was a significant difference between the OC and OM groups in MLC3f/MLC2f ratio of MHCIIB fibers, but not between the OE and OM groups, whereas the MLC3f/MLC2f ratio in MHCIIX/IIB fibers was significantly higher in the OM group compared with the OC and OE groups.

Figure 6 illustrates the relationship between MLC3f/MLC2f ratio and Vo. We found that there was a significant correlation between MLC3f/MLC2f ratio and Vo in all type II fibers (R2 = 0.22, P < 0.001) (Fig. 6A). We also found that there was a positive relationship between these two variables in each MHC isoform (Fig. 6B–D). Combined with MHC data, these results provide strong evidence that the relative contents of MLC3f affect shortening velocity involved in age-induced dysfunction independent of fiber size and force generation.

image

Figure 6.  Relationship between Vo and the MLC3f/MLC2f ratio in MHC type II single fibers. The linear regression line for (A) all MHC type II fibers (5.31x + 1.76, R= 0.22, r = 0.46, P < 0.001), (B) MHCIIB fibers (5.73x + 2.01, R= 0.25, r = 0.50, P < 0.001), (C) MHCIIX/IIB fibers (3.26x + 1.87, R2 = 0.09, r = 0.30, P = 0.020), (D) MHCIIX fibers (5.42x + 1.38, R= 0.23, r = 0.48, P = 0.014) was expressed. Significance was set at < 0.05.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgment
  8. References

The primary purposes of this study were to examine (i) whether aging would decrease the relative MLC3f content (increase the relative MLC1f content) and Vo, and (ii) whether increasing MLC3f content via rAd-MLC3f is able to restore the age-related loss in Vo in MHC type II fibers. There were three major findings in the current study. First, the rAd injection procedure into the SM muscle did not appear to increase cellular inflammation or protein damage. Single fiber diameter and force generation capacity were not altered with the rAd injection. Second, there was a significant age-related decline in diameter, Po, Po/CSA, Vo, and shift in MHC and MLC isoforms in single fibers. More importantly, a decrease in relative MLC3f content (concomitant increase in relative MLC1f content) was highly associated with the age-induced decline in Vo of MHC type II fibers. Third and finally, we demonstrated that rAd-MLC3f injection in SM muscle significantly increased the relative content of MLC3f (i.e., % MLC3f and MLC3f/MLC2f ratio) and attenuated the age-associated decrease in Vo in MHC type II fibers. To the best of our knowledge, these are the first data to indicate that increased MLC3f content via rAd-MLC3f gene transfer has positive effects against age-associated slowing of muscle contraction without changing fiber size, force, damage, and MHC isoforms in permeabilized single muscle fibers. Furthermore, our data offer evidence that gene delivery of MLC3f would be beneficial for muscle aging by providing a selective advantage for enhancing contractile function.

Muscle damage and function with rAd

We used rAd DNA gene transfer technology to overexpress MLC3f in the SM muscle of aged rats because rAd-mediated gene transfer is being widely investigated as a therapeutic vector for a host of muscle-related dysfunction. Although rAd-mediated gene transfer is a suggested therapy, efficient delivery of the specific gene to the target tissue without causing damage and pathogenic or immunogenic responses is a major issue. Macrophages, ED1+ and ED2+, play an important role in the process of inflammation (St Pierre & Tidball, 1994; McLennan, 1996). ED1+ macrophages, which are initially derived from blood monocytes, are the first subpopulation to respond when damage occurs. ED1+ macrophages remove damaged cells via phagocytosis. ED2+ macrophages contribute to satellite cell activation and myogenic proliferation required for repair (Tidball et al., 1999). The adenovirus injection does not appear to induce muscle damage because there was no significant difference in the percentage of ED1+ cells between adenovirus injected groups and age-matched control groups. Interestingly, the degree of ED2+ macrophages without infiltration was higher in the adenovirus-treated groups than in the age-matched control groups. Although it is not possible to identify the mechanism underlying the increase in ED2+ macrophages, one possibility is the injection by itself induces muscle regeneration because both the empty vector and MLC3f DNA injections increase ED2+. On the other hand, it is possible the adenovirus itself might increase ED2+ macrophages through the activation of myogenic regulatory factors such as MyoD, Myf-5, and myogenin (Tsivitse et al., 2003).

To confirm minimal cellular inflammation/damage following injection, we evaluated overall protein damage with the universal marker, protein carbonylation. Consistent with our ED1+ data, rAd injection into the SM muscle did not affect cytosolic and myofibrillar protein carbonylation. Lastly, the adenovirus injection into the SM muscle did not alter individual fiber function. While there was the predicted age-related decline in Po and Po/CSA and changes in MHC distribution, neither rAd-empty vector nor rAd-MLC3f injection caused further reductions in those contractile parameters or alterations in MHC distribution. Most important to our study design is the fact that the rAd-empty vector injection did not change Vo, MHC distribution, or MLC isoform composition compared with age-matched control.

Taken together, the findings described previously support that 7 days postinjection of the rAd does not significantly influence contractile function nor cause cellular damage or protein damage in the SM muscle. These findings are consistent with several studies, demonstrating no detrimental effect of rAd injections into skeletal muscle (Kimura et al., 2001; Lefesvre et al., 2002).

Vo and MLC isoforms with age

An age-induced decline in Vo is documented in humans (Larsson et al., 1997; D’Antona et al., 2003) and rodents (Li & Larsson, 1996; Degens et al., 1998; Thompson & Brown, 1999) using permeabilized single muscle fibers. For example, in elderly humans, MHC type I and MHC type IIA fibers have slower speeds of shortening compared to those fibers from young subjects. Meanwhile, in aged rats, the Vo of MHC type I fibers from the soleus (Li & Larsson, 1996; Degens et al., 1998) and MHC type IIB fibers from the SM decreased by 20–50% relative to those fibers from younger rats.

In the present study, single fibers expressing MHC type II isoforms show a significant age-related slowing of contraction speed consistent with our previous report. Notably, the age-induced decline in contraction speed persists when we compare single fibers containing the same MHC type II isoform. The persistent reduction in contraction speed, within single fibers with the same MHC isoform, suggests the involvement of proteins that regulate contraction speed. The relative content of the MLC isoforms may be one of the molecular mechanisms responsible for the age-related slower velocities in single fibers with the same MHC isoform because it is well documented that a decrease in the relative content of essential MLC3f isoform (parallel with an increase in relative content of essential MLC1f isoform) is associated with slower velocities (Sweeney et al., 1988; Bottinelli et al., 1994; Zhong & Thompson, 2007). Currently, investigations focused on the role of the essential MLCs as the major determinant for age-related slowing of contraction are few. In two rodent studies, the results report a relationship between the relative MLC3f content in MHC type IIB fibers and Vo with age. Our results in the current study are consistent with these studies and document a significant decline in MLC3f isoform content and a concomitant increase in MLC1f isoform content with age.

Vo and MLC isoforms with rAd-MLC3f

The primary goal of the current study was to rescue the age-induced slowing of muscle contraction by increasing the relative content of MLC3f in the SM muscles of old rats. Foremost, the rAd-MLC3f gene transfer into the SM muscle does not alter the MHC isoform distribution; however, increasing MLC3f relative content via rAd-MLC3f injection (i.e., % MLC3f and MLC3f/MLC2f ratio) attenuates the age-related decline in Vo in MHC type II single muscle fibers. Specifically, there is an overall increase in relative MLC3f protein content in MHC type II fibers by 7 days after rAd-MLC3f injection. Moreover, increased % MLC3f content to 7.7% from 4.2–4.6% and MLC3f/MLC2f ratio to 0.19 from 0.10 in MHC type II fibers via rAd gene transfer to SM muscle significantly offset age-induced decline in Vo by 24–61%.

It is also interesting to note that rAd-MLC3f gene transfer improves Vo within the given MHC type II fibers differentially. The observed difference in the slope of the regression line between the ratio of MLC3f to MLC2f and Vo suggests that the influence of MLC3f on Vo is more prominent in MHC type IIB fibers than in MHC type IIX/IIB and MHC type IIX fibers. The strong influence of MLC3f on contraction speed in single fibers with MHC type IIB isoform has been demonstrated before (Bottinelli et al., 1994). As noted previously, the percent increase in relative MLC3f content does not have to be large to have an influence on contraction speed. The rAd-MLC3f gene transfer in aging muscle, which results in increased % MLC3f content to 8.6% from 3.9–6.0% and MLC3f/MLC2f ratio to 0.21 from 0.09, enhances Vo by 44–80% in MHC type IIB fibers.

The mechanisms underlying age-related decline in Vo with regard to MLC regulation are not known. However, the modulatory role of MLCs in Vo with age may be associated with the difference in sequence of MLC isoforms. Evidence suggests that the 45 amino acid N-terminal extension of MLC1f compared with MLC3f which has relatively shorter amino acid sequence can cause unfavorable interactions with actin’s negatively charged C-terminus. The interactions between MLC1f and actin are linked with slower speed of filament sliding on the assumption that they increase the lifetime of the attached state of the myosin heads and delay their dissociation during a cross-bridge cycle (Lowey et al., 2007). The results of the present study are congruent with this theory whereby there was an age-related decline in Vo together with an increase in relative MLC1f content (concomitant decrease in MLC3f content). Moreover, we found that an increase in MLC3f content by rAd gene transfer could ameliorate the age-induced decline in Vo of MHC type II single muscle fibers. The positive relationships between relative MLC3f content and Vo in MHC type II fibers suggest that MLC3f may have therapeutic and clinical value in preserving contractile speed in aging skeletal muscle.

One caveat requiring a notation is the species-dependent variation in MHC gene expression. This species-to-species variation in MHC isoform expression is, perhaps, most notable for MHCIIB, which is highly expressed in rodent muscle, yet effectively not expressed in healthy human skeletal muscle. In contrast to healthy muscle, expression of MHCIIB in human skeletal muscles appears to increase with muscle fiber degeneration/regeneration (Harrison et al., 2011). Because there is evidence of increased degeneration/regeneration in aging muscle and MLC3f is found in both fast and slow human skeletal muscle fibers, it is possible to speculate that the results of the current study in rats have potential to translate into humans (Canepari et al., 2000). However, a more extensive analysis of human skeletal muscle may help to determine whether, in fact, MLC3f has any functional consequences.

In summary, contractile velocity in SM single-MHC type II fibers was slowed with age and the decline of Vo was significant in both MHC type IIB and MHC type IIX/IIB fibers. Our data also indicate that rAd-MLC3f transduction in SM muscle did not increase cellular inflammation nor contractile protein damage. Importantly, this is the first study to demonstrate that the increased relative MLC3f content (i.e., % MLC3f and MLC3f/MLC2f ratio) via rAd-MLC3f increases contraction speed of individual fibers independent of the age-related shift in MHC isoforms. Our data offer evidence that gene delivery of MLC3f would be beneficial for muscle aging by providing a selective advantage for enhancing contractile function.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgment
  8. References

Animals

Adult (10–12 months) and old (24–26 months) male F344 rats were purchased from the NIA colony (Harlan). The experiments were approved by the IACUC (U of Minnesota) and were in accordance with the NIH and APS standards. After acclimatization for 1 week, all rats were randomly assigned to one of the following experimental groups: adult control (AC; n = 8), old control (OC; n = 8), old empty vector treated (OE; n = 14), and old MLC3f vector treated (OM; n = 14).

rAd construction and injection in SM muscle

To construct a rAd vector for rat MLC3f gene, total RNA was isolated from the SM muscle of adult rats using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and cDNA was synthesized using the Transcriptor first strand cDNA synthesis kit (Roche Applied Science, Indianapolis, IN, USA). The amplified MLC3f cDNA (forward primer, 5′-TCT CCA GTC CCG CTG CTG TTT TGC-3′; reverse primer, 5′-ATT TGT GGG ATT GGT GCC CAG AGC-3′) by RT-PCR with Pfx Polymerase (Invitrogen) was first cloned into the pCRII-TOPO vector by TOPO TA cloning (Invitrogen). Plasmid DNAs were purified using QIAprep Miniprep kit (QIAGEN, Valencia, CA, USA) and verified by DNA sequence analysis (BioMedical Genomics Center, University of Minnesota). MLC3f cDNA was excised from pCRII-MLC3f with BamHI and XhoI and cloned into BamHI/SalI site of pDNR-CMV. To confirm MLC3f expression, the expression vector for MLC3f was transfected into primary myoblasts with Lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions. The expression of MLC3f was verified by anti-MLC1f/3f antibody (F310, Developmental Study Hybridoma Bank, Iowa). To create pLP-Adeno-X-MLC3f -DsRed (rAd-MLC3f) and pLP-Adeno-X-DsRed (rAd-empty) adenoviral vector with the Adeno-X™ ViraTrak™ Expression System 2, we followed the manufacturer’s protocol (Clontech, Mountain View, CA, USA). Titration of the adenoviral vectors was determined by the methods descried in the manufacturer’s protocol (Clontech). Briefly, HEK 293 cells were infected with adenoviral vectors. By 48 h after infection, DsRed positive cells were counted under the fluorescent microscope. The ifu (infectious unit) was calculated for the resulting average number of DsRed positive cells/unit dilution. Adenoviral vector solution (250 μL of 1 × 1011 to 1 × 1012 ifu mL−1) was carefully injected into five areas of the SM muscle in vivo via a Kendall syringe equipped with a 28-gauge needle because the SM muscle is very large, 0.25 × 1.5 inches. The SM of the right hindlimb was injected with rAd-MLC3f vector solution (OM), and the SM of the left hindlimb was injected with a same volume of rAd-empty vector solution (OE). Following injection, the rats were allowed to recover from anesthesia and monitored to ensure adequate recovery (i.e., eating, drinking, and cage behavior).

Tissue preparations and determination of protein concentration

After 1 week of adenovirus injection, rats were anesthetized with sodium pentobarbital (50 mg mL−1 i.p.). The SM muscles were rapidly removed and trimmed clean of visible fat and connective tissue. Because the adenovirus vectors were not fully infected in the SM, we identified the fluorescent regions and isolated these regions using fluorescence microscopy. Protein concentration, required for protein carbonylation quantification, was determined using the bicinchoninic acid assay kit (Thermo Scientific, Rockford, IL, USA), with bovine serum albumin as the standard. Cytosolic and myofibrillar fractions were isolated (McDonough et al., 2002; Thompson et al., 2006). The cytosolic and myofibrillar protein content was measured in triplicate and expressed as μg of cytosolic or myofibrillar protein per mg wet weight.

Determination of muscle damage and repair

To determine whether the rAd injection caused muscle damage and repair, we used immunohistochemistry to quantify changes in ED1+ macrophages and ED2+ macrophages, two markers of muscle damage and repair, respectively. Serial cross-sections from one of the isolated fluorescent bundles were cut (−20 °C) and dried (30′). Sections were fixed in ice-cold acetone (4°C, 10′), air-dried (30′), and then rinsed with PBS. After quenching the tissues in 0.3% hydrogen peroxide (30′), the sections were blocked (10% horse serum in Tris–Tween 20 buffer for 30′). The desired monoclonal primary antibody (ED1+ 1:200 and ED2+ 1:200, AbD Serotec) was then applied in blocking buffer and placed on the section for 60′ at room temperature. After rinsing in PBS, biotinylated secondary antibody (Horse anti-mouse IgG, PK-6102, Vector) was applied (1:200) for 30′. The sections were amplified with Vectastain ABC reagent for 30′ and then incubated in DAB development reagent (10′; Vector, SK-4100). Images were captured on a Nikon Eclipse E400 microscope (20X). NIH image J program was used for quantification (http://rsb.info.nih.gov/ij/). After opening the image, the image was separated into the red/green/blue (RGB) windows. Critical components of the analysis include: an automatic threshold function [(average background + average object)/2] and the fractional area of positive stain for ED1+ and ED2+. Additionally, a negative control was used to evaluate nonspecific background staining.

Protein carbonylation

To determine whether protein damage occurred following the adenovirus injection, we measured levels of carbonylation using OxyblotTM detection kits (Millipore, MA) according to the manufacturer’s instructions with slight modifications. Briefly, cytosolic and myofibrillar fractions (110 ng) were denatured with 12% SDS and derivatized with DNPH. Samples were incubated at room temperature (15′) with agitation and treated with neutralization solution. Then, the samples were loaded on a slot-blot apparatus and transferred to a PVDF membrane (12 h). Membranes were blocked in 5% FISH (20 mm Tris, 0.5 m NaCl, 0.1% Tween 20, 5% Fish gelatin) for 1 h and incubated with a 1:150 dilution of anti-DNP polyclonal rabbit antibody for 12 h. Following washing with 1% FISH buffer, membranes were incubated with a HRP-conjugated anti-rabbit IgG secondary antibody diluted in 1:300 ratios (1 h). Each sample was evaluated in triplicate and the intensity of chemiluminescence was quantified by arbitrary optical density. Protein carbonylation was expressed as percentage of the AC group.

Permeabilized single fiber preparations

After dissection, the SM bundles were quickly immersed in ice-cold relaxing solution (pH = 7.0, 200 mm imidazole, 100 mm EGTA, 1 m KCl, 100 mm ATP, 100 mm creatine phosphate, 100 mm CaCl2, 1 m MgCl2, pCa = 9). To maintain optimal length, each end of the bundles was tied with surgical silk (4-0) to glass capillary tubes and stored in skinning solution (pH = 7.0, 50% glycerol: 50% relaxing solution composed of 1 m propionate, 200 mm imidazole, 100 mm EGTA, 1 m MgCl2, 4.4 mm ATP) up to 4–5 weeks (−20 °C).

Single muscle fiber physiology experiments

To determine whether single fiber contractile function, including force and velocity, is influenced by aging and adenovirus injection, we performed single muscle fiber physiology experiments. A fiber segment (1–2 mm) was isolated from a bundle and transferred to an experimental bath filled with relaxing solution (Permeabilized Fiber Apparatus Model 802B; Aurora Scientific, Aurora, Ontario, CAN). The fiber was securely mounted to a force transducer and a high speed length controller by two aluminum T-clips carefully folded over each end of the fiber. Sarcomere length was set to 2.4 μm, and the fiber length (Lo) was measured. The fiber diameter was determined from three places along the length of the fiber. The force transducer and length controller signals were analyzed by custom software program (ASI Model 600A, ver 2.0, Aurora Scientific, Aurora, Ontario, CAN).

Isometric force and shortening velocity

To determine maximal isometric force or Vo, the fiber was transferred into activating solution (pCa 4.5; 200 mm imidazole, 100 mm EGTA, 1 m KCl, 100 mm ATP, 100 mm creatine phosphate, 100 mm CaCl2, 1 m MgCl2). Isometric maximal force (Po) was determined in μN. Specific isometric force (Po/CSA) was calculated in kN m−2. To determine maximal unloaded shortening velocity (Vo), the slack-test technique was utilized (Zhong & Thompson, 2007). Briefly, once the fiber reached Po, the fiber was instantly shortened (slacked) by a specific % of Lo (10%-20%) at which time the force output was zero. The time interval between the slack and when the individual fiber starts to generate force was measured. The fiber was then transferred back into relaxing solution. This procedure was repeated at five to six different length steps. The linear regression line between length steps and time interval was calculated (r2 ≥ 0.98). Vo, represented by the slope of the line divided by Lo, was expressed as fiber length per second (FL s−1). Throughout all single fiber contractility tests, the temperature of experimental solutions was set at 15 °C.

MHC and MLC isoform determination of single muscle fibers

MHC and MLC isoform compositions of the single fibers were determined by SDS-PAGE (Zhong & Thompson, 2007). Ten μl of sample was loaded on an electrophoresis system with 4% stacking gel and either a 5% separating gel for MHC or 12% separating gel for the MLC. The gels were run at 250 V and at constant current (76 Amps) (24 h for MHC and 4 h for MLC). The gels were washed, silver-stained, and scanned using molecular multi-imaging system (GS-800; Bio-Rad, Hercules, CA, USA). To confirm reliability of our densitometry measurements, we performed protein concentration curves (12% SDS-PAGE) in duplicate followed by silver staining using the same myofibrillar SM samples. Briefly, the protein samples ranged from 0.03 to 4.0 μg. The arbitrary density of 96 MLC1f, MLC2f, and MLC3f protein bands was used as a measure of test–retest reliability (R2 > 0.99).

MLC isoform identification using in-gel digestion and mass spectrometry

To identify the essential and regulatory myosin light chain proteins, we used powerful ‘in-gel’ digestion and MS techniques. Single fibers (10-15) were isolated and solubilized in 50 μL of sample buffer. One-dimensional SDS-PAGE was performed (Bio-Rad manufacturer’s protocol). Then, the 17, 19, and 21 kD bands were excised from the gels, transferred into a microcentrifuge, and spun down. The enzymatic digestion protocol and MS were previously described (Shevchenko et al., 2006). Briefly, gel pieces were dehydrated by acetonitrile (ACN) for 10′. The proteins were reduced with DTT (0.1 m) in ammonium bicarbonate (NH4HCO3, 0.1 m) at 56 °C for 30′ followed by alkylation with iodoacetamide (0.055 m) in NH4HCO3 (0.1 m) for 20′ at room temperature in the dark. Then, the gel pieces were dehydrated and shrunk with ACN for 10′. For the ‘in-gel’ digestion with trypsin, gel pieces were kept on ice-cold trypsin buffer containing 0.1 μg μL−1 trypsin, 0.1 m NH4HCO3, and 1.9 m ACN for 30′ and further incubated overnight at 37 °C. Next, samples were desalted using the STAGE tip method (Rappsilber et al., 2003). A LTQ-Orbitrap XL mass spectrometer (Thermo Scientific) was used for protein identification, and the data were analyzed with Scaffold 3 (http://www.proteomesoftware.com/Proteome_software_prod_Scaffold.html).

Statistical analysis

All data from spss software program (ver 18.0, IBM, Armonk, NY, USA) were expressed as mean ± SEM. A one-way ANOVA was used to evaluate statistical difference in diameter, Po, Po/CSA, Vo, MHC and MLC composition in MHC type II single fibers, damage, and protein contents among the four groups. A Tukey’s HSD post hoc test was used to estimate the difference among means. Correlation between MLC3f/MLC2f ratio and Vo in MHC type II fibers (MHCIIB, IIX/IIB, IIX) was examined by linear regression analysis. Because of the disparity in sample size of each MHC type II isoform, the nonparametric one-way ANOVA using Kruskal–Wallis test was applied to analyze the mean difference of Vo, MLC1f, MLC2f, MLC3f, and MLC3f/MLC2f ratio in each MHC isoform. The Mann–Whitney test was used to determine the existence of mean differences in each group.

Acknowledgment

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgment
  8. References

The authors thank Janice Shoeman for invaluable technical assistance. This work was supported by NIA (RO1AG017768).

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgment
  8. References
  • Bottinelli R, Schiaffino S, Reggiani C (1991) Force-velocity relations and myosin heavy chain isoform compositions of skinned fibres from rat skeletal muscle. J. Physiol. 437, 655672.
  • Bottinelli R, Betto R, Schiaffino S, Reggiani C (1994) Unloaded shortening velocity and myosin heavy chain and alkali light chain isoform composition in rat skeletal muscle fibres. J. Physiol. 478(Pt 2), 341349.
  • Canepari M, Rossi R, Pellegrino MA, Bottinelli R, Schiaffino S, Reggiani C (2000) Functional diversity between orthologous myosins with minimal sequence diversity. J. Muscle Res. Cell Motil. 21, 375382.
  • D’Antona G, Pellegrino MA, Adami R, Rossi R, Carlizzi CN, Canepari M, Saltin B, Bottinelli R (2003) The effect of ageing and immobilization on structure and function of human skeletal muscle fibres. J. Physiol. 552, 499511.
  • Degens H, Yu F, Li X, Larsson L (1998) Effects of age and gender on shortening velocity and myosin isoforms in single rat muscle fibres. Acta Physiol. Scand. 163, 3340.
  • Harrison BC, Allen DL, Leinwand LA (2011) IIb or not IIb? Regulation of myosin heavy chain gene expression in mice and men Skelet Muscle 1, 5.
  • Kimura E, Maeda Y, Arima T, Nishida Y, Yamashita S, Hara A, Uyama E, Mita S, Uchino M (2001) Efficient repetitive gene delivery to skeletal muscle using recombinant adenovirus vector containing the Coxsackievirus and adenovirus receptor cDNA. Gene Ther. 8, 2027.
  • Larsson L, Li X, Frontera WR (1997) Effects of aging on shortening velocity and myosin isoform composition in single human skeletal muscle cells. Am. J. Physiol. 272, C638C649.
  • Lefesvre P, Attema J, van Bekkum D (2002) A comparison of efficacy and toxicity between electroporation and adenoviral gene transfer. BMC Mol. Biol. 3, 12.
  • Li X, Larsson L (1996) Maximum shortening velocity and myosin isoforms in single muscle fibers from young and old rats. Am. J. Physiol. 270, C352C360.
  • Lowe DA, Surek JT, Thomas DD, Thompson LV (2001) Electron paramagnetic resonance reveals age-related myosin structural changes in rat skeletal muscle fibers. Am. J. Physiol. Cell Physiol. 280, C540C547.
  • Lowey S, Risby D (1971) Light chains from fast and slow muscle myosins. Nature 234, 8185.
  • Lowey S, Waller GS, Trybus KM (1993a) Function of skeletal muscle myosin heavy and light chain isoforms by an in vitro motility assay. J. Biol. Chem. 268, 2041420418.
  • Lowey S, Waller GS, Trybus KM (1993b) Skeletal muscle myosin light chains are essential for physiological speeds of shortening. Nature 365, 454456.
  • Lowey S, Saraswat LD, Liu H, Volkmann N, Hanein D (2007) Evidence for an interaction between the SH3 domain and the N-terminal extension of the essential light chain in class II myosins. J. Mol. Biol. 371, 902913.
  • McDonough JL, Neverova I, Van EykJE (2002) Proteomic analysis of human biopsy samples by single two-dimensional electrophoresis: coomassie, silver, mass spectrometry, and Western blotting. Proteomics 2, 978987.
  • McLennan IS (1996) Degenerating and regenerating skeletal muscles contain several subpopulations of macrophages with distinct spatial and temporal distributions. J. Anat. 188(Pt 1), 1728.
  • Nalbantoglu J, Larochelle N, Wolf E, Karpati G, Lochmuller H, Holland PC (2001) Muscle-specific overexpression of the adenovirus primary receptor CAR overcomes low efficiency of gene transfer to mature skeletal muscle. J. Virol. 75, 42764282.
  • Periasamy M, Strehler EE, Garfinkel LI, Gubits RM, Ruiz-Opazo N, Nadal-Ginard B (1984) Fast skeletal muscle myosin light chains 1 and 3 are produced from a single gene by a combined process of differential RNA transcription and splicing. J. Biol. Chem. 259, 1359513604.
  • Rappsilber J, Ishihama Y, Mann M (2003) Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal. Chem. 75, 663670.
  • Shevchenko A, Tomas H, Havlis J, Olsen JV, Mann M (2006) In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat. Protoc. 1, 28562860.
  • St Pierre BA, Tidball JG (1994) Differential response of macrophage subpopulations to soleus muscle reloading after rat hindlimb suspension. J. Appl. Physiol. 77, 290297.
  • Sweeney HL, Kushmerick MJ, Mabuchi K, Sreter FA, Gergely J (1988) Myosin alkali light chain and heavy chain variations correlate with altered shortening velocity of isolated skeletal muscle fibers. J. Biol. Chem. 263, 90349039.
  • Thompson LV, Brown M (1999) Age-related changes in contractile properties of single skeletal fibers from the soleus muscle. J. Appl. Physiol. 86, 881886.
  • Thompson LV, Durand D, Fugere NA, Ferrington DA (2006) Myosin and actin expression and oxidation in aging muscle. J. Appl. Physiol. 101, 15811587.
  • Tidball JG, Berchenko E, Frenette J (1999) Macrophage invasion does not contribute to muscle membrane injury during inflammation. J. Leukoc. Biol. 65, 492498.
  • Tsivitse SK, McLoughlin TJ, Peterson JM, Mylona E, McGregor SJ, Pizza FX (2003) Downhill running in rats: influence on neutrophils, macrophages, and MyoD+ cells in skeletal muscle. Eur. J. Appl. Physiol. 90, 633638.
  • Zhong S, Thompson LV (2007) The roles of myosin ATPase activity and myosin light chain relative content in the slowing of type IIB fibers with hindlimb unweighting in rats. Am. J. Physiol. Cell Physiol. 293, C723C728.