Aberrant expression of thyroidal hormone receptor α exasperating mitochondrial dysfunction induced sarcopenia in aged mice

Altered physical parameters of SM in mice during aging

Compared to 6m group, the body weight (BW) of 15m and 24m groups significantly increased (1.2-fold, p<0.05) (Figure 1A), while the Gastrocnemius (Ga) weight of 24m group significantly decreased by 14% compared to 15m group (p<0.05) (Figure 1B). Moreover, we observed age-related decline when Ga weight was corrected by BW (Ga/BW) (Figure 1C). Four-limb grip strength was attenuated in 24m group compared to 6m group (8% lower) (p<0.05) (Figure 1D). Consistently, the ratio of grip strength (Grip) to BW (Grip/BW) significantly reduced by about 20% in 15m and 24m groups compared to 6m group (p<0.001) (Figure 1E). Representative H&E staining images of SM section were shown in Figure 1F, and the myocyte cross sectional area (MCSA) was lower in 15m (28% lower) and 24m (30% lower) groups than in 6m group (P<0.001). As shown in Figure 1G, the mRNA levels of senescence-associated markers p16^ink4a and p21 increased with advancing age. The mRNA expression of MuRF1 and Atrogin-1 involved in muscle degradation showed no significant difference among three groups, while both mRNA (42% lower for Myf5, 53% lower for MyOD1) and protein (35% lower for Myf5, 47% lower for MyOD1) levels of myogenic regulatory factors Myf5 and MyoD1 significantly decreased in 24m group compared to 6m group (Figure 1G–1I).

Figure 1. Changes in body weight (BW), gastrocnemius muscle weight (Ga weight), four-limb grip strength (Grip), MCSA, and expression of myogenesis and senescence-related makers during aging. (A) Body weight of mice; (B) Ga weight of mice; (C) Ga weight normalized by BW (Ga weight/BW); (D) Grip strength of mice; (E) Grip strength normalized by BW (Grip/BW), n=10 for 6m group, n=9 for 15m group, n=11 for 24m group (A–E); (F) Representative H&E staining images of Ga muscle section and quantitative analysis of MCSA level of three groups, Scale bar: 50 μm, n=3; (G) mRNA levels of myogenesis and senescence-related makers, n=7; (H) Representative Western blots and (I) quantification of Myf5, MyoD1, MyoG and Gapdh (loading control), n=3. *p<0.05, **p<0.01, ***p<0.001 vs. 6m group; #p<0.05, ##p<0.01 vs. 15m group.

Increased ROS production and altered mitochondrial regulators of SM in mice with aging

The high-energy SM is composed of a large number of mitochondria and the maintenance of mitochondrial integrity and function is critical for muscle health. Using immunofluorescence staining, we found that the ROS production (stained as red fluorescence) in SM significantly increased with aging (2.2-fold for 15m group, 5.5-fold for 24m group, both compared to 6m group, P<0.05) (Figure 2A). As shown in Figure 2B, the mRNA levels of mitochondrial biogenesis factors Tfam, Nrf-1, and Nrf-2 significantly increased in 24m group compared to 6m and 15m groups. Interestingly, the mRNA expression of mitochondrial dynamics factors Mfn1 (36% lower), Opa1 (40% lower) and mitophagy factors Pink1 (45% lower) and DJ-1 (22% lower) significantly decreased in 24m group compared to 6m group (p<0.01), while the mRNA expression of mitochondrial dynamics factors Mfn2 and Drp1, autophagy-related factors LC3 and p62 showed no difference among three groups (Figure 2B). As shown in Figure 2C, 2D, the protein expression of Mfn1 (62% lower), Pink1 (40% lower), DJ-1 (38% lower), LC3II/I (52% lower), p62 (39% lower) significantly decreased in 24m group, compared with 6m group (p<0.05), and Mfn1 protein expression significantly decreased by 31% in 15m group compared to 6m group (p<0.05).

Figure 2. Increased ROS production and altered mitochondrial regulators of SM in mice during aging. (A) Representative ROS fluorescence staining of Ga muscles and quantification, Scale bar: 100 μm, n=3; (B) mRNA levels of mitochondrial biogenesis, mitochondrial dynamics and mitophagy/autophagy-related markers, n=7; (C) Representative Western blots and (D) quantification of Mfn1, Drp1, Pink1, DJ-1, LC3II/I, p62 and Gapdh (loading control), n=3. *p<0.05, **p<0.01, ***p<0.001 vs. 6m group; #p<0.05, ##p<0.01 vs. 15m group.

In summary, these results demonstrated that mitochondrial regulators which involved in mitochondrial quality control, including mitochondrial dynamics, autophagy, and ROS deteriorated with aging in SM.

Decreased expression of TRα in SM with aging

In our study, TRα mRNA level significantly decreased by 48% in 24m group compared to 6m group (p<0.05) (Figure 3A). Moreover, TRα protein level significantly decreased by 32% in 24m group compared to 6m and 15m groups (p<0.05), yet no significant difference between 6m and 15m groups (Figure 3B, 3C).

Figure 3. TRα mRNA and protein levels of SM in mice during aging. (A) mRNA level of TRα, n=7; (B) Representative Western blots and (C) quantification of TRα and Gapdh (loading control), n=3. *p<0.05 vs. 6m group; #p<0.05 vs. 15m group.

Considering the lower level of TRα observed in our study and its vital role in maintaining mitochondrial function according to previous studies [16, 17], we hypothesized that aging related muscle loss might result from the loss of TRα.

The effects of TRα knockdown on mitochondrial dynamics, autophagy, function and myogenesis

To investigate whether TRα regulates mitochondrial dynamics, autophagy, function or myogenesis, TRα-targeted siRNA (si-TRα) was constructed and transfected into C2C12 cells, with non-targeted siRNA (si-NC) as a control. si-TRα transfection successfully decreased TRα expression by 47% (P<0.01) (Figure 4A). Meanwhile, the mRNA expression of Mfn1 (55% lower), Opa1 (43% lower), and Pink1 (51% lower) significantly decreased (p<0.01) in si-TRα group compared to the control (Figure 4B). Consistently, the protein levels of Mfn1 (45% lower), Pink1 (34% lower), DJ-1 (38% lower), LC3II/I (53% lower), and p62 (21% lower) were significantly down-regulated (Figure 4C, 4D). Interestingly, silencing of TRα also led to a significant decrease (56% lower) in mitochondrial activity measured by MitoTracker immunofluorescence staining, higher (1.6-fold) mitochondrial ROS assessed by MitoSox immunofluorescence staining, and higher (1.6-fold) percent of damaged mitochondria, while no significant difference was observed in mitochondrial number measured by electron microscopy (Figure 4E–4H). Notedly, we also observed decreased (48% lower) oxygen consumption rate (OCR) stimulated by the mitochondrial uncoupler FCCP using a Seahorse Bioscience XF24 respirometry analyzer (Figure 4I). In addition, Myf5 and MyoD1 mRNA (34% lower for Myf5, 55% lower for MyOD1) and protein (24% lower for Myf5, 39% lower for MyOD1) expression significantly decreased in si-TRα group (p<0.05), while MyoG was slightly down-regulated in si-TRα group compared with the control group (Figure 4J–4L).

Figure 4. Knockdown of TRα in C2C12. The cells were transfected with TRα-targeted siRNA or non-targeted siRNA for 24h, 48h or 72h. si-NC negative control, si-TRα siRNA-TRα. (A) mRNA level of TRα, n=3; (B) mRNA levels of Mfn1, Opa1 and Pink1, n=3; (C) Representative Western blots and (D) quantification of Mfn1, Pink1, DJ-1, LC3II/I, p62 and Gapdh (loading control), n=3; (E) Representative MitoTracker immunofluorescence staining of cells, nuclei were counterstained with DAPI, scale bar: 100 μm; (F) Representative MitoSox immunofluorescence staining of cells, nuclei were counterstained with Hoechst, scale bar: 200 μm; (G) Quantification of MitoTracker Green fluorescence intensity (n=8) and MitoSox Red fluorescence intensity (n=5), respectively, fluorescence intensity was quantified using densitometric image analysis software with cell quantity adjustment; (H) Representative transmission electron microscopy images of mitochondria and quantification of mitochondrial number and percentage of abnormal mitochondria, the red ‘*’ represents damaged mitochondria (mitochondria with disrupted membrane, loss of cristae, matrix dissolution and vacuolization), scale bar: 1 μm, n=5; (I) Seahorse analysis of oxygen consumption rate (OCR), OCR was measured continuously throughout the experimental period at baseline and in the presence of the indicated drugs: 1.5 μM oligomycin, 0.5 μM FCCP and 0.5 μM rotenone, n=4; (J) mRNA levels of Myf5, MyoD1 and MyoG, n=3; (K) Representative Western blots and (L) quantification of Myf5, MyoD1, MyoG and Gapdh (loading control), n=3. *p<0.05, **p<0.01 vs. control group (si-NC). All experiments were repeated three independent times.

Together, these data indicated that loss of TRα expression in C2C12 might cause impaired mitochondrial dynamics, autophagy, function and myogenesis.

The effects of TRα overexpression on mitochondrial dynamics, autophagy, function and myogenesis

As shown in Figure 5B, accompanied by the increase of TRα expression (14779-fold, P<0.01) (Figure 5A), the mRNA expression of Mfn1 (1.3-fold), Opa1 (1.4-fold), and Pink1 (4.3-fold) significantly increased, and the protein expression of Mfn1 (1.3-fold), Pink1 (1.4-fold), LC3II/I (2.1-fold), and p62 (1.4-fold) also increased in lv-TRα group compared to the control (p<0.05), while there was only an upward trend for DJ-1 (Figure 5C, 5D). Notedly, overexpression of TRα also led to an increase in mitochondrial activity (1.7-fold) and number (1.2-fold, P<0.05), while there were only downward trends for mitochondrial ROS (22% lower, P=0.052) and percent of damaged mitochondria (43% lower, P=0.412) (Figure 5E–5H). No significant difference was observed in OCR (Figure 5I). Additionally, Myf5, MyoD1 mRNA (1.3-fold for Myf5, 2.0-fold for MyOD1) and protein (1.4-fold for Myf5, 1.5-fold for MyOD1) expression significantly increased when TRα was overexpressed (p<0.05), while MyoG expression showed no significant change (Figure 5J–5L).

Figure 5. Overexpression of TRα in C2C12. Cells were transfected with TRα-targeted overexpression plasmid or non-targeted plasmid for 24h, 48h or 72h. lv-NC negative control, lv-TRα overexpressing plasmid-vector TRα. (A) mRNA level of TRα, n=3; (B) mRNA levels of Mfn1, Opa1 and Pink1, n=3; (C) Representative Western blots and (D) quantification of Mfn1, Pink1, DJ-1, LC3II/I, p62 and Gapdh (loading control), n=3; (E) Representative MitoTracker immunofluorescence staining of cells, nuclei were counterstained with DAPI, scale bar: 100 μm; (F) Representative MitoSox immunofluorescence staining of cells, nuclei were counterstained with Hoechst, scale bar: 200 μm; (G) Quantification of MitoTracker Green fluorescence intensity (n=8) and MitoSox Red fluorescence intensity (n=5), respectively, fluorescence intensity was quantified using densitometric image analysis software with cell quantity adjustment; (H) Representative transmission electron microscopy images of mitochondria and quantification of mitochondrial number and percentage of abnormal mitochondria, the red ‘▲’ is labeling mitochondria inside autophagic vesicles, Scale bar: 1 μm, n=5; (I) Seahorse analysis of oxygen consumption rate (OCR), OCR was measured continuously throughout the experimental period at baseline and in the presence of the indicated drugs: 1.5 μM oligomycin, 0.5 μM FCCP and 0.5 μM rotenone, n=4; (J) mRNA levels of Myf5, MyoD1 and MyoG, n=3; (K) Representative Western blots and (L) quantification of Myf5, MyoD1, MyoG and Gapdh (loading control), n=3. *p<0.05, **p<0.01 vs. control group (lv-NC). All experiments were repeated three independent times.

Taken together, these results indicated that overexpression of TRα in C2C12 might improve mitochondrial dynamics, autophagy, activity and myogenesis.

Animals

Thirty C57BL/6J male mice of different ages were obtained from SPF Biotechnology (Beijing, China) and maintained at 20-25° C with free access to water and food. Young (6 months old, 6m; n=10), middle-aged (15 months old, 15m; n=9), and old (24 months old, 24m; n=11) mice were included, body weight and the four-limb grip strength were recorded. After all animals were euthanized, gastrocnemius muscle tissues were excised from the lower limb and weighted, then fixed in 4% paraformaldehyde or stored at −80° C.

Cell culture

C2C12 myoblasts were cultured in growth media (GM) consisting of high-glucose DMEM containing 10% fetal bovine serum (FBS) with 1% penicillin/streptomycin and incubated at 37°C, 5% CO₂. Myoblast differentiation was induced by replacing GM with differentiation media (DM) consisting of DMEM supplemented with 2% horse serum and 1% penicillin/streptomycin. Cells were collected at 72 h (D3) following the addition of DM.

Grip strength test

The four-limb grip strength was analyzed by a grip strength meter. The tested mouse was first allowed to grasp a grid with four paws, and then the tail of mouse was pulled backwards away from the grid until it released the grid. The pulling speed should be gentle and slow enough to let the mouse resist against the pulling force. The peak force was recorded once the mouse moved away from the grid. Each mouse was tested at least three times.

Histology

Ga muscle tissues were fixed in 4% paraformaldehyde solution, embedded in paraffin, sectioned (8 μm thin), stained with hematoxylin-eosin (HE), and then observed under a microscopy. The myocyte cross sectional area was quantitated from six randomly selected images from three independent muscle samples in each group.

ROS staining

The ROS levels in SM were detected by the dihydroethidium (DHE) staining (Bestbio, Shanghai, China). The images were observed by fluorescence microscopy (Olympus, Tokyo, Japan). Four pictures were taken for each sample, and three independent muscle samples were analyzed for each group.

Small interfering RNA and overexpression plasmid transfection

The TRα-targeting siRNAs and overexpression plasmid are conducted and purchased from GenePharma (Shanghai, China). C2C12 cells were transfected with TRα siRNA/plasmid or non-targeting siRNA/plasmid (negative control, final siRNA concentration 20 μM) using Lipofectamine 3000 (Invitrogen, USA).

Real-time PCR (RT-PCR)

Total RNA was extracted using RNAiso Plus (Takara, Shiga, Japan) and cDNA was synthesized with PrimeScript RT Master Mix Kit (Takara, Japan). RT-PCR was performed with TB-Green Kit (Takara, Japan) on StepOnePlus system (USA). The primer sequences are shown in Supplementary Table 1. Relative mRNA level was analyzed by 2^−ΔΔCT method.

Western blotting

Total protein was isolated using the protein extraction kit (Beyotime, Shanghai, China). Western blot analysis was performed using standard protocol with primary antibodies for Gapdh, Mfn1, Myf5, MyoD1, MyoG (Cell Signaling Technology, USA), and TRα, Drp1, Pink1, DJ-1, LC3, p62 (Abcam, Cambridge, UK). Quantification of protein bands was performed with Image-J software (USA).

Immunofluorescence staining

C2C12 cells (transfected with TRα-targeted siRNA/overexpression plasmid or non-targeting siRNA/plasmid for 24h) were incubated with PBS containing MitoTracker Green FM (200 nM/L, Thermo Fisher Scientific, USA, cod. M7514) or MitoSox Red Mitochondrial Superoxide Indicator (5 uM/L, Thermo Fisher Scientific, USA, cod. M36008) for 20 min at 37° C. 4’,6-Diamidino-2-phenylindole (DAPI, Cell Signaling Technology, USA) or Hoechst was used to counterstain the nuclei. The images were observed by fluorescence microscopy (Olympus, Tokyo, Japan). 5-8 pictures were taken from each group for further analysis. Fluorescence intensity was quantified using densitometric image analysis software with cell quantity adjustment.

Transmission electron microscopy (TEM)

C2C12 cells (transfected with TRα-targeted siRNA/overexpression plasmid or non-targeting siRNA/plasmid for 24h) were fixed with 2.5% glutaraldehyde in sodium phosphate buffer and washed 3 times with phosphate buffered saline, then post-fixed with 1% osmium tetroxide and dehydrated with a series of alcohol with increasing concentration. After embedding samples in Araldite, ultra-thin sections were cut and double-stained with uranyl acetate and lead citrate. Next, ultrastructure of mitochondria and autophagic vesicles was observed using the Hitachi HT7800 transmission electron microscope (Tokyo, Japan). Mitochondria (total and abnormal) were quantified blindly in 5 images from each group.

Measurement of O₂ consumption

The oxygen consumption of C2C12 cells was measured on an XF24 respirometer (Seahorse Bioscience, USA) following the manufacturer's instructions. In brief, C2C12 cells (transfected with TRα-targeted siRNA/overexpression plasmid or non-targeting siRNA/plasmid for 24h) were cultured in 24-well XF cell culture microplates and differentiated as indicated (Seahorse Bioscience). Oxygen consumption rates (OCR) were measured at basal glucose levels, as well as with drugs disrupting the respiratory chain: oligomycin (ATP synthase inhibitor, 1.5 μM) (Agilent Technologies, USA) and FCCP (uncoupler, 0.5 μM) (Agilent Technologies). Finally, the mitochondrial respiration was blocked by 0.5 μM rotenone (Agilent Technologies). The residual OCR was considered non-mitochondrial respiration.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 8.0 software (USA). All results were represented as mean ± standard deviation. The variance of multiple groups was analyzed by one-way ANOVA, followed by Tukey post hoc analysis to specifically test individual differences between groups. And the unpaired Student’s t-test was used to compare two independent groups. P<0.05 indicated statistical significance.