Calpain inhibition rescues troponin T3 fragmentation, increases Cav1.1, and enhances skeletal muscle force in aging sedentary mice

Abstract

Loss of strength in human and animal models of aging can be partially attributed to a well-recognized decrease in muscle mass; however, starting at middle-age, the normalized force (force/muscle cross-sectional area) in the knee extensors and single muscle fibers declines in a curvilinear manner. Strength is lost faster than muscle mass and is a more consistent risk factor for disability and death. Reduced expression of the voltage sensor Ca(2+) channel α1 subunit (Cav1.1) with aging leads to excitation-contraction uncoupling, which accounts for a significant fraction of the decrease in skeletal muscle function. We recently reported that in addition to its classical cytoplasmic location, fast skeletal muscle troponin T3 (TnT3) is fragmented in aging mice, and both full-length TnT3 (FL-TnT3) and its carboxyl-terminal (CT-TnT3) fragment shuttle to the nucleus. Here, we demonstrate that it regulates transcription of Cacna1s, the gene encoding Cav1.1. Knocking down TnT3 in vivo downregulated Cav1.1. TnT3 downregulation or overexpression decreased or increased, respectively, Cacna1s promoter activity, and the effect was ablated by truncating the TnT3 nuclear localization sequence. Further, we mapped the Cacna1s promoter region and established the consensus sequence for TnT3 binding to Cacna1s promoter. Systemic administration of BDA-410, a specific calpain inhibitor, prevented TnT3 fragmentation, and Cacna1s and Cav1.1 downregulation and improved muscle force generation in sedentary old mice.

Keywords: aging; calcium channel; calpain; excitation-contraction coupling; skeletal muscle; troponin T.

Figure 1

TnT3 knockdown in vivo decreases Cav1.1 and Cacna1 expression; its overexpression enhances Cacna1s promoter activity in C2C12 and mouse muscle in vivo; and it is recruited onto the Cacna1s promoter. (A) Representative immunoblot of protein extracts from shC‐ and shT‐electroporated FDB muscles. (B) Quantification of 3 independent immunoblots, showing that shT decreases TnT3 and Cav1.1. (C) Quantitative qRT–PCR showing that shT successfully knocked down Tnnt3 mRNA, which resulted in downregulation of Cacna1s. *P < 0.05, **P < 0.01. (D) Cultured C2C12 cells transfected with shC or shT did not show differences in myotube formation at day 5 day in DM. (E) Immunoblot of total protein extracts. Compared to shC‐, shT‐transfected C2C12 myotubes show strong TnT3, but not MHC, knockdown. PVDF membrane stained with Ponceau S indicates the same total protein loading. (F) Fusion index shows that shT treatment did not prevent C2C12 myoblast fusion at day 5 in DM. (G) Luciferase activity assay, expressed as the firefly/renilla ratio, shows that knocking down TnT3 inhibits Cacna1s promoter activity in C2C12 cells at day 5 in DM. (*P < 0.05, **P < 0.01). (H) Diagram of nuclear localization sequence (NLS) and leucine zipper domain (LZD) deletion in the full‐length TnT3 (TnFL)‐DsRed fusion construct. (I) Raw normalized data comparing luciferase activity for TnFL, TnFL‐ΔLZD/DsRed, TnFL‐ΔNLS/DsRed, and DsRed. Differences between these individual groups and DsRed are noted by their level of significance (*) P < 0.05 or (**) P < 0.005. (J) Primer set used to amplify mouse Cacna1s promoter regions that may interact with TnT3. Numbers indicate distance from the transcription start site. Eight primer pairs were designed to walk along regions P1‐P8. (K) Representative ChIP‐PCR experiment in C2C12 myotubes showing Cacna1s promoter regions recruiting endogenous TnT3. IgG was used as a control.

Figure 2

EMSA mapping of the Cacna1s promoter region that interacts with TnT3. (A) EMSA oligonucleotide designed to test the proximal half of the Cacna1s promoter's P5 region and used in subsequent experiments. (B) Compared to oligos alone (lane 1), TnT3 induces a band shift (lane 2) that is attenuated by unlabeled oligos (lane 3) and pre‐incubation with TnT3 and TnT3 antibody (lane 4). Two oligos, designated control‐ and control‐2 (Table S1), unrelated to Cacna1s, show no binding to TnT3 (lanes 4–8). (C) Compared to P5 oligos alone (lane 1), TnT3 induced a band shift (lane 2) that was attenuated by unlabeled oligos (lane 3). In contrast, TnI, TnC, and Tm antibodies and mouse (m) and rabbit (r) IgG did not attenuate band shift as shown in a separate gel. (D) Multiple EM for Motif Elicitation (MEME) (Bailey et al., 2006) sequence alignment and comparison of P4, P5, and P8 regions. The six consensus motifs identified are highlighted. The blue box encloses the Cacna1s promoter region that interacts with TnT3 in the ChiP assay. (E) Oligonucleotide design, based on MEME, identified motifs in the P5 proximal half region. P5a is the sequence upstream the E‐box (in black); P5b is the sequence downstream of the E‐box; and P5c is the E‐box in the mutated full‐length P5. (F) Representative EMSA data show that P5b has the weakest binding to TnT3, while P5a shows strong binding. The E‐box does not seem required for TnT3 binding because both P5c (E‐box mutant) and P5a (sequence upstream of E‐box) clearly bind to TnT3. (G) Diagrams showing oligos with mutated P5a‐R3 (P5a‐R3 m) or P5a‐R6 (P5a‐R6 m). H) Representative EMSA data showing that both R3 and R6 mutations diminished the gel shift of P5a oligos.

Figure 3

BDA‐410 increases ex vivo isometric contraction force in the soleus muscle. (A) Absolute force production in response to supramaximal stimulation at 1, 5, 10, 20, 50, 100 and 150 Hz. (B) Relative increase in absolute force in BDA‐410 compared to vehicle‐treated mice. (C) Soleus muscle cross‐sectional area (CSA). (D) Soleus muscle‐specific force calculated as the absolute force normalized to muscle CSA. (E) Relative increase in specific force in BDA‐410 compared to vehicle‐treated mice. A and B: *P < 0.01. D and E: *P ≤ 0.003. Left and right soleus muscles were included in the analysis. Data values are expressed as mean ± SEM. n = 6 for each group.

Figure 4

Calpain inhibition does not modify EDL and soleus fiber‐type composition or titin protein levels. Titin and MHC isoforms (IIa, IIx, IIb, and I) in soleus (A) and EDL (B) muscles measured in 5 mice treated with BDA‐410 and 4 treated with vehicle. Differences were not statistically significant (C–F).

Figure 5

BDA‐410 stabilizes nuclear TnT3 integrity in old mice skeletal muscle in vivo. (A) Immunoblot of nuclear protein extractions from old mice treated with BDA‐410 or vehicle. CT‐TnT3 and TnNT were detected with antibodies targeting the C‐ or N‐terminal regions of TnT3. Histone H3 antibody was used to detect H3 as an internal nuclear protein loading control. (B) BDA‐410 effectively reduced the abundance of nuclear CT‐TnT3 (*P < 0.05), but not TnNT normalized to H3 (C) or TnFL normalized to tubulin (D–E).

Figure 6

Cav1.1 protein levels are higher in old mice treated with BDA‐410 than with vehicle. (A) Cav1.1 and GAPDH immunoblot in pooled hindlimb muscles from old mice treated with either BDA‐410 (n = 3) or vehicle (n = 3). (B) Cav1.1 expression, normalized to GAPDH, analyzed by densitometry, is significantly higher in the old mice treated with BDA‐410 (*P < 0.05).