Cardiac troponin T and fast skeletal muscle denervation in ageing

Abstract

Background: Ageing skeletal muscle undergoes chronic denervation, and the neuromuscular junction (NMJ), the key structure that connects motor neuron nerves with muscle cells, shows increased defects with ageing. Previous studies in various species have shown that with ageing, type II fast-twitch skeletal muscle fibres show more atrophy and NMJ deterioration than type I slow-twitch fibres. However, how this process is regulated is largely unknown. A better understanding of the mechanisms regulating skeletal muscle fibre-type specific denervation at the NMJ could be critical to identifying novel treatments for sarcopenia. Cardiac troponin T (cTnT), the heart muscle-specific isoform of TnT, is a key component of the mechanisms of muscle contraction. It is expressed in skeletal muscle during early development, after acute sciatic nerve denervation, in various neuromuscular diseases and possibly in ageing muscle. Yet the subcellular localization and function of cTnT in skeletal muscle is largely unknown.

Methods: Studies were carried out on isolated skeletal muscles from mice, vervet monkeys, and humans. Immunoblotting, immunoprecipitation, and mass spectrometry were used to analyse protein expression, real-time reverse transcription polymerase chain reaction was used to measure gene expression, immunofluorescence staining was performed for subcellular distribution assay of proteins, and electromyographic recording was used to analyse neurotransmission at the NMJ.

Results: Levels of cTnT expression in skeletal muscle increased with ageing in mice. In addition, cTnT was highly enriched at the NMJ region-but mainly in the fast-twitch, not the slow-twitch, muscle of old mice. We further found that the protein kinase A (PKA) RIα subunit was largely removed from, while PKA RIIα and RIIβ are enriched at, the NMJ-again, preferentially in fast-twitch but not slow-twitch muscle in old mice. Knocking down cTnT in fast skeletal muscle of old mice: (i) increased PKA RIα and reduced PKA RIIα at the NMJ; (ii) decreased the levels of gene expression of muscle denervation markers; and (iii) enhanced neurotransmission efficiency at NMJ.

Conclusions: Cardiac troponin T at the NMJ region contributes to NMJ functional decline with ageing mainly in the fast-twitch skeletal muscle through interfering with PKA signalling. This knowledge could inform useful targets for prevention and therapy of age-related decline in muscle function.

Keywords: Cardiac troponin T; Denervation; Neuromuscular junction; Protein kinase A; Sarcopenia.

Figure 1

Cardiac troponin T (cTnT) mRNA and protein expression in skeletal muscle of older adults. (A) qRT‐PCR quantitation of cTnT in young (Y), old (O), and sciatic denervated young (Y‐SCI) mouse gastrocnemius muscles. **P < 0.01 (n = 3) compared with Y muscles. (B) cTnT protein expression in mouse heart (Hrt) and young (Y) and sciatic denervated (Y‐SCI1, Y‐SCI2) skeletal muscles. For 1C11 immunoblotting, 2 μg Hrt and 100 μg gastrocnemius muscle lysates were loaded; for 4B8 immunoblotting, 5 μg Hrt and 15 μg gastrocnemius muscle lysates were loaded. (C) 1C11 immunoblotting of cTnT in mouse heart, young, and old skeletal muscle. 2 μg Hrt and 100 μg gastrocnemius muscle lysates were loaded. (D) 4B8 immunoblotting of cTnT protein expression in old mouse gastrocnemius (m), vervet monkey vastus lateralis (mky), and human vastus lateralis (hu). Five‐μg mouse heart lysates (hrt) were the positive control; Ponceau S staining of membrane shows even loading of 50 μg total skeletal muscle lysates.

Figure 2

Mass spectrometry determination of cardiac troponin T (cTnT) expression in skeletal muscle of old mice. (A) immunoprecipitation (IP) of cTnT from mouse heart lysates with control IgG, 1C11, and 4B8 antibodies. cTnT was detected with 1C11 antibody. Heart lysate was used as input control. HC, antibody heavy chain detected by secondary antibody. (B) IP of cTnT from old tibialis anterior (TA) muscle whole lysates with control IgG and 1C11. cTnT was detected with 1C11 antibody. Heart lysate and old TA lysate were used as input control. LC, antibody light chain detected by secondary antibody. IB, immunoblotting with 1C11. (C) Coomassie blue R‐250‐stained SDS‐PAGE gels with separated IP products from heart and muscle lysates from old mice. Arrow indicates band of cTnT immunoprecipitated from heart lysates. The red box indicates location of cTnT from skeletal muscle. (D) Mass spectrometry of red boxed area on SDS‐PAGE gel detected peptides from cTnT and several other skeletal muscle proteins (Table S1 ). Twenty one peptide fragments of mouse cTnT isoform g were detected, covering about 37% of the full‐length cTnT protein sequence. SDS‐PAGE, sodium dodecyl sulfate‐polyacrylamide gel electrophoresis.

Figure 3

Cardiac troponin T (cTnT) is highly enriched at the neuromuscular junction (NMJ), mainly in fast‐twitch tibialis anterior (TA) skeletal muscle of old mice. Immunofluorescent staining of cTnT (green) with monoclonal antibody 1C11 and nicotinic acetylcholine receptor (nAChR) (red) with Alexa 594‐conjugated bungarotoxin on longitudinal cryosections of young and old C57BL/6 soleus (SOL) and TA muscle. cTnT enrichment at NMJ in old TA was confirmed by two other mouse monoclonal antibodies (1F11 and 4B8) and one rabbit monoclonal antibody (Figure S5 ). Images are representative of five different young and old mice; in each mouse, at least 15 NMJs were analysed. Scale bar, 50 μm.

Figure 4

Protein kinase A (PKA) RIα and RIIα are differentially enriched at the neuromuscular junction (NMJ) in young and old tibialis anterior (TA) and soleus (SOL) muscle. Immunofluorescent staining indicated that (A) PKA RIα is enriched in most NMJs [nicotinic acetylcholine receptor (nAChR)‐positive region] of young TA muscle and less so in old TA muscle; (B) PKA RIIα is enriched at the NMJ of both young and old SOL muscles, with a slight decrease in old SOL muscle; (C) PKA RIIα is not detected at the NMJ of young TA muscle yet is enriched in almost all NMJs in old TA muscle; (D) PKA RIIα is not detected at the NMJs of young SOL muscle and is detected only in a very small portion of NMJ in the old SOL muscles, with a much weaker signal compared with old TA muscles. Three mice in each group were analysed, with at least 15 NMJs counted in each mouse. *P < 0.05; ****P < 0.0001. Scale bars, 50 μm.

Figure 5

Protein kinase A (PKA) RIIα/RIα ratio increases more with ageing in fast extensor digitorum longus (EDL) muscle than in slow soleus (SOL) muscle. (A) Immunoblots of PKA RIα and RIIα in young and old EDL and SOL muscle lysates. GAPDH was used as a loading control. (B–D) PKA RIα, RIIα, and RIIα/RIα ratio after normalization with GAPDH. Y, young; O, old.*P < 0.05; **P < 0.01; ***P < 0.001 (n = 5).

Figure 6

Gene expression levels of muscle denervation markers are inhibited by cardiac troponin T (cTnT) down‐regulation by shRNA electroporation in old tibialis anterior (TA ) muscle. Ipsilateral TA muscle was electroporated with cTnT‐targeting shRNAs (sh_cTnT); the contralateral TA muscle was electroporated with control shRNA (sh_ctrl). (A) Representative immunofluorescent images indicate that 14 days after electroporation, cTnT was largely down‐regulated at the neuromuscular junction [nicotinic acetylcholine receptor (nAChR)‐positive area] in sh_cTnT electroporated TA muscle. qRT‐PCR analyses of total RNA from each TA muscle indicate that cTnT mRNA was down‐regulated by (B) sh_cTnT, whereas (C) chrng, and (D) Runx1 were down‐regulated in sh_cTnT electroporated TA muscle. Two different sh_cTnTs (see Materials and methods section) were tested in four mice. ***P < 0.001; ****P < 0.0001 (n = 4). Scale bar, 50 μm.

Figure 7

Cardiac troponin T (cTnT) down‐regulation led to increased protein kinase A (PKA) RIα and decreased RIIα enrichment at the neuromuscular junction (NMJ) in tibialis anterior (TA) muscle of old mice. Fourteen days after shRNA electroporation, sh_cTnT electroporated TA muscle shows (A) increased PKA RIα enrichment at NMJ [nicotinic acetylcholine receptor (nAChR)‐positive area] and (B) decreased PKA RIIα enrichment at the NMJ. Most NMJs had fainter immunofluorescent signalling for PKA RIIα. Four mice were analysed with at least 15 NMJs counted in each mouse. ***P < 0.001; ****P < 0.0001. Scale bars, 50 μm.

Figure 8

Cardiac troponin T (cTnT) down‐regulation led to increased maximum amplitude of compound muscle action potentials (CMAP) in old tibialis anterior (TA) muscle. (A) Representative recordings in contralateral and ipsilateral TA 14 days after electroporation of sh_cTnT or sh_ctrl, respectively. (B) Fold changes in CMAP between contralateral and ipsilateral TA muscles in each mouse. The mean maximum CMAP amplitude in sh_ctrl electroporated TA muscle was set as ‘1’ in each mouse. *P < 0.05 (n = 4).