Keita Kanzaki

Neuromuscular electrical stimulation prevents skeletal muscle dysfunction in adjuvant-induced arthritis rat

Skeletal muscle weakness is a prominent feature in patients with rheumatoid arthritis (RA). In this study, we investigated whether neuromuscular electrical stimulation (NMES) training protects against skeletal muscle dysfunction in rats with adjuvant-induced arthritis (AIA). AIA was produced by intraarticular injection of complete Freund’s adjuvant into the knees of Wistar rats. For NMES training, dorsiflexor muscles were stimulated via a surface electrode (0.5 ms pulse, 50 Hz, 2 s on/4 s off). NMES training was performed every other day for three weeks and consisted of three sets produced at three min intervals. In each set, the electrical current was set to achieve 60% of the initial maximum isometric torque and the current was progressively increased to maintain this torque; stimulation was stopped when the 60% torque could no longer be maintained. After the intervention period, extensor digitorum longus (EDL) muscles were excised and used for physiological and biochemical analyses. There was a reduction in specific force production (i.e. force per cross-sectional area) in AIA EDL muscles, which was accompanied by aggregation of the myofibrillar proteins actin and desmin. Moreover, the protein expressions of the pro-oxidative enzymes NADPH oxidase, neuronal nitric oxide synthase, p62, and the ratio of the autophagosome marker LC3bII/LC3bI were increased in AIA EDL muscles. NMES training prevented all these AIA-induced alterations. The present data suggest that NMES training prevents AIA-induced skeletal muscle weakness presumably by counteracting the formation of actin and desmin aggregates. Thus, NMES training can be an effective treatment for muscle dysfunction in patients with RA.

l‐arginine ingestion inhibits eccentric contraction‐induced proteolysis and force deficit via S‐nitrosylation of calpain

It has been shown that calpains are involved in the proteolysis of muscle proteins that occurs with eccentric contraction (ECC) and that exogenously applied nitric oxide decreases the calpain‐mediated proteolysis. The aim of this study was to examine the effects of ingestion of l‐arginine (ARG), a nitric oxide precursor, on ECC‐related calpain activation. In the first and second experiments, male Wistar rats were given ARG in water for 7 days starting from 3 days before the ECC protocol (average ingestion, ~600 mg kg‐body wt−1 day−1). Tibialis anterior muscles underwent 200 repeated ECCs and, subsequently, were excised 3 days later. Whole muscle analyses (the first experiment) revealed that ARG attenuated ECC‐induced force deficit and autolysis of calpain‐1, and increased the amounts of S‐nitrosylated calpain‐1. Regarding ryanodine receptor (RyR) and dihydropyridine receptor (DHPR), ECC‐induced proteolysis was completely inhibited by ARG, whereas the inhibition was partial for junctophilin‐1 (JP1). Skinned fiber analyses (the second experiment) showed that ARG also inhibited ECC‐elicited reductions in the ratio of depolarization‐induced to maximum Ca2+‐activated force. In the third experiment, homogenates of rested muscles were treated with S‐nitrosylating agent, S‐nitrosoglutathione (GSNO), and/or high Ca2+ concentration ([Ca2+]). Treatment with high [Ca2+] and without GSNO produced proteolysis of RyR, DHPR, and JP1. On the other hand, treatment with high [Ca2+] and GSNO caused complete inhibition of RyR and DHPR proteolysis and partial inhibition of JP1 proteolysis. These results indicate that ARG ingestion can attenuate ECC‐induced proteolysis of Ca2+ regulatory proteins and force deficit by decreasing calpain activation via S‐nitrosylation.

Role of calpain in eccentric contraction-induced proteolysis of Ca2+-regulatory proteins and force depression in rat fast-twitch skeletal muscle

The aim of this study was to examine the in vivo effects of eccentric contraction (ECC) on calpain-dependent proteolysis of Ca2+-regulatory proteins and force production in fast-twitch skeletal muscles. Rat extensor digitorum longus muscles were exposed to 200 repeated ECC in situ and excised immediately [recovery 0 (REC0)] or 3 days [recovery 3 (REC3)] after cessation of ECC. Calpain inhibitor (CI)-treated rats were intraperitoneally injected with MDL-28170 before ECC and during REC3. Tetanic force was markedly reduced at REC0 and remained reduced at REC3. CI treatment ameliorated the ECC-induced force decline but only at REC3. No evidence was found for proteolysis of dihydropyridine receptor (DHPR), junctophilin (JP)1, JP2, ryanodine receptor (RyR), sarcoplasmic reticulum Ca2+-ATPase (SERCA)1a, or junctional face protein-45 at REC0. At REC3, ECC resulted in decreases in DHPR, JP1, JP2, RyR, and SERCA1a. CI treatment prevented the decreases in DHPR, JP1, and JP2, whereas it had little effect on RyR and SERCA1a. These findings suggest that DHPR, JP1, and JP2, but not RyR and SERCA1a, undergo calpain-dependent proteolysis in in vivo muscles subjected to ECC and that impaired function of DHPR and/or JP might cause prolonged force deficits with ECC.NEW & NOTEWORTHY Calpain-dependent proteolysis is one of the contributing factors to muscle damage that occurs with eccentric contraction (ECC). It is unclear, however, whether calpains account for proteolysis of Ca2+-regulatory proteins in in vivo muscles subjected to ECC. Here, we provide evidence that dihydropyridine receptor and junctophilin, but not ryanodine receptor and sarcoplasmic reticulum Ca2+-ATPase, undergo calpain-dependent proteolysis.

No relationship between enzyme activity and structure of nucleotide binding site in sarcoplasmic reticulum Ca2+‐ATPase from short‐term stimulated rat muscle

Aim:  We examined whether structural alterations to the adenine nucleotide binding site (ANBS) within sarcoplasmic (endo) reticulum Ca2+‐ATPase (SERCA) would account for contraction‐induced changes in the catalytic activity of the enzyme as assessed in vitro.

The Y54(L)W mutation of anti-leukotriene C4 single-chain antibody increases affinity to leukotriene E4

The X-ray crystal structure of an anti-leukotriene (LT) C4 monoclonal antibody (mAbLTC) in complex with LTC4 was determined, however, crystallographic studies alone are not enough to fully understand the structures of the antigen-binding site. To elucidate the individual contribution of Tyr-54 and Asn-58 in the light chain of mAbLTC, both of which formed a hydrogen bond with glutamic acid of LTC4, we examined whether substitution of the residues affects the antigen binding affinity and specificity using an anti-LTC4 single chain variable fragment (scFvLTC). Among the Tyr-54(L) mutants, Y54(L)W showed a dramatic increase in the affinity to LTE4 which was comparable to that to LTD4. Essentially the same results were obtained using the Y54(L)W mutant expressed in Escherichia coli and Pichia pastoris. The structural modeling suggested the formation of a novel hydrogen bond between the substituted tryptophan in the antibody and the cysteine residue in LTE4. The affinity of Y54(L)R, Y54(L)E and Y54(L)L to LTC4 was markedly reduced, whereas other tested Tyr-54(L) mutants as well as Asn-58(L) mutants did not show significant change in LT binding. The results may provide an insight into the molecular basis of specific LT recognition by the antibody.

Ingestion of soy protein isolate attenuates eccentric contraction-induced force depression and muscle proteolysis via inhibition of calpain-1 activation in rat fast-twitch skeletal muscle

OBJECTIVE Eccentric contraction (ECC) is a contraction in which skeletal muscles are stretched while contracting. The aim of this study was to determine how ingestion of soy protein isolate (SPI) or animal-based proteins affect force deficit, calpain activation, and proteolysis of calcium ion (Ca2+)-regulatory proteins in rat fast-twitch muscles subjected to ECC. METHODS In the first experiment, male Wistar rats were randomly assigned to a control and an SPI group, which were fed a 20% casein and a 20% SPI diet, respectively, for 28 d before the ECC protocol. Anterior crural muscles underwent 200 repeated ECCs and were excised 3 d later. In the second experiment, half of the SPI rats were given water containing NG-nitro-l-arginine-methyl ester (L-NAME), an inhibitor of nitric oxide synthase, for 3 d of recovery after ECC. RESULTS SPI ingestion attenuated ECC-induced force deficit, proteolysis of Ca2+-regulatory proteins, and autolysis of calpain-1. Co-ingestion of L-NAME inhibited SPI-associated increases in nitrite and nitrate levels and negated the force recovery effects of SPI. CONCLUSION These results suggest that SPI ingestion inhibits ECC-elicited force deficit and proteolysis of Ca2+ regulatory proteins, which is caused by inhibited activation of calpain-1 via increased nitric oxide production.

Preconditioning contractions prevent the delayed onset of myofibrillar dysfunction after damaging eccentric contractions

We examined the mechanisms underlying the positive effect of preconditioning contractions (PCs) on the recovery of muscle force after damaging eccentric contractions (ECCs). The mechanisms underlying the immediate force decrease after damaging ECCs differ from those causing depressed force with a few days’ delay, where reactive oxygen species (ROS) produced by invading immune cells play an important causative role. PCs counteracted the delayed onset force depression and this could be explained by prevention of immune cell invasion, which resulted in decreased myeloperoxidase‐mediated ROS production, hence avoiding cell membrane disruption, calpain activation and degenerative changes in myosin and actin molecules.

Electrical Stimulation Prevents Preferential Skeletal Muscle Myosin Loss in Steroid-Denervation Rats

Severe muscle weakness concomitant with preferential depletion of myosin has been observed in several pathological conditions. Here, we used the steroid-denervation (S-D) rat model, which shows dramatic decrease in myosin content and force production, to test whether electrical stimulation (ES) treatment can prevent these deleterious changes. S-D was induced by cutting the sciatic nerve and subsequent daily injection of dexamethasone for 7 days. For ES treatment, plantarflexor muscles were electrically stimulated to produce four sets of five isometric contractions each day. Plantarflexor in situ isometric torque, muscle weight, skinned muscle fiber force, and protein and mRNA expression were measured after the intervention period. ES treatment partly prevented the S-D-induced decreases in plantarflexor in situ isometric torque and muscle weight. ES treatment fully prevented S-D-induced decreases in skinned fiber force and ratio of myosin heavy chain (MyHC) to actin, as well as increases in the reactive oxygen/nitrogen species-generating enzymes NADPH oxidase (NOX) 2 and 4, phosphorylation of p38 MAPK, mRNA expression of the muscle-specific ubiquitin ligases muscle ring finger-1 (MuRF-1) and atrogin-1, and autolyzed active calpain-1. Thus, ES treatment is an effective way to prevent muscle impairments associated with loss of myosin.