Preconditioning contractions prevent Ca2+-dependent proteolysis of STAC3 and prolonged force depression after eccentric contractions

Preconditioning contractions (PCs) have been shown to markedly improve recovery from force depression after damaging eccentric contractions (ECCs). Here, we examined the mechanism underlying the effects of PCs with special focus on the SH3 and cysteine rich domain 3 (STAC3) that is essential for the transduction of action potential to the Ca2+ release from the sarcoplasmic reticulum. Rat medial gastrocnemius (MG) muscles were removed immediately (REC0), 1 d (REC1), and 4 d (REC4) after exposure to 100 repeated in vivo damaging ECCs. PCs with 10 repeated nondamaging ECCs were applied 2 d before the damaging ECCs. Damaging ECCs induced in vivo isometric torque depression at 50 and 100 Hz stimulation frequencies at REC1 and REC4, which was accompanied by a significant reduction in the amount of STAC3, an activation of calpain 1, and an increased number of Evans Blue dye positive fibers in MG muscles. Importantly, PCs attenuated all these deleterious alterations induced by damaging ECCs. Moreover, mechanistic experiments performed on normal muscle tissue exposed to various concentration of Ca2+ showed a Ca2+-dependent proteolysis of STAC3, which was prevented by calpain inhibitor MDL-28170. In conclusion, PCs improve recovery from force depression after damaging ECCs, presumably by inhibiting the loss of STAC3 due to the increased permeability of cell membrane and subsequent activation of calpain 1.

Absence of the Z-disc Protein α-actinin-3 Impairs the Mechanical Stability of Actn3KO Mouse Fast- twitch Muscle Fibres without Altering their Contractile Properties or Twitch Kinetics

Background: A common polymorphism (R577X) in the ACTN3 gene results in complete absence of the Z-disc protein α-actinin-3 from fast-twitch muscle fibres in ~16% of the world’s population. This single gene polymorphism has been subject to strong positive selection pressure during recent human evolution. Previously, using an Actn3KO mouse model, we have shown in fast-twitch muscles, eccentric contractions at L0+ 20% stretch did not cause eccentric damage. In contrast, L0+ 30% stretch produced a significant ~40% deficit in maximum force; here we use isolated single fast-twitch skeletal muscle fibres from the Actn3KO mouse to investigate the mechanism underlying this.Methods: Single fast-twitch fibres are separated from the intact muscle by a collagenase digest procedure. We use label-free second harmonic generation (SHG) imaging, ultra-fast video microscopy and skinned fibre measurements from our MyoRobot automated biomechatronics system to study the morphology, visco-elasticity, force production and mechanical strength of single fibres from the Actn3KO mouse. Data are presented as means ± SD and tested for significance using ANOVA.Results: We show that the absence of α-actinin-3 does not affect the unloaded maximum speed of contraction, visco-elastic properties or myofibrillar force production. Eccentric contractions demonstrated that chemically skinned Actn3KO fibres are mechanically weaker being prone to breakage when eccentrically contracted. Furthermore, SHG images reveal disruptions in the myofibrillar alignment of Actn3KO fast-twitch fibres with an increase in Y-shaped myofibrillar lattice shifts. Conclusions: Absence of α-actinin-3 from the Z-disc in fast-twitch fibres disrupts the organisation of the myofibrillar proteins, leading to structural weakness. This provides a mechanistic explanation for our earlier findings that, in vitro intact Actn3KO fast-twitch muscles are significantly damaged by L0+ 30%, but not, L0+ 20%, eccentric contraction strains. Our study also provides a possible mechanistic explanation as to why α-actinin-3 deficient humans have been reported to have a faster decline in muscle function with increasing age, that is; as sarcopenia reduces muscle mass and force output, the eccentric stress on the remaining functional α-actinin-3 deficient fibres will be increased, resulting in fibres breakages.

Ryanodine receptors mediate high intracellular Ca2+ and some myocyte damage following eccentric contractions in rat fast twitch skeletal muscle

Eccentric contractions (ECC) facilitate cytosolic calcium ion (Ca2+) release from the sarcoplasmic reticulum (SR) and Ca2+ influx from extracellular space. Ca2+ is a vital signaling messenger that regulates multiple cellular processes via its spatial and temporal concentration ([Ca2+]i) dynamics. We hypothesized that: 1) a specific pattern of spatial/temporal intramyocyte Ca2+ dynamics portends muscle damage following ECC, and 2) these dynamics would be regulated by the ryanodine receptor (RyR). [Ca2+]i in the tibialis anterior muscles of anesthetized adult Wistar rats was measured by ratiometric (i.e. ratio, R, 340/380 nm excitation) in vivo bioimaging with Fura-2 pre-ECC and at 5 and 24 hours post-ECC (5 x 40 contractions). Rats received RyR inhibitor dantrolene (DAN; 10 mg/kg i.p.) immediately post-ECC (+DAN). Muscle damage was evaluated by histological analysis on hematoxylin-eosin stained muscle sections. Compared to control (CONT, no ECC), [Ca2+]i distribution was heterogeneous with increased % total area of high [Ca2+]i sites (operationally defined as R > 1.39 i.e., > 1 SD of mean control) 5 hours post-ECC (CONT, 14.0 ± 8.0; ECC5h: 52.0 ± 7.4%, p < 0.01). DAN substantially reduced the high [Ca2+]i area 5 hours post-ECC (ECC5h+DAN: 6.4 ± 3.1%, p < 0.01) and myocyte damage (ECC24h, 63.2 ± 1.0%; ECC24h+DAN, 29.1 ± 2.2%, p < 0.01). Temporal and spatially-amplified [Ca2+]i fluctuations occurred regardless of DAN (ECC vs ECC+DAN, p > 0.05). These results suggest that the RyR-mediated local high [Ca2+]i itself is related to the magnitude of muscle damage while the [Ca2+]i fluctuation is an RyR-independent phenomenon.

Absence of the Z-disc protein α-actinin-3 impairs the mechanical stability of Actn3KO mouse fast-twitch muscle fibres without altering their contractile properties or twitch kinetics

Background: A common polymorphism (R577X) in the ACTN3 gene results in complete absence of the Z-disc protein α-actinin-3 from fast-twitch muscle fibres in ~16% of the worlds population. This single gene polymorphism has been subject to strong positive selection pressure during recent human evolution. Previously, using an Actn3KO mouse model, we have shown in fast-twitch muscles, eccentric contractions at L0+ 20% stretch did not cause eccentric damage. In contrast, L0+ 30% stretch produced a significant ~40% deficit in maximum force; here we use isolated single fast-twitch skeletal muscle fibres from the Actn3KO mouse to investigate the mechanism underlying this. Methods: Single fast-twitch fibres are separated from the intact muscle by a collagenase digest procedure. We use label-free second harmonic generation (SHG) imaging, ultra-fast video microscopy and skinned fibre measurements from our MyoRobot automated biomechatronics system to study the morphology, visco-elasticity, force production and mechanical strength of single fibres from the Actn3KO mouse. Data are presented as means ± SD and tested for significance using ANOVA. Results: We show that the absence of α-actinin-3 does not affect the unloaded maximum speed of contraction, visco-elastic properties or myofibrillar force production. Eccentric contractions demonstrated that chemically skinned Actn3KO fibres are mechanically weaker being prone to breakage when eccentrically contracted. Furthermore, SHG images reveal disruptions in the myofibrillar alignment of Actn3KO fast-twitch fibres with an increase in Y-shaped myofibrillar lattice shifts. Conclusions: Absence of α-actinin-3 from the Z-disc in fast-twitch fibres disrupts the organisation of the myofibrillar proteins, leading to structural weakness. This provides a mechanistic explanation for our earlier findings that, in vitro intact Actn3KO fast-twitch muscles are significantly damaged by L0+ 30%, but not, L0+ 20%, eccentric contraction strains. Our study also provides a possible mechanistic explanation as to why α-actinin-3 deficient humans have been reported to have a faster decline in muscle function with increasing age, that is; as sarcopenia reduces muscle mass and force output, the eccentric stress on the remaining functional α-actinin-3 deficient fibres will be increased, resulting in fibres breakages.

Phytoecdysteroids Accelerate Recovery of Skeletal Muscle Function Following in vivo Eccentric Contraction-Induced Injury in Adult and Old Mice

Background: Eccentric muscle contractions are commonly used in exercise regimens, as well as in rehabilitation as a treatment against muscle atrophy and weakness. If repeated multiple times, eccentric contractions may result in skeletal muscle injury and loss of function. Skeletal muscle possesses the remarkable ability to repair and regenerate after an injury or damage; however, this ability is impaired with aging. Phytoecdysteroids are natural plant steroids that possess medicinal, pharmacological, and biological properties, with no adverse side effects in mammals. Previous research has demonstrated that administration of phytoecdysteroids, such as 20-hydroxyecdysone (20E), leads to an increase in protein synthesis signaling and skeletal muscle strength.Methods: To investigate whether 20E enhances skeletal muscle recovery from eccentric contraction-induced damage, adult (7–8 mo) and old (26–27 mo) mice were subjected to injurious eccentric contractions (EC), followed by 20E or placebo (PLA) supplementation for 7 days. Contractile function via torque-frequency relationships (TF) was measured three times in each mouse: pre- and post-EC, as well as after the 7-day recovery period. Mice were anesthetized with isoflurane and then electrically-stimulated isometric contractions were performed to obtain in vivo muscle function of the anterior crural muscle group before injury (pre), followed by 150 EC, and then again post-injury (post). Following recovery from anesthesia, mice received either 20E (50 mg•kg−1 BW) or PLA by oral gavage. Mice were gavaged daily for 6 days and on day 7, the TF relationship was reassessed (7-day).Results: EC resulted in significant reductions of muscle function post-injury, regardless of age or treatment condition (p &lt; 0.001). 20E supplementation completely recovered muscle function after 7 days in both adult and old mice (pre vs. 7-day; p &gt; 0.05), while PLA muscle function remained reduced (pre vs. 7-day; p &lt; 0.01). In addition, histological markers of muscle damage appear lower in damaged muscle from 20E-treated mice after the 7-day recovery period, compared to PLA.Conclusions: Taken together, these findings demonstrate that 20E fully recovers skeletal muscle function in both adult and old mice just 7 days after eccentric contraction-induced damage. However, the underlying mechanics by which 20E contributes to the accelerated recovery from muscle damage warrant further investigation.

Preconditioning contractions prevent prolonged force depression and Ca2+-dependent proteolysis of STAC3 after damaging eccentric contractions

Preconditioning contractions (PCs) have been shown to markedly improve recovery from eccentric contractions (ECCs)-induced force depression. We here examined the mechanism behind the effects of PCs with focusing on the SH3 and cysteine rich domain 3 (STAC3) that is essential for coupling membrane depolarization to Ca2+ release from the sarcoplasmic reticulum. Rat medial gastrocnemius (MG) muscles were excised immediately (REC0), 1 day (REC1), and 4 days (REC4) after exposure to 100 repeated damaging ECCs in vivo. PCs with 10 repeated non-damaging ECCs were applied 2 days before the damaging ECCs. Damaging ECCs induced in vivo isometric torque depression at 50 and 100 Hz stimulation frequencies, which was accompanied by a significant decrease in the amount of full-length STAC3, an activation of calpain 1, and an increased number of Evans Blue dye positive fibers in MG muscles at REC1 and REC4. Interestingly, PCs attenuated all these deleterious alterations induced by damaging ECCs. Moreover, mechanistic experiments performed on normal muscle samples exposed to various concentration of Ca2+ showed a Ca2+-dependent proteolysis of STAC3, which was prevented by calpain inhibitor MDL-28170. In conclusion, PCs may improve recovery from force depression after damaging ECCs, in part by inhibiting the loss of STAC3 due to the increased permeability of cell membrane and subsequent activation of calpain 1.