Background: Astaxanthin (AX) a marine carotenoid is a powerful natural antioxidant which protects against oxidative stress and improves muscle performance. Retinol and its derivatives were described to affect lipid and energy metabolism. Up to date, the effects of AX and retinol on excitation-contraction coupling (ECC) in skeletal muscle are poorly described. Methods: 18 C57Bl6 mice were divided into two groups: Control and AX supplemented in rodent chow for 4 weeks (AstaReal A1010). In vivo and in vitro force and intracellular calcium homeostasis was studied. In some experiments acute treatment with retinol was employed. Results: The voltage activation of calcium transients (V₅₀) were investigated in single flexor digitorum brevis isolated fibers under patch clamp and no significant changes were found following AX supplementation. Retinol shifted V₅₀ towards more positive values and decreased the peak F/F₀ of the calcium transients. The amplitude of tetani in the extensor digitorum longus was significantly higher in AX than in control group. Lastly, the mitochondrial calcium uptake was found to be less prominent in AX. Conclusion: AX supplementation increases in vitro tetanic force without affecting ECC and exerts a protecting effect on the mitochondria. Retinol treatment has an inhibitory effect on ECC in skeletal muscle.

The endocannabinoid system (ECS) refers to a widespread signaling system and its alteration is implicated in a growing number of human diseases. Cannabinoid receptors (CBRs) are highly expressed in the central nervous system and many peripheral tissues. Evidence suggests that CB1Rs are expressed in human and murine skeletal muscle mainly in the cell membrane, but a subpopulation is present also in the mitochondria. However, very little is known about the latter population. To date, the connection between the function of CB1Rs and the regulation of intracellular Ca²⁺ signaling has not been investigated yet. Tamoxifen-inducible skeletal muscle-specific conditional CB1 knock-down (skmCB1-KD, hereafter referred to as Cre^+/−) mice were used in this study for functional and morphological analysis. After confirming CB1R down-regulation on the mRNA and protein level, we performed in vitro muscle force measurements and found that peak twitch, tetanus, and fatigue were decreased significantly in Cre^+/− mice. Resting intracellular calcium concentration, voltage dependence of the calcium transients as well as the activity dependent mitochondrial calcium uptake were essentially unaltered by Cnr1 gene manipulation. Nevertheless, we found striking differences in the ultrastructural architecture of the mitochondrial network of muscle tissue from the Cre^+/− mice. Our results suggest a role of CB1Rs in maintaining physiological muscle function and morphology. Targeting ECS could be a potential tool in certain diseases, including muscular dystrophies where increased endocannabinoid levels have already been described.

The remodelling of the extracellular matrix plays an important role in skeletal muscle development and regeneration. Syndecan-4 is a cell surface proteoglycan crucial for muscle differentiation. Syndecan-4^−/− mice have been reported to be unable to regenerate following muscle damage. To investigate the consequences of the decreased expression of Syndecan-4, we have studied the in vivo and in vitro muscle performance and the excitation–contraction coupling machinery in young and aged Syndecan-4^+/− (SDC4) mice. In vivo grip force was decreased significantly as well as the average and maximal speed of voluntary running in SDC4 mice, regardless of their age. The maximal in vitro twitch force was reduced in both EDL and soleus muscles from young and aged SDC4 mice. Ca²⁺ release from the sarcoplasmic reticulum decreased significantly in the FDB fibres of young SDC4 mice, while its voltage dependence was unchanged regardless of age. These findings were present in muscles from young and aged mice as well. On C2C12 murine skeletal muscle cells, we have also found altered calcium homeostasis upon Syndecan-4 silencing. The decreased expression of Syndecan-4 leads to reduced skeletal muscle performance in mice and altered motility in C2C12 myoblasts via altered calcium homeostasis. The altered muscle force performance develops at an early age and is maintained throughout the life course of the animal until old age.

Aging and frailty are associated with a decline in muscle force generation, which is a direct consequence of reduced muscle quantity and quality. Among the leading contributors to aging is the generation of reactive oxygen species, the byproducts of terminal oxidation. Their negative effects can be moderated via antioxidant supplementation. Krill oil and astaxanthin (AX) are nutraceuticals with a variety of health promoting, geroprotective, anti-inflammatory, anti-diabetic and anti-fatigue effects. In this work, we examined the functional effects of these two nutraceutical agents supplemented via pelleted chow in aging mice by examining in vivo and in vitro skeletal muscle function, along with aspects of intracellular and mitochondrial calcium homeostasis, as well as cognition and spatial memory. AX diet regimen limited weight gain compared to the control group; however, this phenomenon was not accompanied by muscle tissue mass decline. On the other hand, both AX and krill oil supplementation increased force production without altering calcium homeostasis during excitation-contraction coupling mechanism or mitochondrial calcium uptake processes. We also provide evidence of improved spatial memory and learning ability in aging mice because of krill oil supplementation. Taken together, our data favors the application of antioxidant nutraceuticals as geroprotectors to improve cognition and healthy aging by virtue of improved skeletal muscle force production.

Skeletal muscle, constituting 40–50% of total body mass, is vital for mobility, posture, and systemic homeostasis. Muscle contraction heavily relies on ATP, primarily generated by mitochondrial oxidative phosphorylation. Mitochondria play a key role in decoding intracellular calcium signals. The endocannabinoid system (ECS), including CB₁ receptors (CB₁Rs), broadly influences physiological processes and, in muscles, regulates functions like energy metabolism, development, and repair. While plasma membrane CB₁Rs (pCB₁Rs) are well-established, a distinct mitochondrial CB₁R (mtCB₁R) population also exists in muscles, influencing mitochondrial oxidative activity and quality control. We investigated the role of mtCB₁Rs in skeletal muscle physiology using a novel systemic mitochondrial CB₁ deletion murine model. Our in vivo studies showed no changes in motor function, coordination, or grip strength in mtCB₁ knockout mice. However, in vitro force measurements revealed significantly reduced specific force in both fast-twitch (EDL) and slow-twitch (SOL) muscles following mtCB₁R ablation. Interestingly, knockout EDL muscles exhibited hypertrophy, suggesting a compensatory response to reduced force quality. Electron microscopy revealed significant mitochondrial morphological abnormalities, including enlargement and irregular shapes, correlating with these functional deficits. High-resolution respirometry further demonstrated impaired mitochondrial respiration, with reduced oxidative phosphorylation and electron transport system capacities in knockout mitochondria. Crucially, mitochondrial membrane potential dissipated faster in mtCB₁ knockout muscle fibers, whilst mitochondrial calcium levels were higher at rest. These findings collectively establish that mtCB₁Rs are critical for maintaining mitochondrial health and function, directly impacting muscle energy production and contractile performance. Our results provide new insights into ECS-mediated regulation of skeletal muscle function and open therapeutic opportunities for muscle disorders and aging.

The endocannabinoid system (ECS) is known to regulate crucial bodily functions, including healthy muscle activity. However, its precise roles in normal skeletal muscle function and the development of muscle disorders remain unclear. Previously, we developed a tamoxifen-inducible, skeletal muscle-specific CB₁ receptor knockdown (skmCB1-KD) mouse model using the Cre/LoxP system. In this study, we aimed to clarify the mechanisms behind the observed reduction in muscle force generation in these mice. To investigate this, we analyzed calcium dynamics following electrical stimulation-induced muscle fatigue, assessed store-operated calcium entry (SOCE), and performed functional analysis of mitochondrial respiration. Our findings suggest that the reduced muscle performance observed in vivo likely arises from interconnected alterations in ATP production by mitochondria. Moreover, in skmCB1-KD mice, we detected a significant decrease in a component of the respiratory chain (complex IV) and a slowed dissipation of mitochondrial membrane potential upon the addition of an un-coupler (FCCP).