Carnosine, an endogenous cytoplasmic dipeptide (ß-alanyl-l-histidine), has been shown to be important in a myriad of cellular processes such as pH buffering, membrane stabilization, acting as an osmotic shock protector, anti-oxidant and anti-aging agent (see reviews Derave et al., 2010; Sale et al., 2010). However, its effects on excitation-contraction (EC) coupling are not well defined. There is considerable interest into the potential ergogenic and therapeutic effects of carnosine supplementation (Kendrick et al., 2009; Baguet et al., 2010a & b; Derave et al., 2010; Sale et al., 2010). Consequently, we sought to characterize what effect carnosine, at levels attained by supplementation, has on human muscle function, using a skinned fibre preparation in which all key EC coupling proteins are in their in situ positions.
All protocols and procedures were approved by the Human Research Ethics Committee at Victoria University. A muscle needle biopsy was taken from the middle third of the vastus lateralis muscle from four healthy subjects who had given written informed consent. After injection of a local anaesthetic into the skin and fascia (1% lidocaine (Xylocaine)), a small incision was made and a muscle sample taken (∼150 mg) using a Bergstrom biopsy needle. Individual fibre segments, obtained from the biopsy, were mechanically skinned and their sarcoplasmic reticulum (SR)-Ca2+-handling and contractile apparatus properties were characterized. Thereafter, western blotting was performed on the same fibre segments to precisely determine their fibre-type. All solutions (including carnosine-containing solutions) used in this study are identical to those described in Dutka & Lamb (2004). A carnosine stock was made similar to the standard K-HDTA solution but with 80mM carnosine and reduced [HEPES]. When used at the final [carnosine] changes to osmolality and ionic strength were minor.
The effects of carnosine on the properties of the contractile apparatus were determined by exposing each fibre segment to a series of heavily Ca2+-buffered solutions containing progressively higher free [Ca2+] until maximum Ca2+-activated force was produced. Hill curves were fitted and the mean change (Δ) in pCa50 (where pCa50 = -log10[Ca2+]) determined. Compared to control levels, the Ca2+-sensitivity of the contractile apparatus was significantly increased by the presence of 8 and 16 mM carnosine (Δ pCa50 for six type I fibres: 0.073±0.007 and 0.116±0.006 pCa units for 8 and 16 mM respectively, and 0.063±0.018 and 0.103±0.013 pCa units for 8 and 16 mM respectively in five type II fibres). This equates to an increase in absolute force of ∼20% (e.g. 50% force would be 70% in the presence of carnosine). Caffeine (8 mM)-induced responses were potentiated by 8 mM carnosine in both type I and II fibres, with the level of potentiation in type II fibres being entirely explicable by the increase in Ca2+-sensitivity of the contractile apparatus caused by carnosine. However, the potentiation of caffeine-induced responses caused by carnosine in type I fibres was beyond that expected from the associated increase in Ca2+-sensitivity of the contractile apparatus and suggestive that carnosine potentiated Ca2+-induced Ca2+-release.
These findings suggest that increasing muscle carnosine content via supplementation or by dietary means could confer benefits on muscle performance in both type I and type II fibres based on the increase in Ca2+-sensitivity of the contractile apparatus. However, the potentiation of caffeine-induced SR Ca2+ release caused by carnosine in type I fibres may not be manifested in vivo when Ca2+ release is strictly controlled by dihydropyridine receptors (Lamb et al., 2003). Increasing Ca2+-sensitivity of the contractile apparatus and potentiating Ca2+ release might help to lessen the decline in force output observed during muscle fatigue.
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