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The Effects of Calcium-β-Hydroxy-β-Methylbutyrate on Aging-Associated Apoptotic Signaling and Muscle Mass and Function in Unloaded but Nonatrophied Extensor Digitorum Longus Muscles of Aged Rats.

Oxidative medicine and cellular longevity (2020-08-11)
Brian T Bennett, Junaith S Mohamed, Stephen E Alway
ABSTRACT

Beta-hydroxy-beta-methylbutyrate (HMB), a naturally occurring leucine metabolite, has been shown to attenuate plantar flexor muscle loss and increase myogenic stem cell activation during reloading after a period of significant muscle wasting by disuse in old rodents. However, it was less clear if HMB would alter dorsiflexor muscle response to unloading or reloading when there was no significant atrophy that was induced by unloading. In this study, we tested if calcium HMB (Ca-HMB) would improve muscle function and alter apoptotic signaling in the extensor digitorum longus (EDL) of aged animals that were unloaded but did not undergo atrophy. The EDL muscle was unloaded for 14 days by hindlimb suspension (HS) in aged (34-36 mo.) male Fisher 344 × Brown Norway rats. The rats were removed from HS and allowed normal cage ambulation for 14 days of reloading (R). Throughout the study, the rats were gavaged daily with 170 mg of Ca-HMB or water 7 days prior to HS, then throughout 14 days of HS and 14 days of recovery after removing HS. The animals' body weights were significantly reduced by ~18% after 14 days of HS and continued to decline by ~22% during R as compared to control conditions; however, despite unloading, EDL did not atrophy by HS, nor did it increase in mass after R. No changes were observed in EDL twitch contraction time, force production, fatigue resistance, fiber cross-sectional area, or markers of nuclear apoptosis (myonuclei + satellite cells) after HS or R. While HS and R increased the proapoptotic Bax protein abundance, BCL-2 abundance was also increased as was the frequency of TUNEL-positive myonuclei and satellite cells, yet muscle mass and fiber cross-sectional area did not change and Ca-HMB treatment had no effect reducing apoptotic signaling. These data indicate that (i) increased apoptotic signaling preceded muscle atrophy or occurred without significant EDL atrophy and (ii) that Ca-HMB treatment did not improve EDL signaling, muscle mass, or muscle function in aged rats, when HS and R did not impact mass or function.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Protease Inhibitor Cocktail, for use with mammalian cell and tissue extracts, DMSO solution
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Phosphatase Inhibitor Cocktail 2, aqueous solution (dark coloration may develop upon storage, which does not affect the activity)
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In Situ Cell Death Detection Kit, Fluorescein, sufficient for ≤50 tests, suitable for detection
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Phosphatase Inhibitor Cocktail 1, DMSO solution
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Ethyl oleate, tested according to Ph. Eur.