Improving the preservation of isolated rat skeletal muscles stored for 16 hours at 4 degrees C

Background. Limiting factors for long-term cold preservation of isolated sk eletal muscles are increased intracellular calcium levels, the occurrence o f hypercontraction, and the overproduction of oxygen free radicals. In the present study, we investigated whether muscle preservation during cold stor age could be improved by additives that can protect against such processes or by oxygen supply. Methods. The soleus (SOL) and a strip of the cutaneus trunci muscle (CT) fr om the rat were isolated and stored for 16 hr at 4 degrees C in Bretschneid er's Histidine Tryptophane Ketoglutarate (HTK) and subsequently acclimatize d in Krebs-Henseleit solution for 90 min at room temperature. The protectiv e effects of 2,3-butanedione monoxime (BDM; reduces intracellular calcium r elease and inhibits fiber contraction) and of the following antioxidants we re investigated: N-tert-butyl-alpha-phenylnitrone (PBN), trolox, desferal, and deferione. The antioxidants and BDM were added to both HTK and Krebs-He nseleit solution. Dose-response curves were made for each of the additives (n greater than or equal to 4 for each dose). To evaluate the effect of oxy gen supply, HTR was aerated with 95% O-2/5% CO2. Muscle function (P-0), ene rgy metabolism (ATP), and cytoarchitecture were analyzed. The measured valu es were compared with those of fresh unstored muscles (% of control) and wi th those of muscles stored in HTK without any additive (multivariate analys is of variance, P<0.05). Results. We found a significant protection of the contractile function (P-0 ) of both muscles after the addition of 1 mM of trolox (SOL: 46% of control ; CT: 53%) and after the addition of 3 mM or 0.3 mM of deferione to the SOL and CT, respectively (P-0 for both muscles: 55%), whereas no protection wa s found with PEN (0.03-1 mM) and Desferal (0.001-1 mM). The addition of BDM (10 or 30 mM) resulted in the highest increase of P-0 (84% and 60% for the SOL and CT, respectively). The combinations BDM-trolox and BDM-deferione d id not further improve the preservation of the SOL function, but P-0 values (88% and 91% of control, respectively) were not different from those found for control muscles, Oxygenation of HTK was only beneficial for the SOL (P -0: 83%). The improved preservation of muscle function was accompanied by a reduction of the twitch threshold current, increased by storage, suggestin g a protective effect of the intervention on the preservation of the muscle cell membrane integrity. Biochemical and histological data corresponded we ll with the functional data. Conclusions. The results showed that the addition of BDM and antioxidants ( trolox and deferione) to the bathing solutions improved the preservation of the function, metabolism, and cytoarchitecture of isolated skeletal muscle s after cold storage for 16 hr.