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 skeletal muscles are increased intracellular calcium levels, the occurrence of hypercontraction, and the overproduction of oxygen free radicals. In the present study, we investigated whether muscle preservation during cold storage 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) from the rat were isolated and stored for 16 hr at 4 degrees C in Bretschneider's Histidine Tryptophane Ketoglutarate (HTK) and subsequently acclimatized in Krebs-Henseleit solution for 90 min at room temperature. The protective effects of 2,3-butanedione monoxime (BDM; reduces intracellular calcium release and inhibits fiber contraction) and of the following antioxidants were investigated: N-tert-butyl-alpha-phenylnitrone (PBN), trolox, desferal, and deferione. The antioxidants and BDM were added to both HTK and Krebs-Henseleit solution. Dose-response curves were made for each of the additives (n> or =4 for each dose). To evaluate the effect of oxygen supply, HTK was aerated with 95% O2/5% CO2. Muscle function (P0), energy metabolism (ATP), and cytoarchitecture were analyzed. The measured values were compared with those of fresh unstored muscles (% of control) and with those of muscles stored in HTK without any additive (multivariate analysis of variance, P<0.05).


RESULTS
We found a significant protection of the contractile function (P0) 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 (P0 for both muscles: 55%), whereas no protection was found with PBN (0.03-1 mM) and Desferal (0.001-1 mM). The addition of BDM (10 or 30 mM) resulted in the highest increase of P0 (84% and 60% for the SOL and CT, respectively). The combinations BDM-trolox and BDM-deferione did not further improve the preservation of the SOL function, but P0 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 (P0: 83%). The improved preservation of muscle function was accompanied by a reduction of the twitch threshold current, increased by storage, suggesting a protective effect of the intervention on the preservation of the muscle cell membrane integrity. Biochemical and histological data corresponded well 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 muscles after cold storage for 16 hr.
van der Heijden
E P
EP
Department of Plastic, Reconstructive and Handsurgery, University Medical Center of Utrecht, The Netherlands. b.heijden@chir.azu.nl
Kroese
A B
AB
Werker
P M
PM
de With
M C
MC
de Smet
M
M
Kon
M
M
Bär
D P
DP
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D002614:Chelating Agents / Q000494:pharmacology; D002839:Chromans / Q000494:pharmacology; D015925:Cryopreservation / Q000592:standards*; D003497:Cyclic N-Oxides; D003676:Deferoxamine / Q000494:pharmacology; D003931:Diacetyl / Q000031:analogs & derivatives / Q000494:pharmacology; D004338:Drug Combinations; D004558:Electric Stimulation; D004734:Energy Metabolism; D005947:Glucose / Q000494:pharmacology; D008297:Male; D008353:Mannitol / Q000494:pharmacology; D009119:Muscle Contraction / Q000187:drug effects; D018482:Muscle, Skeletal / Q000166:cytology / Q000187:drug effects; D009589:Nitrogen Oxides / Q000494:pharmacology; D010100:Oxygen / Q000494:pharmacology; D011189:Potassium Chloride / Q000494:pharmacology; D011343:Procaine / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D013997:Time Factors
D000975:Antioxidants; C039984:Bretschneider cardioplegic solution; D002614:Chelating Agents; D002839:Chromans; D003497:Cyclic N-Oxides; D004338:Drug Combinations; D009589:Nitrogen Oxides; C004717:diacetylmonoxime; C029217:phenyl-N-tert-butylnitrone; D008353:Mannitol; D011343:Procaine; D011189:Potassium Chloride; D005947:Glucose; D003676:Deferoxamine; D003931:Diacetyl; C010643:6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; D010100:Oxygen