Hemin enhances differentiation and maturation of cultured regenerated skeletal myotubes

Satellite cells, isolated from marcaine‐damaged rat skeletal muscle, differentiate in culture to form contracting, cross‐striated myotubes. Addition of 20 μM hemin (ferriprotoporphyrin IX chloride) to the culture medium resulted in increases in the number, size, and alignment of myotubes; in the number of myotubes that exhibited cross‐striations; and in the strength and frequency of myotube contractions. Hemin increased satellite cell fusion by 27%, but decreased cell proliferative rate by 30%. Hemin increased the specific activity of creatine kinase (CK), a sensitive indicator of muscle differentiation, by 157%. Separation of CK isoenzymes by agarose gel electrophoresis showed that hemin increased only the muscle‐specific CK isoenzymes (MM‐CK and MB‐CK). Thus, hemin seems to duplicate some of the effects of innervation on cultured myotubes by increasing contraction frequency and strength, appearance of cross‐striations, and muscle‐specific isoenzymes. In contrast, 3‐amino‐1,2,4‐triazole, an inhibitor of heme biosynthesis, decreased the number of cross‐striated myotubes, the strength and frequency of myotube contractions, and CK activity. These inhibitory effects were reversed by hemin. Collectively, these results demonstrate a physiologically significant role for heme in myotube maturation.