Adult fast myosin pattern and Ca ²⁺ -induced slow myosin pattern in primary skeletal muscle culture

Abstract

A primary muscle cell culture derived from newborn rabbit muscle and growing on microcarriers in suspension was established. When cultured for several weeks, the myotubes in this model develop the completely adult pattern of fast myosin light and heavy chains. When Ca 2؉ ionophore is added to the culture medium on day 11, raising intracellular [Ca 2؉ ] about 10-fold, the myotubes develop to exhibit properties of an adult slow muscle by day 30, expressing slow myosin light as well as heavy chains, elevated citrate synthase, and reduced lactate dehydrogenase. The remarkable plasticity of these myotubes becomes apparent, when 8 days after withdrawal of the ionophore a marked slow-to-fast transition, as judged from the expression of isomyosins and metabolic enzymes, occurs.While the alterations that occur in mammalian muscle during fast-to-slow transition in vivo are known in great detail, mostly from chronic electrostimulation experiments with fast hindlimb muscles (1), the information on the mechanism initiating this process is sparse. A reduced intracellular phosphorylation potential and an elevated intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ), as they occur during sustained contractile activity, have been discussed as possible trigger events (2-5). It was our aim to test whether an increase in [Ca 2ϩ ] i imposed on the muscle cell is indeed able to induce a fast-to-slow transition. Because such an experiment cannot be performed in vivo, we were searching for a suitable myogenic cell culture system in which this would become feasible.Thus, this paper has two goals: (i) to report how a primary muscle culture can be grown that develops into an adult-like state, expressing adult myosin of the fast type only, and (ii) to study whether manipulation of [Ca 2ϩ ] i induces a shift between ''fast'' and ''slow'' fiber properties in the cultured myotubes. We show, first, that myotubes derived from newborn rabbit muscles and grown for 4 weeks on microcarriers in suspension possess a purely adult fast myosin pattern. Second, we show that these myotubes exhibit a similarly remarkable plasticity as it is known for adult rabbit muscles (1). This plasticity becomes apparent during exposure of the myotubes to high [Ca 2ϩ ] i , which causes development of the slow isoforms of myosin light chains (MLCs) and myosin heavy chains (MHCs) instead of their fast counterparts, of an increased aerobic metabolic capacity accompanied by a decreased anaerobic capacity as evidenced from elevated citrate synthase (CS) and reduced lactate dehydrogenase (LDH) levels, and of an increased activity ratio of the carbonic anhydrases CA III/CA II. Lowering [Ca 2ϩ ] i back to normal levels is followed by a reversal of all these changes within a few days. EXPERIMENTAL PROCEDURESCulture and Harvesting of Muscle Cells. Newborn White New Zealand rabbits were killed by decapitation. Hindlimb muscles were cut in small pieces and incubated in BSS, pH 7.0 (4.56 mM KCl͞0.44 mM KH 2 PO 4 ͞0.42 mM Na 2 HPO 4 ͞25 mM NaHCO 3 ͞ 119.8 mM NaCl͞50 mg/...

Fiber Types in Mammalian Skeletal Muscles

“…In rodent muscles, values close to 50 -60 nM have been reported in slow fibers versus 30 nM in fast fibers. The influence of resting calcium concentration on the determination and maintenance of fiber phenotype has been first hypothesized by Sreter (757), and experiments on rabbit myotube cultures (443) have confirmed the hypothesis showing that the increase of resting calcium concentration induced by a calcium ionophore is followed by a fast-to-slow transformation (see sect. VII).…”

Fiber type conversion alters inactivation of voltage-dependent sodium currents in murine C2C12 skeletal muscle cells

“…Kubis et al (27) showed that a modest but sustained rise in [Ca 2ϩ ] i levels induced by 400 nM of the Ca 2ϩ ionophore A-23187 caused marked fast-to-slow fiber type conversion in a rabbit primary skeletal muscle cell culture. This crucial study not only emphasized the importance of [Ca 2ϩ ] i for phenotypic adaptations in mammalian skeletal muscle but also provided an easy method by which to induce fast-to-slow fiber type conversion in muscle cell cultures.…”

“…However, changes in intracellular free Ca 2ϩ concentration ([Ca 2ϩ ] i ) seem to be essentially involved. Importantly, Kubis et al (27) showed that a modest but sustained rise in [Ca 2ϩ ] i caused by low concentrations of the Ca 2ϩ ionophore A-23187 in the culture medium induced fast-to-slow fiber type conversion in a rabbit primary skeletal muscle cell culture. This clearly emphasizes the importance of Ca 2ϩ for phenotypic adaptations in skeletal muscle.…”

The p38α/β Mitogen-activated Protein Kinases Mediate Recruitment of CREB-binding Protein to Preserve Fast Myosin Heavy Chain IId/x Gene Activity in Myotubes

“…Fiber type shifts are also observed during aging and disease. The importance of changes in intracellular Ca 2ϩ as a trigger for fiber type transformation has clearly been demonstrated by a Ca 2ϩ -ionophore-induced fast-to-slow transformation in primary skeletal muscle cells (4,5). The involvement of calcineurin, a Ca 2ϩ -calmodulin-regulated serine/threonine phosphatase, in transducing the Ca 2ϩ -signal into altered gene expression in skeletal muscle has been established (6,7).…”

“…We used myotubes of the C2C12 cell line and of a rabbit primary skeletal muscle cell culture that has been shown previously to develop exclusively adult MyHC isoforms (4,5). We provide evidence that inhibition of p38␣/␤ MAPKs resulted in the down-regulation of fast adult MyHCIId/x gene activity.…”