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

Abstract

. Fiber type conversion alters inactivation of voltage-dependent sodium currents in murine C2C12 skeletal muscle cells. Am J Physiol Cell Physiol 287: C270 -C280, 2004. First published March 24, 2004 10.1152/ ajpcell.00015.2004.-Each skeletal muscle of the body contains a unique composition of "fast" and "slow" muscle fibers, each of which is specialized for certain challenges. This composition is not static, and the muscle fibers are capable of adapting their molecular composition by altered gene expression (i.e., fiber type conversion). Whereas changes in the expression of contractile proteins and metabolic enzymes in the course of fiber type conversion are well described, little is known about possible adaptations in the electrophysiological properties of skeletal muscle cells. Such adaptations may involve changes in the expression and/or function of ion channels. In this study, we investigated the effects of fast-to-slow fiber type conversion on currents via voltage-gated Na ϩ channels in the C2C12 murine skeletal muscle cell line. Prolonged treatment of cells with 25 nM of the Ca 2ϩ ionophore A-23187 caused a significant shift in myosin heavy chain isoform expression from the fast toward the slow isoform, indicating fast-to-slow fiber type conversion. Moreover, Na ϩ current inactivation was significantly altered. Slow inactivation less strongly inhibited the Na ϩ currents of fast-to-slow fiber type-converted cells. Compared with control cells, the Na ϩ currents of converted cells were more resistant to block by tetrodotoxin, suggesting enhanced relative expression of the cardiac Na ϩ channel isoform Nav1.5 compared with the skeletal muscle isoform Na v1.4. These results imply that fast-toslow fiber type conversion of skeletal muscle cells involves functional adaptation of their electrophysiological properties. muscle plasticity; myosin heavy chain expression; sodium channel expression ADULT SKELETAL MUSCLE has a remarkable capacity to adapt in response to altered functional demands such as enhanced activity or disuse. On the basis of contraction kinetics, a distinction is drawn between "fast" and "slow" skeletal muscles, which contain predominantly fast-and slow-type muscle fibers, respectively. These fiber types express specific sets of fast and slow protein isoforms (for review, see Refs. 5,36,47) and are generally categorized according to the specific myosin heavy chain (MHC) isoforms that they express. In adaptation to altered functional demands, muscle fibers can switch from the fast to the slow fiber type and vice versa (i.e., fiber type conversion). Fiber type conversion involves morphological and biochemical changes that result in altered contractile properties and endurance capacities. It is well established that motor neuron firing patterns control the expression of isoforms of contractile proteins and metabolic enzymes of muscle fibers in vivo (6, 44). These firing patterns have been mimicked successfully in vitro in cell culture studies by imposed electrical stimulation (28, 58). The cellular s...

C2C12 skeletal muscle cells adopt cardiac-like sodium current properties in a cardiac cell environment

“…A detailed description of our MATERIALS AND METHODS is given in a previous report by our group (49).…”

“…Mouse C 2C12 skeletal muscle cells (CRL-1772; American Type Culture Collection, Manassas, VA) were grown and differentiated on Matrigel (Becton Dickinson, Schwechat, Austria)-coated culture dishes as previously described (49). Cardiac primary cultures for the conditioning procedure were prepared from healthy neonatal Wistar rat (2-3 days old) hearts in accordance with a protocol described by Simpson and Savion (38) and coinciding with the rules of the University Animal Welfare Committee.…”

“…Primers for the PCR reaction were designed in the 3Ј untranslated regions of the transcripts where the isoforms share almost no homology (skeletal muscle Nav1.4 forward, 5Ј-TGAAGATCCCGCCTCCTGA-3Ј; Nav1.4 reverse, 5Ј-AGTTTGTCTTTGTCCTGGCTA-3Ј; cardiac Nav1.5 forward, 5Ј-CGTGCCCTGTTGTATCCTG-3Ј; Nav1.5 reverse, 5Ј-CCAGCCAGGGTTGCTCGA-3Ј). These primers were used in a previous study by our group (49). PCR amplification of cDNAs (25 cycles, i.e., within the linear range of these PCRs) was performed with the Expand High Fidelity PCR System (Roche).…”

“…Na ϩ currents were recorded from differentiated spherically shaped C2C12 cells (C2C12 myoballs) at room temperature (22 Ϯ 1.5°C) using an Axoclamp 200B patch-clamp amplifier (Axon Instruments, Union City, CA) as described previously (49). Recording was begun ϳ10 min after whole cell access was attained to minimize time-dependent shifts in gating.…”

“…Na ϩ current density was calculated by dividing the maximum peak inward current amplitude (normally at Ϫ25 mV) of a cell by its membrane capacitance (49). Resting membrane potentials were measured using the whole cell patch-clamp technique in the current-clamp mode.…”