Disuse of rat muscle in vivo reduces protein kinase C activity controlling the sarcolemma chloride conductance

Abstract

Muscle disuse produced by hindlimb unloading (HU) induces severe atrophy and slow-to-fast fibre type transition of the slow-twitch soleus muscle (Sol). After 2 weeks HU, the resting ClC-1 chloride conductance (g(Cl)) of sarcolemma, which controls muscle excitability, increases in Sol toward a value typical of the fast-twitch EDL muscle. After 3 days of HU, the g(Cl) increases as well before initiation of fibre type transition. Since ClC-1 channels are acutely silenced by PKC-dependent phosphorylation, we studied the modulation of g(Cl) by PKC and serine-threonine phosphatase in Sol during HU, using a number of pharmacological tools. We show that a fraction of ClC-1 channels of control Sol are maintained in an inactive state by PKC basal activity, which contributes to the lower g(Cl) in control Sol compared to EDL. After 14 days of HU, PKC/phosphatase manipulation produces effects on Sol g(Cl) that corroborate the partial slow-to-fast transition. After 3 days of HU, the early increase of g(Cl) in Sol is entirely attributable to a reduction of PKC activity and/or activation of phosphatase, maintaining ClC-1 channels in a fully active state. Accordingly, we found that HU reduces expression of PKCalpha, epsilon, and isoenzymes in Sol and EDL muscles and reduces total PKC activity. Moreover, we show that the rheobase current is increased in Sol muscle fibres as soon as after 3 days of HU, most probably in relation to the increased g(Cl). In conclusion, Sol muscle disuse is characterized by a rapid reduction of PKC activity, which reduces muscle excitability and is likely to contribute to disuse-induced muscle impairment.

Figure 1

Effects of disuse on the protein kinase C-dependent modulation of sarcolemma chloride conductance in fast-twitch and slow-twitch muscles A, effects of 50 or 100 nm 4-β-phorbol-dibutyrate (PDB), an activator of PKC, on the total resting conductance of sarcolemma measured in control EDL muscle, control Sol muscle, and Sol muscles after 3 or 14 days' hindlimb unloading (HU3 or HU14). B, effects of 1 μm chelerythrine, an inhibitor of PKC, on the resting chloride conductance of sarcolemma measured in the same experimental conditions as in A. Each bar represent the mean value ±s.e.m. from at least 11 fibres/2 muscles. Symbols indicate significant difference (P < 0.05, Student's unpaired t test) between treated fibres and control (*), between fibres treated with 50 and 100 nm PDB (#), between Sol and EDL muscle fibres (†), and between HU and control Sol muscle fibres (‡).

Figure 2

Effects of disuse on the modulation of sarcolemma chloride conductance by okadaic-sensitive protein phosphatase in fast-twitch and slow-twitch muscles A, effects of 0.25 μm okadaic acid, an inhibitor of phosphatase, on the resting chloride conductance of sarcolemma measured in control EDL, control Sol muscle, and Sol muscles after 3 or 14 days' hindlimb unloading (HU3 or HU14). B, effects of 3.3 nm IGF-1, an activator of protein phosphatase, on the resting chloride conductance of sarcolemma in the same experimental conditions as in A. Each bar represents the mean value ±s.e.m. from at least 22 fibres/2 muscles. Symbols indicate significant difference (P < 0.05, Student's unpaired t test) between treated fibres and control (*), between Sol and EDL muscle fibres (†), and between HU and control Sol muscle fibres (‡).

Figure 3

Effects of disuse on protein expression levels of PKCα, PKCɛ, and PKCθ isoenzymes in fast-twitch and slow-twitch muscles Cell extracts were prepared from EDL and Sol muscle of control rats and after 3 (HU3) or 14 (HU14) days' hindlimb unloading (2 rats for each condition). Western blots were immunolabelled using antibodies against PKCα (A), PKCɛ (B), and PKCθ (C) isoenzymes, as well as muscle creatine kinase. For each muscle, the measure was repeated in three independent experiments. PKC protein levels were quantified by densitometry, normalized with respect to creatine kinase signal, and expressed as percentage ±s.e.m. of control muscle. Symbols indicate significant difference (P < 0.05, Student's paired t test) between HU and control (*), and between HU3 and HU14 (#),

Figure 4

Effects of disuse on total PKC activity in fast-twitch and slow-twitch muscles PKC total activity was evaluated in cell extracts from EDL muscle and Sol muscle of control rats and after 3 (HU3) or 14 (HU14) days' hindlimb unloading (2 rats for each condition). For each muscle, the measure was repeated in three independent experiments. Data are expressed as percentage ±s.e.m. of the activity detected in control muscles. Symbols indicate significant difference (P < 0.05, Student's paired t test) between HU and control (*), and between HU3 and HU14 (#).

Figure 5

Schematic diagram of Sol muscle chloride conductance modulation by hindlimb unloading- induced disuse In control conditions, slow-twitch fibres (red dashed) in the Sol muscle express a lower amount of ClC-1 channels than the fast-twitch fibres (grey dashed) in the EDL muscle. In addition, a fraction of Sol muscle ClC-1 channels are in an inactive state due to basal PKC activity, which contributes to the lower g_Cl in the slow-twitch muscle. There are a number of pieces of evidence suggesting that the phosphorylation site is the ClC-1 channel protein itself, as shown in the diagram, but we cannot exclude an indirect effect of PKC on ClC-1 channel function (see Discussion). After 3 days of HU, PKC activity is reduced in the whole Sol muscle, thus leading to inactivation of ClC-1 channels and elevated g_Cl. A similar effect can be obtained acutely by application of chelerythrine (PKC inhibition) or IGF-1 (phosphatase activation) in control Sol muscle fibres. After 14 days of HU, part of the Sol muscle fibres acquire a fast phenotype characterized by expression of myosin heavy chain type II isoform. These fast fibres show an increased expression of ClC-1 channels, which are fully active owing to a low basal activity of PKC. The remaining slow-twitch fibres of HU14 Sol muscle conserve the characteristics of control Sol muscle fibres, with a low expression of ClC-1 channels and a partial PKC-dependent ClC-1 channel inactivation.