The KATP channel is a molecular sensor of atrophy in skeletal muscle

Abstract

The involvement of ATP-sensitive K(+) (K(ATP)) channels in the atrophy of slow-twitch (MHC-I) soleus (SOL) and fast-twitch (MHC-IIa) flexor digitorum brevis (FDB) muscles was investigated in vivo in 14-day-hindlimb-unloaded (14-HU) rats, an animal model of disuse, and in vitro in drug-induced muscle atrophy. Patch-clamp and gene expression experiments were performed in combination with measurements of fibre diameters used as an index of atrophy, and with MHC labelling in 14-HU rats and controls. A down-regulation of K(ATP) channel subunits Kir6.2, SUR1 and SUR2B with marked atrophy and incomplete phenotype transition were observed in SOL of 14-HU rats. The observed changes in K(ATP) currents were well correlated with changes in fibre diameters and SUR1 expression, as well as with MHC-IIa expression. Half of the SOL fibres of 14-HU rats had reduced diameter and K(ATP) currents and were labelled by MHC-I antibodies. Non-atrophic fibres were labelled by MHC-IIa (22%) antibodies and had enhanced K(ATP) currents, or were labelled by MHC-I (28%) antibodies but had normal current. FDB was not affected in 14-HU rats and this is related to the high expression/activity of Kir6.2/SUR1 subunits characterizing this muscle phenotype. The long-term incubation of the control muscles in vitro with the K(ATP) channel blocker glibenclamide (10(6)m) reduced the K(ATP) currents with atrophy and these effects were prevented by the K(ATP) channel opener diazoxide (10(4)m). The in vivo down-regulation of SUR1, and possibly of Kir6.2 and SUR2B, or their in vitro pharmacological blockade activates atrophic signalling in skeletal muscle. All these findings suggest a new role for the K(ATP) channel as a molecular sensor of atrophy.

Figure 1. Example of electrophoretic (SDS-PAGE) separation of myosin heavy chain (MHC) isoforms in bioptic samples of isolated fibres from 14-HU rats

All samples were pure myosin extracted from single muscle fibre. Gels were Coomassie stained. Lane 1 shows a mixed rat fibre sample used as a reference; lane 2, pure slow type I fibre; lane 3, pure fast type IIa fibre; lane 4, hybrid type I–IIa fibre. No type IIx fibres were detected in our experiments.

Figure 2. Percentage changes in myosin heavy chain (MHC) expression levels, fibre diameters and muscle-to-body weight ratio of slow-twitch soleus (SOL) muscle from 14-day-hindlimb-unloaded (14-HU) rats

A, the mean muscle-to-body weight ratio was calculated for 14-HU rats (n= 10 rats) and normalized with respect to that of control rats (n= 6 rats). The body weights of the 14-HU rats and controls were 355 ± 30 g and 385 ± 30 g, respectively, at the end of the unloading period. The change to fibre diameter was calculated (n= 90 fibres) for the 14-HU rats and normalized with respect to that of control rats (n= 110 fibres). Disuse leads to a significant reduction of the muscle-to-body weight ratio and of fibre diameter. The muscles were rapidly excised from the bones of anaesthetized rats and carefully dried before weighing. B, percentage MHC isoform expression levels were evaluated on muscles from 14-HU and control (CTRL) rats. C, D, E and F, immunofluorescence staining for specific MHC isoforms expressed in SOL muscle sections from 14-HU rats and controls. Disuse leads to a significant increase in the expression levels of the fast type IIa MHC isoform; while no changes were observed in the expression levels of the slow type I MHC isoform. The numbers above the columns indicate the number of sampled muscles/rats. *Significant differences between data are evaluated by an unpaired Student's t test for P < 0.05 or less.

Figure 3. K_ATP channel currents of SOL muscle fibres from control and 14-day-hindlimb-unloaded (14-HU) rats

Sample traces of K_ATP channel currents recorded in excised macropatches from SOL fibres of 14-HU rats and controls during voltage steps from 0 mV holding potential to −60 mV (V_m) with 150 mm KCl on both sides of the membrane, at 20°C. C indicates closed channel levels; O indicates open channel levels. ATP applied on the internal side of the patches inhibited all types of currents. Three types of currents are represented from SOL fibres of 14-HU rats: the first is a sample trace of an atrophic fibre with diameter of 45 μm and K_ATP current amplitude of > −20 pA characterizing the fibre group named A; the second was not atrophic showing a diameter of 80 μm and a K_ATP current amplitude of −150 pA characterizing the fibre group named B; the third was not atrophic showing a K_ATP current amplitude of −85 pA and diameter of 76 μm characterizing the fibre group named C. The K_ATP current of a control fibre had an amplitude of −84 pA and fibre diameter of 76 μm.