S. Pierno¹, A. Liantonio¹, G.M. Camerino¹, A. Scaramuzzi¹, M.M. Dinardo¹, M. De Bellis¹, C. Digennaro¹, L. Madaro², M. Bouchè², J.-F. Desaphy¹ and D. Conte Camerino¹

¹Dept. of Pharmacobiology, Faculty of Pharmacy, Aldo Moro University of Bari, Bari, Italy; ²Dept. of Histology and Medical Embryology, Sapienza University of Rome, Rome, Italy.

In skeletal muscle the resting chloride conductance (gCl), maintained by the ClC-1 chloride channel, controls the sarcolemma electrical stability and its reduction may produce myotonia-like symptoms. By using pharmacological tools we previously showed that gCl is negatively regulated in vitro by Protein Kinase C (PKC). Slow-twitch muscles are characterized by lower value of gCl compared to fast-twitch muscles, due in part to a higher basic activity of PKC (Pierno et al., 2007) and in part to reduced ClC-1 expression. In skeletal muscle different PKC isoforms have been found to be expressed, including the novel isoform PKC theta (Serra et al., 2003). To better characterize the modulation of gCl by PKC and the possible involvement in muscle diseases we measured resting gCl and muscle excitability in soleus and extensor digitorum longus (EDL) muscles of PKC theta null mice (Sun et al., 2000). The electrophysiological experiments were done by using the two intracellular microelectrode technique. We found a significant increase of gCl in soleus muscle of null mice with respect to control, being 1876±53 µS/cm2 (n=41) and 1356±37 µS/cm2 (n=19), respectively. A slight increase of gCl was also found in EDL muscle. Muscle excitability was reduced accordingly to gCl increase. Chelerythrine, a non-specific PKC inhibitor, further increase gCl (by 25%) in soleus muscle showing that other PKC isoforms are involved in the control of gCl. In contrast chelerythrine have minor effect on EDL of PKC theta null mice. We used fluvastatin, a hypocholesterolemic drug, as a tool able to stimulate PKC activity (Pierno et al., 2009). This drug significantly reduced gCl in EDL muscle of PKC null mice by 30% confirming that also in EDL other PKC isoforms contribute to ClC-1 channel modulation. To gain insight into the mechanism of regulation we evaluate the expression of ClC-1 channel by real time-PCR. We found no modification of ClC-1 expression either in soleus or EDL muscle of PKC theta null mice with respect to healthy mice, demonstrating that the ClC-1 expression was not involved. Moreover we evaluated the resting cytosolic (restCa) calcium amount in the PKC null mice by cytofluorimetric analysis. As already described (Fraysse et al., 2003), we found a higher concentration of restCa in soleus muscle with respect to EDL. Interestingly, we found a significant reduction of restCa in both muscle types as compared to the wild-type. In particular, the restCa was 40±1.7 nM and 87±2.5 nM, in EDL and soleus of PKC null mice, respectively, while was 52±3.7 nM and 113±9.1 nM, in EDL and soleus of control mice. To evaluate the causes of calcium reduction, by means of the Mn2+ quenching technique we demonstrate a decreased sarcolemmal permeability in PKC null mice. Those results indicate that PKC theta contribute to the regulation of ClC-1 channel in both muscle types and that this PKC isoform can also modulate calcium homeostasis likely by interacting with calcium channels. (Supported by ASI-OSMA)

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