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Protein kinase C theta (PKCθ) modulates the ClC-1 chloride channel activity and skeletal muscle phenotype: a biophysical and gene expression study in mouse models lacking the PKCθ

  • Ion channels, receptors and transporters
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Abstract

In skeletal muscle, the resting chloride conductance (gCl), due to the ClC-1 chloride channel, controls the sarcolemma electrical stability. Indeed, loss-of-function mutations in ClC-1 gene are responsible of myotonia congenita. The ClC-1 channel can be phosphorylated and inactivated by protein kinases C (PKC), but the relative contribution of each PKC isoforms is unknown. Here, we investigated on the role of PKCθ in the regulation of ClC-1 channel expression and activity in fast- and slow-twitch muscles of mouse models lacking PKCθ. Electrophysiological studies showed an increase of gCl in the PKCθ-null mice with respect to wild type. Muscle excitability was reduced accordingly. However, the expression of the ClC-1 channel, evaluated by qRT-PCR, was not modified in PKCθ-null muscles suggesting that PKCθ affects the ClC-1 activity. Pharmacological studies demonstrated that although PKCθ appreciably modulates gCl, other isoforms are still active and concur to this role. The modification of gCl in PKCθ-null muscles has caused adaptation of the expression of phenotype-specific genes, such as calcineurin and myocyte enhancer factor-2, supporting the role of PKCθ also in the settings of muscle phenotype. Importantly, the lack of PKCθ has prevented the aging-related reduction of gCl, suggesting that its modulation may represent a new strategy to contrast the aging process.

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Abbreviations

PKCθ:

Protein kinase C theta

PKCα:

Protein kinase C alpha

gCl:

Chloride conductance

gK:

Potassium conductance

EDL:

Extensor digitorum longus

Sol:

Soleus

WT:

Wild type

CN:

Calcineurin

MEF2:

Myocyte enhancer factor-2

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Acknowledgments

The support of ASI-OSMA is gratefully acknowledged.

Author information

Author notes

  1. Luca Madaro

    Present address: IRCCS Fondazione Santa Lucia Epigenetics & Regenerative Pharmacology, Via del Fosso di Fiorano, 64, 00143, Rome, Italy

  2. Piera Smeriglio

    Present address: Department Orthopaedic & Surgery, Stanford University, 300 Pasteur Dr, Edwards Bldg. R166, Stanford, CA, 94305-534, USA

Authors and Affiliations

  1. Section of Pharmacology, Department of Pharmacy & Drug Sciences, University of Bari - Aldo Moro, 70125, Bari, Italy

    Giulia Maria Camerino, Michela De Bellis, Maria Cannone, Antonella Liantonio, Kejla Musaraj, Rossella Romano, Annamaria De Luca, Jean-François Desaphy, Diana Conte Camerino & Sabata Pierno

  2. Section of Histology and Medical Embryology, Department of Anatomy, Histology, Forensic Medicine & Orthopedics, Sapienza University of Rome, Rome, Italy

    Marina Bouchè, Piera Smeriglio & Luca Madaro

  3. Department of Biomedical Sciences and Human Oncology, University of Bari, Medical School, Bari, Italy

    Arcangela Giustino

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Supplementary Figure S1

Resting chloride conductance (gCl) and resting potassium conductance (gK) measured in Soleus (Sol) muscle of Wild-Type (WT) and of mPKCθ K/R (K/R) transgenic mice. a. Representative traces of the electrotonic potentials recorded in Sol muscle fibres by standard two microelectrodes technique at 0.05 mm distance between electrodes, in response to hyperpolarizing square-wave current pulse. The electrotonic potential recorded in normal physiological solution allows to measure of membrane resistance Rm and its reciprocal, the total membrane conductance (gm). The electrotonic potential recorded in chloride free solution allows to measure the potassium conductance (gK). The chloride conductance (gCl) is the mean gm minus the mean gK. b. Measure of resting component conductances for Cl- and K+. Each bar represents the mean value ± S.E.M. of 15-57 fibers from 3–7 animals. Statistical analysis was performed using ANOVA followed by Bonferroni’s t-test. Significantly different *with respect to WT (at least P<0.05). (PDF 20 kb)

Supplementary Figure S2

Resting chloride conductance (gCl) and resting potassium conductance (gK) measured in Extensor Digitorum Longus (EDL) muscle of Wild-Type (WT) and of mPKCθ K/R (K/R) transgenic mice. a. Representative traces of the electrotonic potentials recorded in EDL muscle fibres by standard two microelectrodes technique at 0.05 mm distance between electrodes, in response to hyperpolarizing square-wave current pulse. The electrotonic potential recorded in normal physiological solution allows to measure of membrane resistance Rm and its reciprocal, the total membrane conductance (gm). The electrotonic potential recorded in chloride free solution allows to measure the potassium conductance (gK). The chloride conductance (gCl) is the mean gm minus the mean gK. b. Measure of resting component conductances for Cl- and K+. Each bar represents the mean value ± S.E.M. of 15-57 fibers from 3–7 animals. Statistical analysis was performed using ANOVA followed by Bonferroni’s t-test. Significantly different *with respect to WT (at least P<0.05). (PDF 20 kb)

Supplementary Figure S3

Effects of chelerythrine and statin in vitro application on resting chloride conductance (gCl) measured in Soleus (Sol) and Extensor Digitorum Longus (EDL) muscles of Wild-Type (WT) and mPKC K/R (K/R) transgenic mice. a, Chelerythrine (1 μM), a PKC inhibitor, was applied acutely on muscle bath 30 min before the electrophysiological recordings in all the experimental conditions. Each bar represents the mean value ± S.E.M. of 10-27 fibers from 3–7 animals. Statistical analysis was performed using ANOVA followed by Bonferroni’s t-test. Significantly different *with respect to the value recorded in the absence of chelerythrine (at least P<0.05). b, Simvastatin (10 μM), previously demonstrated to stimulate PKC activity [39], was applied acutely on muscle bath 30 min before the electrophysiological recordings in all the experimental conditions. Each bar represents the mean value ± S.E.M. of 9-21 fibers from 3–7 animals. Statistical analysis was performed using ANOVA followed by Bonferroni’s t-test. Significantly different *with respect to the value recorded in the absence of drug (at least P<0.05). (PDF 16 kb)

Supplementary Figure S4

Excitability parameters measured in Soleus (Sol) and Extensor Digitorum Longus (EDL) muscles of Wild-Type (WT), PKCθ knock-out (KO) and mPKCθ K/R (K/R) transgenic mice. a, The excitability parameters evaluated were the amplitude of the first action potential (AP1) obtained with a minimal current (Ith) amplitude needed to obtain one action potential. Action potentials were recorded in skeletal muscle fibers using two-microelectrode current clamp method. Each bar represents the mean value ± S.E.M. of 8-21 fibers from 3–7 animals. Statistical analysis was performed using ANOVA followed by Bonferroni’s t-test. No significant differences were found. (PDF 13 kb)

Supplementary Figure S5

Effects of chelerythrine in vitro application on the excitability parameters measured in Soleus (Sol) muscle of Wild-Type (WT) and mPKCθ K/R (K/R) transgenic mice. Chelerythrine (1 μM) was applied and the following parameters measured: the amplitude of the first action potential (AP1) obtained with a minimal current (Ith) needed to obtain one action potential; the latency (Lat) of the first action potential and the maximum number of spikes (N spikes) elicitable with a maximal stimulation. Each bar represents the mean value ± S.E.M. of 8-18 fibers from 3–7 animals. Statistical analysis was performed using ANOVA followed by Bonferroni’s t-test. Significantly different *with respect to WT (at least P<0.05). (PDF 16 kb)

Supplementary Figure S6

Effects of chelerythrine in vitro application on the excitability parameters measured in Extensor Digitorum Longus (EDL) muscle of Wild-Type (WT) and mPKCθ K/R (K/R) transgenic mice. Chelerythrine (1 μM) was applied and the following parameters measured: the amplitude of the first action potential (AP1) obtained with a minimal current (Ith) needed to obtain one action potential; the latency (Lat) of the first action potential and the maximum number of spikes (N spikes) elicitable with a maximal stimulation. Each bar represents the mean value ± S.E.M. of 8-18 fibers from 3–7 animals. Statistical analysis was performed using ANOVA followed by Bonferroni’s t-test. No significant modifications were found. (PDF 16 kb)

Supplementary Figure S7

Resting cytosolic calcium concentration (restCa) measured in soleus (Sol) and extensor digitorum longus (EDL) muscles of Wild-Type (WT) and mPKCθ K/R (K/R) transgenic mice. RestCa was measured in skeletal muscle by using Fura-2 fluorescence method. Each bar represents the mean value ± S.E.M. of 9-55 fibers from 3–7 animals. Statistical analysis was performed using ANOVA followed by Bonferroni’s t-test. Significantly different *with respect to WT (at least P<0.05). (PDF 11 kb)

Supplementary Figure S8

Gene expression modification in Soleus (Sol) and extensor digitorum longus (EDL) muscles of Wild-Type (WT) and mPKCθ K/R (K/R) transgenic mice. Transcript levels were determined by real-time PCR for selected genes indicated with the abbreviation on the left. The numbers on the abscissa indicate the fold change in gene expression normalized for housekeeping gene. The bars indicate the fold change in gene expression in K/R vs. WT. The number of animals examined was 3-7 for each group. Statistical analysis was performed using ANOVA followed by Bonferroni’s t-test. Significantly different *with respect to WT (at least P<0.05). Abbreviations: ClC-1, ClC-1 chloride channel; MyHC1, myosin heavy chain type-1; CN, calcineurin; MEF2D, Myocyte enhancer factor 2d; HDAC5, histone deacetylase; NFAT, nuclear factor of activated T cells; PKM2, piruvate kinase M2; IRS1, insulin receptor substrate 1; MURF-1, muscle RING-finger protein-1; MSTN, myostatin; NFKB1, nuclear factor kappa-light-chain-enhancer of activated B cells. (PDF 185 kb)

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Camerino, G.M., Bouchè, M., De Bellis, M. et al. Protein kinase C theta (PKCθ) modulates the ClC-1 chloride channel activity and skeletal muscle phenotype: a biophysical and gene expression study in mouse models lacking the PKCθ. Pflugers Arch - Eur J Physiol 466, 2215–2228 (2014). https://doi.org/10.1007/s00424-014-1495-1

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