Changes in Expression and Cellular Localization of Rat Skeletal Muscle ClC-1 Chloride Channel in Relation to Age, Myofiber Phenotype and PKC Modulation

Figures

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  Figure 1. ClC-1 gene and protein expression analysis in EDL and SOL muscles of rats from birth to old age. (A) Histograms show the transcription level of Clcn1 determined by qPCR and normalized with Actb in EDL and SOL muscles. (B) SDS-PAGE Western blot of ClC-1 and β-actin in EDL and SOL muscles at different ages. (C) Histograms show the densitometric analysis of ClC-1 bands expressed as a fraction of the respective β-actin band level in EDL and Sol muscles. Bars represent means ± SEM of the number of animals indicated in brackets. For graphical reasons, the values of single data were normalized on the mean of the M8 group. Significant differences between groups were assessed using one-way ANOVA (mRNA content of Clcn1 in EDL muscles F=22.11, R=0.811; mRNA content of Clcn1 in SOL muscles F=16.13, R=0.768; protein content of ClC-1 in EDL muscles F=58.78, R=0.947; protein content of ClC-1 in SOL muscles F=39.77, R=0.92). Fisher’s test was used to evaluate the individual differences between groups. Significant differences (P < 0.05) are expressed for * vs. P1, ° vs. P7, • vs. P12, □ vs. P33, Δ vs. P47, ∇ vs. M2, and # vs. M8.

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  Figure 2. The difference of CLC-1 gene expression in EDL and SOL muscles from birth to old age. Histograms show the transcription level of Clcn1 determined by qPCR and normalized with Actb in EDL and SOL muscles. Bars represent means ± SEM of the number of animals indicated in brackets. Significant differences between groups were evaluated using Student’s unpaired t-test. The significant difference was evaluated between EDL and SOL muscles in the same age-stage using Student’s unpaired t-test, with P <0.05 and expressed with *.

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  Figure 3. Pkca and Pkct gene expression analysis in EDL and SOL muscles of rats from birth to old age. Histograms show the transcription level of Pkct (A) and Pkca (B) performed by qPCR and normalized with Actb. Bars represent means ± SEM of the number of animals indicated in brackets. For graphical reasons, the values of the single data were normalized on the mean of the M8 group. Significant differences between groups were evaluated using one-way ANOVA (mRNA content of Pkct in EDL muscles F=48.35, R=0.89; mRNA content of Pkct in SOL muscles F=25.61, R=0.84; mRNA content of Pkca in EDL muscles F=2.96, R=0.36; mRNA content of Pkca in SOL muscles F=9.57, R=0.93). Fisher’s test was used to evaluate the individual differences between groups. Significant differences (P <0.05) are expressed for * vs. P1, ° vs. P7, • vs. P12, □ vs. P33, Δ vs. P47, ∇ vs. M2, and # vs. M8.

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  Figure 4. Subcellular localization of ClC-1 in EDL and SOL muscle fibers in relation to rat age obtained by epifluorescence microscopy analysis (16× magnification). The images show double-immunofluorescence with β-DG (red) and ClC-1 (green) in EDL (A) and SOL (B) fibers.

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  Figure 5. Quantification of colocalization of ClC-1 and β-DG in the fibers of EDL and SOL muscles in relation to age obtained using Pearson’s correlation coefficient and epifluorescence microscopy. The histograms show the mean ± SEM of Pcc-R obtained by comparing the distribution of ClC-1 and β-DG in three different images measured using epifluorescence microscopy (16× magnification) for each age analyzed. Significant differences between groups were evaluated using one-way ANOVA (Pcc-R in EDL muscles F=25.34, R=0.91; Pcc-R in SOL muscles F=34.72, R=0.92). Fisher’s test was used to evaluate the individual differences between groups. Significant differences (P <0.05) are expressed for ° vs. 7 Days and • vs. 12 Days.

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  Figure 6. Subcellular localization of ClC-1 in EDL and SOL muscle fibers in relation to rat age using high-resolution confocal microscopy (20× magnification). Images show a transversal section of (EDL (A) and SOL (B) rat muscles 7P and 2M old obtained by double-immunofluorescence with ClC-1 (green) and β-DG (red).

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  Figure 7. Subcellular localization of ClC-1 in EDL and SOL muscle fibers in relation to rat age using high-resolution confocal microscopy (100× magnification). Images show a transversal section of EDL (A) and SOL (B) rat muscles 7P and 2M old obtained by double-immunofluorescence with ClC-1 (green) and β-DG (red).

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  Figure 8. Quantification of colocalization of ClC-1 and β-DG in the fibers of EDL and SOL muscles in relation to age obtained using Pearson’s correlation coefficient and confocal microscopy. The histograms show the mean ± SEM of Pcc-R obtained by comparing the distribution of ClC-1 and β-DG in three different images measured using confocal microscopy (20× magnification) for each age analyzed. Significant differences between groups were evaluated using Student’s t-test (P <0.0001) are expressed for ° vs. 7 Days.