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Diverse Action of Selected Statins on Skeletal Muscle Cells-An Attempt to Explain the Protective Effect of Geranylgeraniol (GGOH) in Statin-Associated Myopathy (SAM)

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Diverse Action of Selected Statins on Skeletal Muscle Cells-An Attempt to Explain the Protective Effect of Geranylgeraniol (GGOH) in Statin-Associated Myopathy (SAM)

Anna Jaśkiewicz et al. J Clin Med.

Abstract

The present study is centered on molecular mechanisms of the cytoprotective effect of geranylgeraniol (GGOH) in skeletal muscle harmed by statin-associated myopathy (SAM). GGOH via autophagy induction was purportedly assumed to prevent skeletal muscle viability impaired by statins, atorvastatin (ATR) or simvastatin (SIM). The C2C12 cell line was used as the 'in vitro' model of muscle cells at different stages of muscle formation, and the effect of ATR or SIM on the cell viability, protein expression and mitochondrial respiration were tested. Autophagy seems to be important for the differentiation of muscle cells; however, it did not participate in the observed GGOH cytoprotective effects. We showed that ATR- and SIM-dependent loss in cell viability was reversed by GGOH co-treatment, although GGOH did not reverse the ATR-induced drop in the cytochrome c oxidase protein expression level. It has been unambiguously revealed that the mitochondria of C2C12 cells are not sensitive to SIM, although ATR effectively inhibits mitochondrial respiration. GGOH restored proper mitochondria functioning. Apoptosis might, to some extent, explain the lower viability of statin-treated myotubes as the pan-caspase inhibitor, N-Benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (Z-VAD-FMK), partly reversed ATR- or SIM-induced cytotoxic effects; however, it does not do so in conjunction with caspase-3. It appears that the calpain inhibitor, N-Acetyl-L-leucyl-L-leucyl-L-norleucinal (ALLM), restored the viability that was reduced by ATR and SIM (p < 0.001). GGOH prevents SAM, in part, as a consequence of a caspase-3 independent pathway, probably by calpain system inactivation.

Keywords: geranylgeraniol; mitochondrial bioenergetics; myotoxicity; skeletal muscle cell viability; statin-associated myopathy; statins; water-soluble cholesterol.

Conflict of interest statement

The authors declare that there is no conflict of interests regarding the publication of this paper.

Figures

Figure 1
Figure 1
Effect of geranylgeraniol (GGOH, 10 µM) on cell viability (MTT assay) affected by mevalonate (MEV) pathway modulators (atorvastatin—ATR, simvastatin—SIM) or water-soluble cholesterol (Chol-PEG) in the presence or absence of chloroquine (CQ, 10 mM). Differentiating C2C12 myoblasts were exposed for 24, 72, or 120 h to statins (IC50) or water-soluble cholesterol (Chol-PEG), (Day 1—proliferating myoblasts; Day 3—differentiating myotubes; Day 5—differentiated myotubes). (a) ATR diminished fraction of viable cells (IC50). Neither CQ nor Chol-PEG or GGOH affected cell viability in comparison to control untreated cells (p > 0.05); GGOH but not Chol-PEG improved cell viability that had been reduced by ATR in proliferating myoblasts (p < 0.001). The effect of GGOH was not affected by CQ administration (p > 0.05); (b) SIM diminished the fraction of viable cells (IC50). Neither CQ nor water-soluble cholesterol (Chol-PEG) affected cell viability in comparison to control untreated cells (p > 0.05); GGOH but not Chol-PEG recovered cell viability reduced by SIM in proliferating myoblasts, differentiating and differentiated myotubes (p < 0.001). The effect of GGOH was not affected by CQ administration (p > 0.05); ** p < 0.01, *** p < 0.001, for comparison between the means. Statistically significant differences from untreated control cells are marked by # (at least at the level of p < 0.05). The results are indicative of three independent experiments performed in eight replicates.
Figure 2
Figure 2
Effect of geranylgeraniol (GGOH, µ10 M) on the protein content (sulforhodamine B (SRB) incorporation) affected by MEV pathway modulators (atorvastatin—ATR, simvastatin—SIM) or water-soluble cholesterol (Chol-PEG) in the presence or absence of chloroquine (CQ, 10 mM). Differentiating C2C12 myoblasts were exposed for 24, 72 or 120 h to statins (IC50) or water-soluble cholesterol (Chol-PEG), (Day 1—proliferating myoblasts; Day 3—differentiating myotubes; Day 5—differentiated myotubes). (a) ATR diminished the protein content in viable cells (IC50). Neither CQ nor Chol-PEG or GGOH affected the protein content in comparison to control untreated cells (p > 0.05); GGOH but not Chol-PEG recovered cellular protein content reduced by ATR in proliferating myoblasts (p < 0.001). The effect of GGOH was not affected by CQ administration (p > 0.05); (b) SIM diminished the protein content in viable cells (IC50). Neither CQ nor Chol-PEG or GGOH affected the protein content in comparison to control untreated cells (p > 0.05); GGOH but not Chol-PEG recovered the cellular protein content reduced by SIM in proliferating myoblasts (p < 0.001). GGOH worsened the protein content that had been diminished by SIM in differentiated myotubes (p < 0.01). The effect of GGOH was not affected by CQ administration (p > 0.05); *** p < 0.001, for comparison between the means. Statistically significant differences from untreated control cells are marked by # (at least at the level of p < 0.05, ns—non significant). The results are indicative of three independent experiments performed in eight replicates.
Figure 3
Figure 3
Top. Autophagy flux. Western blots of MAPLC-Ib/IIb with regard to actin. Effect of geranylgeraniol (GGOH, 10 µM) on the protein expression levels affected by MEV pathway modulators (atorvastatin—ATR, simvastatin—SIM) or water-soluble cholesterol (Chol-PEG) in the presence or absence of chloroquine (CQ, 10 mM). (a) In untreated myoblasts, the expression levels of MAPLC3-Ib/IIb protein increased accordingly with the differentiation stage. MAPLC3-Ib/IIb expression levels increased considerably upon CQ treatment. (b) ATR markedly reduced MAPLC3-Ib/IIb protein expression levels in proliferating myoblasts but differentiating and differentiated myotubes. Irrespective of co-treatment, CQ administration elevated MAPLC3-Ib/IIb protein expression levels equally, with no differences observed between the treatments. (c) SIM markedly reduced MAPLC3-Ib/IIb protein expression levels in proliferating myoblasts but not differentiating and differentiated myotubes. Irrespective of co-treatment, CQ administration elevated MAPLC3-Ib/IIb protein expression levels equally, with no differences observed between the treatments. Representative blots. The results are indicative of three independent experiments. Bottom. Autophagy flux. Densitometry analysis of MAPL3-IIb protein expression levels calculated in relation to house-keeping protein (actin). The effect of non-sterol isoprenoid, GGOH, and soluble cholesterol treatments on MAP LC3-IIb in C2C12 myoblasts affected by statins (atorvastatin—ATR; simvastatin—SIM) or water-soluble cholesterol (Chol-PEG). Differentiating C2C12 myoblasts exposed for 24, 72, or 120 h to statins (IC50), (Day 1—proliferating myoblasts; Day 3—differentiating myotubes; Day 5—differentiated myotubes). Different small letters indicate statistical significance (p < 0.05), whereas different capital letters represent values with high statistically significant (p < 0.01) differences between the means, respectively. The results are indicative of three independent experiments.
Figure 4
Figure 4
Effect of pan-caspase inhibitor Z-VAD-FMK on the cell viability (MTT assay) affected by MEV pathway modulators (atorvastatin—ATR, simvastatin—SIM). Differentiating C2C12 myoblasts were exposed for 24, 72, or 120 h to statins (IC50), (Day 1—proliferating myoblasts; Day 3—differentiating myotubes; Day 5—differentiated myotubes). ATR diminished the fraction of viable cells (IC50). Z-VAD-FMK itself did not affect cell viability, moreover, it could not recover ATR- or SIM-treated proliferating myoblasts (p > 0.05), but it significantly increased viable differentiating and differentiated myotubes treated with ATR or SIM (p < 0.05 and p < 0.001, respectively). * p < 0.05, *** p < 0.001, for comparison between the means. Statistically significant differences for untreated control cells are marked by # (at least at the level of p < 0.05). The results are indicative of three independent experiments performed in eight replicates.
Figure 5
Figure 5
Top. Western blots of cleaved caspase-3 with regard to actin. Effect of geranylgeraniol (GGOH, 10 µM) on cleaved caspase-3 protein expression levels affected by MEV pathway modulators (atorvastatin—ATR, simvastatin—SIM) or water-soluble cholesterol (Chol-PEG). In untreated myoblasts, the expression levels of cleaved caspase-3 protein (cleaved cas 3) decreased accordingly with the differentiation stage. ATR and SIM markedly reduced cleaved cas 3 protein expression levels in proliferating and differentiating but not in differentiated myotubes. Irrespective of co-treatment, GGOH administration elevated cleaved cas 3 protein expression levels and, again, without observing the differences found in differentiated myotubes. In contrast to GGOH, the Chol-PEG co-treatment did not affect cleaved cas 3 protein expression levels regardless of co-treatment. Representative blots. The results are indicative of three independent experiments. Bottom. Densitometry analysis of cleaved caspase-3 (cleaved cas 3) protein expressions levels calculated in relation to house-keeping protein (actin). The effect of non-sterol isoprenoid GGOH and soluble cholesterol treatments on cleaved cas 3 in C2C12 myoblasts affected by statins (atorvastatin—ATR, simvastatin—SIM) or water-soluble cholesterol (Chol-PEG). Differentiating C2C12 myoblasts exposed for 24, 72, or 120 h to statins (IC50), ((a) Day 1—proliferating myoblasts; (b) Day 3—differentiating myotubes; (c) Day 5—differentiated myotubes). Different small letters indicate statistically significant (p < 0.05), whereas different capital letters indicate statistically highly significant (p < 0.01) differences between the means, respectively. The results are indicative of three independent experiments.
Figure 6
Figure 6
Effect of pan-caspase inhibitor Z-VAD-FMK or calpain inhibitor ALLM on cell viability (MTT assay) affected by MEV pathway modulators (atorvastatin—ATR, simvastatin—SIM) in the presence or absence of chloroquine (CQ, 10 mM). Differentiating C2C12 myoblasts were exposed for 24, 72, or 120 h to statins (IC50), (Day 1—proliferating myoblasts; Day 3—differentiating myotubes; Day 5—differentiated myotubes). (a) Both ATR and SIM diminished cell viability (IC50). Z-VAD-FMK itself had no effect on cell viability (p > 0.05). However, it overruled the myotoxicity evoked by ATR but not SIM administration in proliferating myoblasts only (p < 0.05). Upon CQ addition, the cell viability that had already been reduced by ATR or SIM co-treatment shrunk considerably further (p < 0.01). (b) ALLM itself had no effect on cell viability (p > 0.05). In contrast to Z-VAD-FMK, ALLM (25 μM) markedly recovered cells from ATR- or SIM-dependent myotoxicity in proliferating myoblasts, differentiating and differentiated myotubes (p < 0.001). ALLM recovered muscle cell viability less efficiently in ATR- than in SIM-dependent SAM. Statistically significant differences from untreated control cells are marked by # (at least at the level of p < 0.05, ns—non significant). * p < 0.05, ** p < 0.01, *** p < 0.001. The results are indicative of three independent experiments performed in eight replicates.
Figure 7
Figure 7
Top. The effect of GGOH or Chol-PEG co-treatment upon mitochondrial cytochrome c oxidase protein expression levels in statin-treated C2C12 muscle cells. In untreated myoblasts, the expression levels of cytochrome c oxidase (COX) protein remained high throughout muscle differentiation. COX expression levels dropped considerably upon ATR treatment. ATR markedly reduced COX protein expression levels in proliferating myoblasts and differentiating and differentiated myotubes. Irrespective of co-treatment with GGOH or Chol-PEG protein, expression levels of COX remained low in proliferating myoblasts and differentiating and differentiated myotubes. There was no such effect observed upon SIM treatment, whereby COX expression levels did not change either to SIM, GGOH or Chol-PEG co-treatment. Representative blots. The results are indicative of three independent experiments. Bottom. Densitometry analysis of mitochondrial cytochrome c oxidase subunit I (mtCOX1) protein expressions levels calculated in relation to house-keeping protein (actin). Effect of non-sterol isoprenoid GGOH and soluble cholesterol treatments on mtCOX1 in C2C12 myoblasts affected by statins (atorvastatin—ATR, simvastatin—SIM) or water-soluble cholesterol (Chol-PEG). Differentiating C2C12 myoblasts exposed for 24, 72, or 120 h to statins (IC50), ((a) Day 1—proliferating myoblasts; (b) Day 3—differentiating myotubes; (c) Day 5—differentiated myotubes). Different small letters indicate statistical significance (p < 0.05), whereas different capital letters indicate high statistically significant (p < 0.01) differences between the means, respectively. The results are indicative of three independent experiments.
Figure 7
Figure 7
Top. The effect of GGOH or Chol-PEG co-treatment upon mitochondrial cytochrome c oxidase protein expression levels in statin-treated C2C12 muscle cells. In untreated myoblasts, the expression levels of cytochrome c oxidase (COX) protein remained high throughout muscle differentiation. COX expression levels dropped considerably upon ATR treatment. ATR markedly reduced COX protein expression levels in proliferating myoblasts and differentiating and differentiated myotubes. Irrespective of co-treatment with GGOH or Chol-PEG protein, expression levels of COX remained low in proliferating myoblasts and differentiating and differentiated myotubes. There was no such effect observed upon SIM treatment, whereby COX expression levels did not change either to SIM, GGOH or Chol-PEG co-treatment. Representative blots. The results are indicative of three independent experiments. Bottom. Densitometry analysis of mitochondrial cytochrome c oxidase subunit I (mtCOX1) protein expressions levels calculated in relation to house-keeping protein (actin). Effect of non-sterol isoprenoid GGOH and soluble cholesterol treatments on mtCOX1 in C2C12 myoblasts affected by statins (atorvastatin—ATR, simvastatin—SIM) or water-soluble cholesterol (Chol-PEG). Differentiating C2C12 myoblasts exposed for 24, 72, or 120 h to statins (IC50), ((a) Day 1—proliferating myoblasts; (b) Day 3—differentiating myotubes; (c) Day 5—differentiated myotubes). Different small letters indicate statistical significance (p < 0.05), whereas different capital letters indicate high statistically significant (p < 0.01) differences between the means, respectively. The results are indicative of three independent experiments.
Figure 8
Figure 8
Selected parameters (routine, OXPHOS and electron transport system (ETS)) of mitochondrial respiration in permeabilized C2C12 myoblasts after treatment with statins at IC50 (ATR, atorvastatin; SIM, simvastatin) and geranylgeraniol (GGOH; 10 µM) or cholesterol (Chol-PEG; 1 mM). Data were expressed as the mean ± standard deviation (SD) and the raw data, n = 5. The significance of differences was estimated based on: One-way ANOVA test and Tukey’s test as post hoc; (A) p < 0.05 for ATR vs. ATR + GGOH, ATR vs. SIM, ATR + GGOH vs. ATR + Chol; p < 0.001 for CTRL vs. ATR; CTRL vs. ATR + Chol; (B) p < 0,001 for CTRL vs. ATR, p < 0,05 for CTRL vs. ATR + Chol; (C) p < 0,01 for CTRL vs. ATR, CTRL vs. ATR+Chol; more experimental details see ‘Material and Methods’.
Figure 9
Figure 9
The effect of geranylgeraniol (GGOH, 10 µM) on cell viability (MTT assay) affected by geranylgeranyl transferase I (GGTI-286, IC50) in the presence or absence of chloroquine (CQ, 10 mM). Differentiating C2C12 myoblasts were exposed for 24, 72, or 120 h to GGTI-286 (IC50), (Day 1—proliferating myoblasts; Day 3—differentiating myotubes; Day 5—differentiated myotubes). Neither CQ nor GGOH affected the cell viability in comparison to control untreated cells (p > 0.05); GGOH could not rescue the muscle cell viability reduced by GGTI-286 in proliferating myoblasts and differentiating and differentiated myotubes (p > 0.05). The effect of GGOH was not affected by CQ administration (p > 0.05); CQ effectively reversed the negative effect of GGTI-286 on cell viability. GGOH blocked the effect of CQ on the GGTI-286-dependent drop in cell viability in differentiating and differentiated myotubes (p < 0.001) but not in proliferating myoblasts (p > 0.05). * p < 0.05, ** p < 0.01, *** p < 0.001, for comparison between the means. Statistically significant differences from untreated control cells are marked by # (at least at the level of p < 0.05). The results are indicative of three independent experiments performed in eight replicates.

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