The role of acid-base imbalance in statin-induced myotoxicity

Abstract

Disturbances in acid-base balance, such as acidosis and alkalosis, have potential to alter the pharmacologic and toxicologic outcomes of statin therapy. Statins are commonly prescribed for elderly patients who have multiple comorbidities such as diabetes mellitus, cardiovascular, and renal diseases. These patients are at risk of developing acid-base imbalance. In the present study, the effect of disturbances in acid-base balance on the interconversion of simvastatin and pravastatin between lactone and hydroxy acid forms have been investigated in physiological buffers, human plasma, and cell culture medium over pH ranging from 6.8-7.8. The effects of such interconversion on cellular uptake and myotoxicity of statins were assessed in vitro using C2C12 skeletal muscle cells under conditions relevant to acidosis, alkalosis, and physiological pH. Results indicate that the conversion of the lactone forms of simvastatin and pravastatin to the corresponding hydroxy acid is strongly pH dependent. At physiological and alkaline pH, substantial proportions of simvastatin lactone (SVL; ∼87% and 99%, respectively) and pravastatin lactone (PVL; ∼98% and 99%, respectively) were converted to the active hydroxy acid forms after 24 hours of incubation at 37°C. At acidic pH, conversion occurs to a lower extent, resulting in greater proportion of statin remaining in the more lipophilic lactone form. However, pH alteration did not influence the conversion of the hydroxy acid forms of simvastatin and pravastatin to the corresponding lactones. Furthermore, acidosis has been shown to hinder the metabolism of the lactone form of statins by inhibiting hepatic microsomal enzyme activities. Lipophilic SVL was found to be more cytotoxic to undifferentiated and differentiated skeletal muscle cells compared with more hydrophilic simvastatin hydroxy acid, PVL, and pravastatin hydroxy acid. Enhanced cytotoxicity of statins was observed under acidic conditions and is attributed to increased cellular uptake of the more lipophilic lactone or unionized hydroxy acid form. Consequently, our results suggest that comorbidities associated with acid-base imbalance can play a substantial role in the development and potentiation of statin-induced myotoxicity.

Supplementary Fig 1

Interconversion of statins between lactone and hydroxy acid forms in phosphate buffer saline at different pH levels. Simvastatin lactone (A), simvastatin hydroxy acid (B), pravastatin lactone (C), and pravastatin hydroxy acid (D) were incubated with PBS of modified pH (6.8–7.8) at a concentration of 50 μmol/L for 24 hours at 37°C. The percentages of the lactone and hydroxy acid form recovered after 24 hours are expressed as mean ± standard deviation, (n = 6). Differences between samples of different pH were analyzed by one-way analysis of variance followed by Tukey's post hoc test (^∗∗∗P < 0.001; **P < 0.01). SVL, simvastatin lactone; SVA, simvastatin hydroxy acid; PVL, pravastatin lactone; PVA, pravastatin hydroxy acid.

Supplementary Fig 2

Interconversion of statins between lactone and hydroxy acid forms in Dulbecco's modified eagle medium culture medium at different pH levels. Simvastatin lactone (A), simvastatin hydroxy acid (B), pravastatin lactone (C), and pravastatin hydroxy acid (D) were incubated with Dulbecco's modified eagle medium culture medium of modified pH (6.8–7.8) at a concentration of 50 μmol/L for 24 hours at 37°C. The percentages of the lactone and hydroxy acid form recovered after 24 hours are expressed as mean ± standard deviation, (n = 6). Differences between samples of different pH were analyzed by one-way analysis of variance followed by Tukey's post hoc test (^∗∗∗P < 0.001). SVL, simvastatin lactone; SVA, simvastatin hydroxy acid; PVL, pravastatin lactone; PVA, pravastatin hydroxy acid.

Supplementary Fig 3

Time course interconversion of simvastatin lactone (SVL) and pravastatin lactone (PVL) in phosphate buffer saline at different pH levels. The lactone forms of simvastatin and pravastatin were incubated with phosphate buffer saline of modified pH (6.8–7.8) at a concentration of 50 μmol/L for 48 hours at 37°C. The percentages of lactone and hydroxy acid form recovered at different time points are expressed as mean ± standard deviation, (n = 6). Differences between samples of different pH were analyzed by 2-way analysis of variance followed by Bonferroni's post hoc test. Time course interconversion of SVL shows disappearance of SVL (A) and formation of simvastatin hydroxy acid form (B). a = pH 7.8 vs pH 6.8, 7.0 (P < 0.001); pH 7.8 vs pH 7.2 (P < 0.05). b = pH 6.8 vs pH 7.2, 7.4, 7.6, 7.8; pH 7.0 vs pH 7.2, 7.4, 7.6, 7.8; pH 7.2 vs pH 7.6, 7.8; pH 7.4 vs pH 7.6, 7.8; pH 7.6 vs pH 7.8 (P < 0.001); pH 7.2 vs pH 7.4 (P < 0.05). c = pH 6.8 vs pH 7.0, 7.2, 7.4, 7.6, 7.8; pH 7.0 vs pH 7.2, 7.4, 7.6, 7.8; pH 7.2 vs pH 7.4, 7.6, 7.8; pH 7.4 vs pH 7.6, 7.8; pH 7.6 vs pH 7.8 (P < 0.001). d = pH 6.8 vs pH 7.0, 7.2, 7.4, 7.6, 7.8; pH 7.0 vs pH 7.2, 7.4, 7.6, 7.8; pH 7.2 vs pH 7.4, 7.6, 7.8; pH 7.4 vs pH 7.8 (P < 0.001); pH 7.4 vs pH 7.6 (P < 0.01); pH 7.6 vs pH 7.8 (P < 0.05). e = pH 6.8 vs pH 7.0, 7.2, 7.4, 7.6, 7.8; pH 7.0 vs pH 7.2, 7.4, 7.6, 7.8; pH 7.2 vs pH 7.4, 7.6, 7.8 (P < 0.001); pH 7.4 vs pH 7.8 (P < 0.05). f = pH 6.8 vs pH 7.0, 7.2, 7.4, 7.6, 7.8; pH 7.0 vs pH 7.2, 7.4, 7.6, 7.8; pH 7.2 vs pH 7.4, 7.6, 7.8 (P < 0.001). g = pH 6.8 vs pH 7.0, 7.2, 7.4, 7.6, 7.8; pH 7.0 vs pH 7.2, 7.4, 7.6, 7.8 (P < 0.001). Time course interconversion of PVL shows disappearance of PVL (C) and formation of pravastatin hydroxy acid form (D). a = pH 6.8 vs pH 7.6, 7.8; pH 7.0 vs pH 7.6, 7.8; pH 7.2 vs pH 7.8 (P < 0.001); pH 7.4 vs pH 6.8, 7.8; pH 7.2 vs pH 7.6 (P < 0.01); pH 7.0 vs pH 7.4 (P < 0.05). b = pH 6.8 vs pH 7.0, 7.2, 7.4, 7.6, 7.8; pH 7.0 vs pH 7.4, 7.6, 7.8; pH 7.2 vs pH 7.4, 7.6, 7.8; pH 7.4 vs pH 7.6, 7.8 (P < 0.001); pH 7.0 vs pH 7.2 (P < 0.01). c = pH 6.8 vs pH 7.2, 7.4, 7.6, 7.8; pH 7.0 vs 7.2, 7.4, 7.6, 7.8; pH 7.2 vs pH 7.4, 7.6, 7.8 (P < 0.001); pH 6.8 vs pH 7.0 (P < 0.01); pH 7.4 vs pH 7.6 (P < 0.05). d = pH 6.8 vs pH 7.0, 7.2, 7.4, 7.6, 7.8; pH 7.0 vs pH 7.4, 7.6, 7.8; pH 7.2 vs pH 7.4, 7.6, 7.8 (P < 0.001). e = 6.8 vs pH 7.0, 7.2, 7.4, 7.6, 7.8; pH 7.0 vs pH 7.4, 7.6, 7.8; pH 7.2 vs pH 7.6, 7.8, (P < 0.001); pH 7.4 vs pH 7.8 (P < 0.01); pH 7.2 vs pH 7.4 (P < 0.05). f = pH 6.8 vs pH 7.0, 7.2, 7.4, 7.6, 7.8; pH 7.0 vs pH 7.4, 7.6, 7.8 (P < 0.001). g = pH 6.8 vs pH 7.0, 7.2, 7.4, 7.6, 7.8 (P < 0.001).

Supplementary Fig 4

Time course interconversion of simvastatin lactone (SVL) and pravastatin lactone (PVL) in Dulbecco's modified eagle medium culture medium at different pH levels. The lactone forms of simvastatin and pravastatin were incubated with Dulbecco's modified eagle mediumculture medium of modified pH (6.8–7.8) at a concentration of 50 μmol/L for 48 hours at 37°C. The percentages of lactone and hydroxy acid form recovered at different time points are expressed as mean ± standard deviation, (n = 6). Differences between samples of different pH were analyzed by 2-way analysis of variance followed by Bonferroni's post hoc test. Time course interconversion of SVL shows disappearance of SVL (A) and formation of simvastatin hydroxy acid form (B). a = pH 7.8 vs pH 6.8, 7.2 (P < 0.01). b = pH 6.8 vs pH 7.2 (P < 0.05); pH 6.8 vs pH 7.4, 7.8; pH 7.2 vs pH 7.4, 7.8; pH 7.4 vs pH 7.8 (P < 0.001). c = pH 6.8 vs pH 7.2, 7.4, 7.8; pH 7.2 vs pH 7.4, 7.8; pH 7.4 vs 7.8 (P < 0.001). d = pH 6.8 vs pH 7.2, 7.4, 7.8; pH 7.2 vs pH 7.4, 7.8 (P < 0.001); pH 7.4 vs 7.8 (P < 0.01). e = pH 6.8 vs pH 7.2, 7.4, 7.8; pH 7.2 vs pH 7.4, 7.8 (P < 0.001). f = pH 6.8 vs pH 7.2, 7.4, 7.8 (P < 0.001). Time course interconversion of PVL shows disappearance of PVL (C) and formation of pravastatin hydroxy acid form (D). a = pH 6.8 vs pH 7.8 (P < 0.01); pH 7.2 vs pH 7.8 (P < 0.05). b = pH 6.8 vs pH 7.2, 7.4, 7.8; pH 7.8 vs pH 7.2, 7.4 (P < 0.001). c = 6.8 vs pH 7.2, 7.4, 7.8; pH 7.2 vs 7.8 (P < 0.001); pH 7.2 vs pH 7.4 (P < 0.05). d = pH 6.8 vs pH 7.2, 7.4, 7.8 (P < 0.001). e = pH 6.8 vs pH 7.4, 7.8 (P < 0.001); pH 6.8 vs pH 7.2 (P < 0.05).

Supplementary Fig 5

Uptake of simvastatin by undifferentiated and differentiated C2C12 cells. The uptake was determined after incubation of the cells with 1 μmol/L of simvastatin lactone (SVL) or simvastatin hydroxy acid (SVA) in Dulbecco's modified eagle medium of different pH levels (6.8–7.8) at 37°C for 6 hours. Results are expressed as nanomoles per milligram of protein ± standard deviation, (n = 5). Data were analyzed by 2-way analysis of variance followed by Bonferroni's post hoc test. (A) Intracellular concentration of simvastatin acid and lactone recovered from undifferentiated cells after treatment with 1 μmol/L SVL. a = SVL, pH 6.8 vs SVA, pH 6.8; SVL, pH 7.4 vs SVA, pH 7.4 (P < 0.001); b = SVL, pH 6.8 vs SVA, pH 6.8; SVL, pH 7.4 vs SVA, pH 7.4; SVL, pH 7.8 vs SVA, pH 7.8 (P < 0.001); c = SVL, pH 6.8 vs SVA, pH 6.8; SVL, pH 7.4 vs SVA, pH 7.4 (P < 0.001); SVL, pH 7.8 vs SVA, pH 7.8 (P < 0.01). (B) Intracellular concentration of simvastatin acid and lactone recovered from differentiated cells after treatment with 1 μmol/L SVL. a = SVL, pH 6.8 vs SVA, pH 6.8 (P < 0.001). (C) Intracellular concentration of simvastatin acid and lactone recovered from differentiated cells after treatment with 1 μmol/L SVA. a = SVA, pH 6.8 vs SVL, pH 6.8; SVA, pH 7.4 vs SVL, pH 7.4 (P < 0.001); b = SVA, pH 6.8 vs SVL, pH 6.8; SVA, pH 7.4 vs SVL, pH 7.4; SVA, pH 7.8 vs SVL, pH 7.8 (P < 0.001); c = SVA, pH 6.8 vs SVL, pH 6.8; SVA, pH 7.4 vs SVL, pH 7.4 (P < 0.001); SVA, pH 7.8 vs SVL, pH 7.8 (P < 0.01).

Supplementary Fig 6

HPLC chromatograms of (A) SVA spiked into Dulbecco's modified eagle medium (pH 7.4) with internal standard (IS = 4,4-dichlorodiphenyltrichloroethane), (B) SVL spiked into Dulbecco's modified eagle medium (pH 7.4) with internal standard (IS = 4,4-dichlorodiphenyltrichloroethane), (C) blank culture medium. SVA, simvastatin hydroxy acid; SVL, simvastatin lactone; IS, internal standard.

Supplementary Fig 7

HPLC chromatograms of (A) PVA spiked into Dulbecco's modified eagle medium (pH 7.4) with internal standard (IS = griseofulvin), (B) PVL spiked into Dulbecco's modified eagle medium (pH 7.4) with internal standard (IS = griseofulvin), (C) blank culture medium. PVA, pravastatin hydroxy acid; PVL, pravastatin lactone; IS, internal standard.

Supplementary Fig 8

LC-MS/MS chromatograms of a mixture of measured analytes from cell lysate samples. LOV-A, lovastatin hydroxy acid (IS); SVA, simvastatin hydroxy acid; LOV-L, lovastatin lactone (IS); SVL, simvastatin lactone; IS, internal standard.

Supplementary Fig 9

LC MS/MS chromatograms of a mixture of measured analytes from cell lysate samples. PVA, pravastatin hydroxy acid; PVL, pravastatin lactone; LOV-A, lovastatin hydroxy acid (IS); LOV-L, lovastatin lactone (IS); IS, internal standard.

Supplementary Fig 10

Messenger RNA expression of (A) MRP1, (B) MRP4, and (C) MRP5 in undifferentiated and differentiated C2C12 cells maintained at different pH levels. Results are expressed as fold changes in gene expression relative to the baseline level observed with undifferentiated C2C12 maintained at physiological pH and were normalized relative to Gapdh. Data are presented as mean ± standard deviation, (n = 4) and were analyzed by 2-way analysis of variance followed by Bonferroni's post hoc test, ^∗∗∗P < 0.001, ^∗∗P < 0.01, ^∗P < 0.05. Solid lines denote differences in relative messenger RNA levels between undifferentiated and differentiated cells, whereas dashed lines denote differences within each cell type maintained at different pH levels.

Supplementary Fig 11

Messenger RNA expression of (A) ribosomal protein S12, (B) hypoxanthine phosphoribosyltransferase, (C) TATA box-binding protein, and (D) glyceraldehyde-3-phosphate dehydrogenase reference genes. Results are expressed as fold changes in gene expression relative to the baseline level observed with undifferentiated C2C12 maintained at physiological pH. Each reference gene was normalized to the other 3 reference genes. Data are presented as mean ± standard deviation, (n = 4) and were analyzed 2-way analysis of variance followed by Bonferroni's post hoc test, ^{∗∗∗, §§§, †††}P < 0.001, ^{∗∗, §§}P < 0.01, ^{∗, §, †}P < 0.05 compared with messenger RNA expression level in undifferentiated cell samples of pH 7.4.

Fig 1

Schematic diagram for the possible mechanisms of statin interconversion between lactone and hydroxy acid forms and potential effect on membrane permeability. The interconversion between the 2 forms is mediated by pH and enzyme-dependent process. The higher lipophilicity of statins in the lactone form can potentially facilitate their penetration into muscle cells and consequently induce high local drug concentrations within skeletal muscle tissues.

Fig 2

Interconversion of simvastatin and pravastatin between lactone and hydroxy acid forms in human plasma of different pH levels. Simvastatin lactone (A), simvastatin hydroxy acid (B), pravastatin lactone (C), and pravastatin hydroxy acid (D) were incubated with human plasma of modified pH (6.8–7.8) at a concentration of 50 μmol/L for 24 hours at 37°C. The percentages of lactone and hydroxy acid form recovered after 24 hours are expressed as mean ± standard deviation, (n = 6). Differences between samples of different pH were analyzed by one-way analysis of variance followed by Tukey's post hoc test (***P < 0.001). SVL, simvastatin lactone; SVA, simvastatin hydroxy acid; PVL, pravastatin lactone; PVA, pravastatin hydroxy acid.

Fig 3

Time course interconversion of simvastatin lactone (SVL) and pravastatin lactone (PVL) in human plasma at different pH levels. The lactone forms of simvastatin and pravastatin were incubated with human plasma of modified pH (6.8–7.8) at a concentration of 50 μmol/L for 48 hours at 37°C. The percentages of lactone and hydroxy acid form recovered at different time points are expressed as mean ± standard deviation, (n = 6). Differences between samples of different pH were analyzed by 2-way analysis of variance followed by Bonferroni's post hoc test. Time course interconversion of SVL shows disappearance of SVL (A) and formation of simvastatin hydroxy acid form (B). a = pH 7.0 vs pH 7.6 (P < 0.05); pH 7.4 vs pH 6.8, 7.8 (P < 0.01); pH 6.8 vs pH 7.6, 7.8; pH 7.8 vs pH 7.0, 7.2 (P < 0.001). b = pH 6.8 vs pH 7.0, 7.2, 7.4, 7.6, 7.8; pH 7.0 vs pH 7.2, 7.4, 7.6, 7.8; pH 7.2 vs pH 7.4, 7.6, 7.8; pH 7.4 vs pH 7.6, 7.8; pH 7.6 vs pH 7.8 (P < 0.001). c = pH 6.8 vs pH 7.0, 7.2, 7.4, 7.6, 7.8; pH 7.0 vs pH 7.2, 7.4, 7.6, 7.8; pH 7.2 vs pH 7.4, 7.6, 7.8; pH 7.4 vs pH 7.6, 7.8 (P < 0.001); pH 7.6 vs pH 7.8 (P < 0.05). d = pH 6.8 vs pH 7.0, 7.2, 7.4, 7.6, 7.8; pH 7.0 vs pH 7.2, 7.4, 7.6, 7.8; pH 7.2 vs pH 7.4, 7.6, 7.8 (P < 0.001); pH 7.4 vs pH 7.6 (P < 0.05). e = pH 6.8 vs pH 7.0, 7.2, 7.4, 7.6, 7.8; pH 7.0 vs pH 7.2, 7.4, 7.6, 7.8; pH 7.2 vs pH 7.6, 7.8 (P < 0.001); pH 7.4 vs pH 7.2, 7.8 (P < 0.05). Time course interconversion of PVL shows disappearance of PVL (C) and formation of pravastatin hydroxy acid form (D). a = pH 7.6 vs pH 6.8, 7.4 (P < 0.01); pH 7.8 vs pH 6.8, 7.0, 7.2, 7.4; pH 7.2 vs pH 7.6 (P < 0.001). b = pH 6.8 vs pH 7.2, 7.4, 7.6, 7.8; pH 7.0 vs pH 7.2, 7.4, 7.6, 7.8; pH 7.2 vs pH 7.4, 7.6, 7.8; pH 7.4 vs pH 7.6, 7.8; pH 7.6 vs pH 7.8 (P < 0.001). c = pH 6.8 vs pH 7.0, 7.2, 7.4, 7.6, 7.8; pH 7.0 vs pH 7.2, 7.4, 7.6, 7.8; pH 7.2 vs pH 7.4, 7.6, 7.8; pH 7.4 vs pH 7.6, 7.8 (P < 0.001). d = pH 6.8 vs pH 7.0, 7.2, 7.4, 7.6, 7.8; pH 7.0 vs pH 7.4, 7.6, 7.8 (P < 0.001); pH 7.0 vs pH 7.2 (P < 0.01).

Fig 4

Liver microsomal stability of simvastatin lactone at 3 different pH levels. (A) Microsomal stability of simvastatin lactone as a function of time at different pH levels; (B) concentration-time profiles of simvastatin hydroxy acid form detected in liver microsomal stability reaction mixtures at 3 different pH levels. Results are expressed as mean ± standard deviation, (n = 3). Solid line denotes exponential regression of samples at pH 6.8; dashed line denotes exponential regression of samples at pH 7.4; whereas dotted line denotes exponential regression samples at pH 7.8.

Fig 5

qPCR expression profiles of (A) myogenin and myosin heavy chain at days 0–9 of C2C12 differentiation; (B) glyceraldehyde-3-phosphate dehydrogenase, hypoxanthine-guanine phosphoribosyltransferase and ribosomal protein S12 reference genes at days 0–9 of C2C12 differentiation. Analysis of gene expression was done after normalization to single reference gene (TATA box-binding protein) and the fold changes in gene expression were expressed relative to gene expression in undifferentiated cells. Results are expressed as mean ± standard deviation, (n = 3). Data were analyzed by Kruskal-Wallis nonparametric test followed by Dunn's test for multiple comparisons. a = significant difference in myogenin expression between day 0 and day 3 (P < 0.01); b = significant difference in myosin heavy chain expression between day 0 and day 7 (P < 0.01); c = significant difference in ribosomal protein S12 expression between day 0 and day 5 (P < 0.05). mRNA, messenger RNA.

Fig 6

Uptake of simvastatin lactone (SVL) and simvastatin hydroxy acid (SVA) by undifferentiated and differentiated C2C12 cells. The uptake was determined after incubation of the cells with 1 μmol/L of SVL or SVA in Dulbecco's modified eagle medium culture medium of different pH levels (6.8–7.8) at 37°C for 6 hours. Results are expressed as nanomoles per milligram of protein ±standard deviation, (n = 5). Data were analyzed by 2-way analysis of variance followed by Bonferroni's post hoc test. (A) Total simvastatin (lactone + hydroxy acid) recovered by undifferentiated C2C12 cells after treatment with 1 μmol/L SVL. a = pH 6.8 vs pH 7.4 (P < 0.01); pH 6.8 vs pH 7.8 (P < 0.001); b = pH 6.8 vs pH 7.4, 7.8 (P < 0.001); c = pH 6.8 vs pH 7.8 (P < 0.05). (B) Total simvastatin recovered by differentiated C2C12 cells after treatment with 1 μmol/L SVL. a = pH 6.8 vs pH 7.4, 7.8 (P < 0.001); pH 7.4 vs pH 7.8 (P < 0.01); b = pH 6.8 vs pH 7.4 (P < 0.01); pH 6.8 vs pH 7.8 (P < 0.001); c = pH 6.8 vs pH 7.4, 7.8 (P < 0.01). (C) SVA recovered by undifferentiated C2C12 cells after treatment with 1 μmol/L SVA (no SVL was recovered in this experiment). a = pH 6.8 vs pH 7.4 (P < 0.01); pH 6.8 vs pH 7.8 (P < 0.001); b = pH 6.8 vs pH 7.4, 7.8 (P < 0.001). (D) Total simvastatin recovered by differentiated C2C12 cells after treatment with 1 μmol/L SVA. a = pH 6.8 vs pH 7.4, 7.8 (P < 0.001); b = pH 6.8 vs pH 7.4, 7.8 (P < 0.001); pH 7.4 vs pH 7.8 (P < 0.05); c = pH 6.8 vs pH 7.4, 7.8 (P < 0.001); pH 7.4 vs pH 7.8 (P < 0.01); d = pH 6.8 vs pH 7.4, 7.8; pH 7.4 vs pH 7.8 (P < 0.001).

Fig 7

Effect of simvastatin lactone (SVL) and simvastatin hydroxy acid (SVA) on the viability of undifferentiated and differentiated C2C12. Cells were cultured at a density of 4,000 cells/well and allowed to attach for 24 hours or to differentiate for 4 days, then exposed to increasing concentrations of SVL or SVA under acidic, neutral, and alkaline medium pH for 72 hours. Results are presented as mean ± standard deviation of 3 experiments, 8 replicates per experiment. Data were analyzed by 2-way analysis of variance followed by Bonferroni's post hoc test. (A) Effects of SVL on cell viability of undifferentiated C2C12 myoblasts. a = pH 7.8 vs pH 6.8, 7.4 (P < 0.001); b = pH 7.8 vs pH 6.8, 7.4; pH 6.8 vs pH 7.4 (P < 0.001). (B) Effects of SVL on cell viability of differentiated C2C12 myocytes. a = pH 6.8 vs pH 7.4 (P < 0.01); pH 6.8 vs pH 7.8 (P < 0.05); b = pH 6.8 vs pH 7.4, 7.8; pH 7.4 vs pH 7.8 (P < 0.001); c = pH 6.8 vs pH 7.4, 7.8 (P < 0.001); d = pH 6.8 vs pH 7.4, 7.8 (P < 0.001); pH 7.4 vs pH 7.8 (P < 0.01); e = pH 7.8 vs pH 6.8, 7.4 (P < 0.001). (C) Effects of SVA on cell viability of undifferentiated C2C12 myoblasts. a = pH 6.8 vs pH 7.4 (P < 0.001); pH 7.4 vs pH 7.8 (P < 0.05); b = pH 7.8 vs pH 6.8, 7.4 (P < 0.05); c = pH 7.4 vs pH 7.8 (P < 0.01); d = pH 7.8 vs pH 6.8, 7.4 (P < 0.001); e = pH 6.8 vs pH 7.4, 7.8; pH 7.4 vs pH 7.8 (P < 0.001). (D) Effects of SVA on cell viability of differentiated C2C12 myocytes. a = pH 6.8 vs pH 7.8 (P < 0.001); b = pH 7.4 vs pH 6.8, 7.8 (P < 0.05); pH 6.8 vs pH 7.8 (P < 0.001); c = pH 6.8 vs pH 7.4, 7.8 (P < 0.001); pH 7.4 vs pH 7.8 (P < 0.05); d = pH 6.8 vs pH 7.4, 7.8 (P < 0.001); pH 7.4 vs pH 7.8 (P < 0.01); e = pH 6.8 vs pH 7.4, 7.8; pH 7.4 vs pH 7.8 (P < 0.001).

Fig 8

Effect of pravastatin lactone (PVL) and pravastatin hydroxy acid (PVA) on the viability of undifferentiated and differentiated C2C12. Cells were cultured at a density of 4,000 cells/well and allowed to attach for 24 hours or to differentiate for 4 days, then exposed to increasing concentrations of PVL or PVA under acidic, neutral, and alkaline medium pH for 72 hours. Results are presented as mean ± standard deviation of 3 experiments, 8 replicates per experiment. Data were analyzed by 2-way analysis of variance followed by Bonferroni's post hoc test. (A) Effects of PVL on cell viability of undifferentiated C2C12 myoblasts. a = pH 6.8 vs pH 7.8 (P < 0.001); b = pH 6.8 vs pH 7.4 (P < 0.05); pH 7.8 vs pH 6.8, 7.4 (P < 0.001); c = pH 6.8 vs pH 7.4, 7.8, pH 7.4 vs pH 7.8 (P < 0.001). (B) Effects of PVL on cell viability of differentiated C2C12 myocytes. a = pH 7.4 vs pH 7.8 (P < 0.05); b = pH 6.8 vs pH 7.8 (P < 0.01); c = pH 6.8 vs pH 7.4, 7.8 (P < 0.001); d = pH 6.8 vs pH 7.4, 7.8 (P < 0.001), pH 7.4 vs pH 7.8 (P < 0.01); e = pH 6.8 vs pH 7.4, 7.8, pH 7.4 vs pH 7.8 (P < 0.001). (C) Effect of PVA on cell viability of undifferentiated C2C12 myoblasts. a = pH 6.8 vs pH 7.8 (P < 0.01); b = pH 6.8 vs pH 7.8 (P < 0.001). (D) Effect of PVA on cell viability of differentiated C2C12 myocytes. a = pH 6.8 vs pH 7.8 (P < 0.001); pH 7.4 vs pH 7.8 (P < 0.01); b = pH 7.8 vs pH 6.8, 7.4 (P < 0.001); c = pH 7.8 vs pH 6.8, 7.4 (P < 0.001); pH 6.8 vs pH 7.4 (P < 0.05).

Fig 9

Effect of simvastatin lactone (SVL) and simvastatin hydroxy acid (SVA) on lactate dehydrogenase (LDH) release from undifferentiated and differentiated C2C12 cells maintained under different pH levels. C2C12 cells were cultured at a density of 4,000 cells/well and allowed to attach for 24 hours or to differentiate for 4 days, then exposed to increasing concentrations of SVL or SVA under acidic, neutral, and alkaline medium pH. Undifferentiated cells were treated for 72 hours, whereas differentiated cells were maintained for 24 hours. Data are presented as mean ± standard deviation, (n = 3) and analyzed by 2-way analysis of variance followed by Bonferroni's post hoc test. (A) LDH release from undifferentiated C2C12 cells treated with SVL. a = pH 6.8 vs pH 7.4 (P < 0.001); b = pH 6.8 vs pH 7.4, 7.8 (P < 0.001); c = pH 6.8 vs pH 7.4, 7.8; pH 7.4 vs pH 7.8 (P < 0.001). (B) LDH release from differentiated C2C12 cells treated with SVL. a = pH 6.8 vs pH 7.4, 7.8; pH 7.4 vs pH 7.8 (P < 0.001). (C) LDH release from undifferentiated C2C12 cells treated with SVA. a = pH 6.8 vs pH 7.8 (P < 0.05); b = pH 6.8 vs pH 7.4, 7.8; pH 7.4 vs pH 7.8 (P < 0.001); c = pH 6.8 vs pH 7.4 (P < 0.01); pH 7.8 vs pH 6.8, 7.4 (P < 0.001). (D) LDH release from differentiated C2C12 cells treated with SVA. a = pH 6.8 vs pH 7.4 (P < 0.01); pH 6.8 vs pH 7.8 (P < 0.05); b = pH 6.8 vs pH 7.4, 7.8 (P < 0.0001).

Fig 10

Effect of pravastatin lactone (PVL) and pravastatin hydroxy acid (PVA) on lactate dehydrogenase (LDH) release from undifferentiated and differentiated C2C12 cells maintained under different pH levels. C2C12 cells were cultured at a density of 4,000 cells/well and allowed to attach for 24 hours or to differentiate for 4 days, then exposed to increasing concentrations of PVL or PVA under acidic, neutral, and alkaline medium pH. Undifferentiated cells were treated for 72 hours, whereas differentiated cells were maintained for 24 hours. Data are presented as mean ± standard deviation, (n = 3) and analyzed by 2-way analysis of variance followed by Bonferroni's post hoc test. (A) LDH release from undifferentiated C2C12 cells treated with PVL. a = pH 7.4 vs pH 7.8 (P < 0.05); b = pH 6.8 vs pH 7.8 (P < 0.001); c = pH 6.8 vs pH 7.4, 7.8 (P < 0.001); pH 7.4 vs pH 7.8 (P < 0.05); d = pH 6.8 vs pH 7.4, 7.8 (P < 0.001); e = pH 6.8 vs pH 7.4, 7.8; pH 7.4 vs pH 7.8 (P < 0.001). (B) LDH release from differentiated C2C12 cells treated with PVL. a = pH 6.8 vs pH 7.4, 7.8 (P < 0.01); b = pH 6.8 vs pH 7.4, 7.8 (P < 0.001); c = pH 6.8 vs pH 7.4, 7.8 (P < 0.001); pH 7.4 vs pH 7.8 (P < 0.05). (C) LDH release from undifferentiated C2C12 cells treated with PVA. a = pH 6.8 vs pH 7.8 (P < 0.05); b = pH 6.8 vs pH 7.4 (P < 0.01); pH 6.8 vs pH 7.8 (P < 0.001); c = pH 6.8 vs pH 7.4, 7.8 (P < 0.001); d = pH 6.8 vs pH 7.4, 7.8 (P < 0.001); pH 7.4 vs pH 7.8 (P < 0.05); e = pH 6.8 vs pH 7.4, 7.8; pH 7.4 vs pH 7.8 (P < 0.001). (D) LDH release from differentiated C2C12 cells treated with PVA. a = pH 6.8 vs pH 7.8 (P < 0.05).