Risk of Myopathy in Patients in Therapy with Statins: Identification of Biological Markers in a Pilot Study

Figures

• 

  FIGURE 1. Muscle biopsy of a patient (MM) with statin myopathy showing SDH hyper-reactive and COX negative fibers. (A,B) 20× SDH staining showing muscle fibers with hyper-reactive sub-sarcolemmal rims (arrows). (C,D) COX staining with several COX negative fibers (arrows). (E,F) COX and SDH staining from a control subject.

• 

  FIGURE 2. Activity of mitochondrial enzymes as a biochemical markers was measured in muscle biopsies of statin-treated myopathic (statin) and untreated (ctrl) subjects. Bars represent the mean value ± SEM of succinate dehydrogenase (SDH), cytochrome oxidase (COX) and citrate synthetase (CS) activity measured in 10 statin-treated subjects and 9 untreated subjects. ∗Significantly different with respect to control value (p < 0.05) by Student’s t-test.

• 

  FIGURE 3. (A) Real-time PCR analysis of mRNA expression level of ClC-1 chloride channel and of PKCα and PKCθ in VL muscle biopsies of patients in therapy with statin showing sign of myopathy. Histograms show quantification of transcript levels normalized by the beta-2 microglobulin gene. Mean values ± SEM in myopathic subjects (Statin) were compared with those of untreated subjects (Ctrl). ∗Significantly different with respect to the own control (P < 0.05 or less) by Student’s t-test. (B) Representative Western blot showing the expression levels of ClC-1 and PKCθ proteins in the unphosphorylated and phosphorylated form in muscle tissues. The blots were reacted with specific antibodies. β-actin was used to normalize the blot. The densitometric analysis of each experimental band was performed using ImageLab software. Histograms show quantification of relative protein levels. Relative intensity was calculated by normalization of the absolute intensity of target protein with the absolute intensity of β-actin, as reference standard, and are represented as arbitrary units (AU). Each bar represents the mean ± SEM from the number of muscle biopsies as indicated. ∗Significantly different with…

• 

  FIGURE 4. Real-time PCR analysis of mRNA expression level of genes involved in ClC-1 chloride channel regulation and calcium signaling in VL muscle biopsies of patients in therapy with statin showing sign of myopathy. Histograms show quantification of transcript levels for MEF2D, HDAC5, CN, NFix, RyR1, SERCA1, Troponin T and Parvalbumin normalized by the beta-2 microglobulin gene. In each graph, the bars represent the mean values ± SEM in myopathic subjects (Statin) compared with those of untreated subjects (Ctrl) from the number of subjects indicated in brackets. ∗Significantly different with respect to the own control (P < 0.05 or less) by Student’s t-test.

• 

  FIGURE 5. Real-time PCR analysis of mRNA expression level of genes involved in muscle atrophy and oxidative-metabolism related genes in VL muscle biopsies of patients in therapy with statin showing sign of myopathy. Histograms show quantification of transcript levels for MuRF1, MyoD, Notch1, PGC1α and IDH normalized by the beta-2 microglobulin gene. In each graph, the bars represent the mean values ± SEM in myopathic subjects (Statin) compared with those of untreated subjects (Ctrl) from the number of subjects indicated in brackets. ∗Significantly different with respect to the own control (P < 0.05 or less) by Student’s t-test.

• 

  FIGURE 6. (A) Real-time PCR analysis of mRNA expression level of AMP Kinase (AMPK) gene involved in muscle energetic metabolism in VL muscle biopsies of patients in therapy with statin showing sign of myopathy. Histograms show quantification of transcript levels for AMPK normalized by the beta-2 microglobulin gene. In each graph, the bars represent the mean values ± SEM in myopathic subjects (Statin) compared with those of untreated subjects (Ctrl) from the number of subjects indicated in brackets. (B) Representative Western blot showing the expression level of total AMPK protein and its phosphorylated form in muscle tissues. The blots were reacted with specific antibodies. β-actin was used to normalize the blot. Histograms show quantification of relative protein levels of total AMPK (C) and pAMPK (D) obtained by densitometric analysis of each protein normalized with β-actin, as reference standard, and represented as AU. Each bar represents the mean ± SEM from the number of muscle biopsies as indicated. ∗Significantly different with respect to the own control (P < 0.05) by Student’s t-test.

• 

  FIGURE 7. Real-time PCR analysis of mRNA expression level of genes involved in glucose metabolism and transporter genes in VL muscle biopsies of patients in therapy with statin showing sign of myopathy. Histograms show quantification of transcript levels for IRS1, and GLUT-4 normalized by the beta-2 microglobulin gene. In each graph, the bars represent the mean values ± SEM in myopathic subjects (Statin) compared with those of untreated subjects (Ctrl) from the number of subjects indicated in brackets. ∗Significantly different with respect to the own control (P < 0.05) by Student’s t-test.