Association between statin-associated myopathy and skeletal muscle damage

Ask your doctor about statin side effects, From a review of the article came this:
Since the heart is a muscle there was a concern that there maybe some underlying damage being done to the heart. I asked the lead author, Dr. Annette Draeger, if it was possible that a certain amount of damage maybe occurring in the heart muscle even in patient not experiencing any symptoms? She replied that "That is a very good question. Cardiomyopathy is not a prominent feature in statin users.
http://www.cmaj.ca/content/181/1-2/E11.full?sid=16f82ed0-6ed0-43e0-8e5f-de9d7d9da4b6

Background: Many patients taking statins often complain of muscle pain and weakness. The extent to which muscle pain reflects muscle injury is unknown.

Methods: We obtained biopsy samples from the vastus lateralis muscle of 83 patients. Of the 44 patients with clinically diagnosed statin-associated myopathy, 29 were currently taking a statin, and 15 had discontinued statin therapy before the biopsy (minimal duration of discontinuation 3 weeks). We also included 19 patients who were taking statins and had no myopathy, and 20 patients who had never taken statins and had no myopathy. We classified the muscles as injured if 2% or more of the muscle fibres in a biopsy sample showed damage. Using reverse transcriptase polymerase chain reaction, we evaluated the expression levels of candidate genes potentially related to myocyte injury.

Results: Muscle injury was observed in 25 (of 44) patients with myopathy and in 1 patient without myopathy. Only 1 patient with structural injury had a circulating level of creatine phosphokinase that was elevated more than 1950 U/L (10× the upper limit of normal). Expression of ryanodine receptor 3 was significantly upregulated in patients with biopsy evidence of structural damage (1.7, standard error of the mean 0.3).

Interpretation: Persistent myopathy in patients taking statins reflects structural muscle damage. A lack of elevated levels of circulating creatine phosphokinase does not rule out structural muscle injury. Upregulation of the expression of ryanodine receptor 3 is suggestive of an intracellular calcium leak.

Statins are among the most widely prescribed medications worldwide. Although their overall safety profile is excellent, myalgia without functional muscle impairment commonly affects patients taking statins. The clinical manifestations of statin-associated myopathy include pain and muscle weakness. Observational studies have shown a myalgia rate of 10%–15% among patients taking statins. 1^,2 Fulminant and potentially fatal rhabdomyolysis may also occur. Myalgia is typically considered by patients and physicians to be a minor adverse effect. 3^–5 Current consensus guideline support continuation of the statin therapy as long as circulating levels of creatine phosphokinase are less than 1950 U/L (10× the upper limit of normal). 6

We sought to determine whether statin-associated myopathy is associated with underlying structural muscle damage. We investigated whether the extent of muscle damage is reflected by the level of circulating creatine phosphokinase. We also sought to identify alterations in the expression of genes expressed in myocytes, which could provide insight into the cause of statin-associated myopathy.

Methods

Patients

Samples of the vastus lateralis muscle were collected from 83 people in 5 groups. The first control group comprised 10 healthy male volunteers who had never taken statins and who had no complaints of muscle pain (biopsy samples from these patients were obtained for a previous study 7). The second control group included 10 patients who were age-matched to patients in the myopathy group. These patients were each receiving treatment for a condition not related to their muscles. These patients were recruited from a pool of patients in the Department of Nephrology and Hypertension, and they had no history of statin use or muscle complaints. Several patients in this group had hypercholesterolemia but had never taken statins. Biopsy samples were obtained from these patients before they began statin therapy as part of an ongoing longitudinal study.

The third group comprised 15 patients with clinically diagnosed statin-associated myopathy who had discontinued statin therapy (discontinued for a minimum of 3 [median 12] weeks before biopsy). These patients were referred to the Department of Nephrology and Hypertension for alternative treatment options for hypercholesterinemia because they refused to continue statin therapy. These patients reported persistent myalgia or muscle weakness, or both, in the absence of other reasons for muscle disease.

The fourth group included 29 patients who had a history of clinically diagnosed statin-associated myopathy and who were receiving statin therapy at the time of the biopsy. These patients were recruited from the pool of patients in the Department of Nephrology and Hypertension

The fifth group included 19 patients who had received statin therapy for hypercholesterolemia for 4–20 years and were taking statins at the time of the biopsy. These patients had no muscle complaints.

Patients were identified as having statin-associated myopathy by clinical criteria consistent with the recommendations of the Muscle Safety Expert Panel. 3 These patients had persistent complaints of myalgia, cramps or muscle weakness predominately in the trunk or proximal muscle groups. Some reported that their pain worsened with exertion while taking statins and in the absence of other reasons for myopathy. Some patients had elevated serum concentrations of creatine phosphokinase (585–975 U/L [3–5× the upper limit of normal]).

Patients with myopathy (with or without statin use) were referred to the Department of Nephrology and Hypertension by local hospitals or physicians for management of their muscle symptoms. The diagnosis of statin-associated myopathy and decisions about the continued use of statins were made by the clinical team caring for each patient. Patients taking statins without muscle complaints were recruited via advertisements in local newspapers.

The vastus lateralis muscle was biopsied at midthigh level by use of the technique of Bergström. 8 The samples were processed within 10 minutes after excision. Although the vastus lateralis muscle is rarely affected by myalgia, it serves as a reference muscle and is routinely biopsied when systemic muscle pathology is investigated.

This study was conducted with the approval of the ethics committee of the Canton of Bern Switzerland. All patients gave informed consent.

Tissue processing

An average of 3 tissue blocks (about 3 mm − 6 mm) per patient were fixed and processed for semi-thin sectioning and ultrastructural analysis by electron microscopy. All samples were fixed in 6.25% glutaraldehyde solution and embedded in epoxy resin. 9

Immunohistochemistry and ultracryomicrotomy

One or more cylinders of some tissue samples were fixed in 4% paraformaldehyde and processed for ultra-thin frozen sectioning. 10 We labelled the T tubules with a monoclonal antibody against annexin A6 and a secondary polyclonal antibody coupled to Cy3. 10 We viewed the T tubules using a ZEISS Axiophot 2 fluorescence microscope, and we captured photomicrographs using a digital CCD camera (Ultra-pix, Astrocam).

RNA isolation and gene expression

We isolated total RNA from muscle biopsy samples using the RNeasy Fibrous Tissue Kit (Qiagen), according to the manufacturer’s instructions. The samples were treated with DNase I (Qiagen) to avoid genomic DNA contamination. We measured the total RNA concentration by ultraviolet spectrophotometry at 260 nm (DU 530, Beckman Coulter). The purity of the RNA was determined by the ratio of the absorbance at 260 nm to the absorbance at 280 nm and confirmed by agarose gel electrophoresis. We synthesized cDNA using random hexamer primers and the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems).

We analyzed the expression of the following 8 genes that code for proteins located in the T-tubule membrane and adjacent sarcoplasmic reticulum and that are involved in the regulation of intracellular calcium homeostasis: Inositol 1,4,5-triphosphate receptor, type 1 (ITPR1-Hs00181881_m1); inositol 1,4,5-triphosphate receptor, type 2 (ITPR2-Hs00181916_m1); inositol 1,4,5-triphosphate receptor, type 3 (ITPR3-Hs00609908_m1); ryanodine receptor 1 (RYR1-Hs00166991_m1); ryanodine receptor 3 (RYR3-Hs00168821_m1); sarco-endoplasmic reticulum transporting Ca²⁺ ATPase 1 (ATP2A1-Hs00188877_m1); sarco-endoplasmic reticulum transporting Ca²⁺ ATPase 2 (ATP2A2-Hs00544877_m1); and sarco-endoplasmic reticulum transporting Ca²⁺ ATPase 3 (ATP2A3-Hs00193090_m1).

We performed expression studies using validated TaqMan Gene Expression Assays with 18S rRNA as a reference gene. The reactions were performed using the 7900HT Fast Real-Time PCR System (Applied Biosystems). The default program was used (40 cycles, each consisting of 15 seconds at 95°C followed by 1 minute at 60°C). Data acquisition was performed according to the manufacturer’s instructions.

For analysis of the expression data, we carried out a relative quantity study. The expression values of the target genes were normalized to the concentration of 18S rRNA. We calculated gene expression values based on the comparative threshold cycle (Ct) method. The threshold cycle for each gene and 18S rRNA in each sample was determined and used to calculate Ct values. The Ct values were calculated by subtracting the Ct of the calibrator from the Ct value of each target (Ct = Ct_gene − Ct_{18S rRNA}). The relative quantities were calculated with the equation: relative quantity = 2^−Ct. To calculate the relative quantity, we designated as calibrators the average Ct values from the group without structural damage. All gene amplification reactions were performed in triplicate.

Tissue analysis

An observer (A.D.) skilled in light and electron microscopy evaluated the specimens. The observer was unaware of the patient groups. The number of muscle fibres with structural abnormalities in each section was determined. Significant injury was defined as 2% or more damaged fibres per biopsy sample. This arbitrary value reflected what we considered to be a clinically meaningful level of damage. Smaller amounts of damage may be found in patients with no symptoms of myopathy.

Statistical analysis

The data are expressed as mean and standard error of the mean (SEM). We compared the prevalence of muscle injury among the groups by the use of the Fisher exact test. We performed multiple group comparisons for categorical variables by nonparametric analysis of variance by ranks. For continuous variables, we performed analysis of variance followed by the Dunn Multiple Comparison Test. We evaluated the correlation between the circulating level of creatine phosphokinase and the extent of injury using the Pearson correlation coefficient. For mRNA expression analysis, normality was evaluated with the Kolmogorov–Smirnov test (α = 0.05), with the Lilliefors significance correction. Because the expression of the genes did not have a normal distribution, we performed a Mann–Whitney U test. The level of significance was set at p < 0.05.

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Results

Most of the patients with statin-associated myopathy were men with moderate hypercholesterolemia (Table 1). The average age was 56.5 years. Simvastatin and pravastatin were the most commonly reported statins used, and 25% of patients had taken more than 1 statin.