Molecular mechanisms of statin-associated myotoxicity

Statins, hydroxyl-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitors, are cholesterol-lowering drugs that are majorly used to treat hypercholesterolaemia and dyslipidaemia implicated in the pathogenesis of coronary heart disease and atherosclerosis [1]. They are generally considered safe drugs, but there are a number of reports of skeletal muscle damage associated with their use [2]. The myotoxicity ranges from a mild clinical syndrome consisting of benign myalgia to rare but life-threating rhabdomyolysis [3]. These side-effects can impact on quality of life and compliance, and in extreme cases lead to death [4]. Because millions of people in the world are currently taking statins every day, it is an urgent task to uncover the mechanism by which statins lead to side effects [5]. This thesis includes two published papers and one still in preparation. Our first paper presents a comparison between three different statins on the market: simvastatin, atorvastatin and rosuvastatin. Since there are differences among statins in terms of their efficacy and toxicity, we aimed to analyze the different molecular mechanisms that may contribute to the diverse grade of toxicity between simvastatin, atorvastatin and rosuvastatin. Simvastatin and atorvastatin appear to have a higher than average risk of myotoxicity contributing to the highest number of cases of rhabdomyolysis among statins [6] [7]. On the contrary rosuvastatin, the most hydrophilic statin, appears to have a reduced myotoxicity [8] [7]. C2C12 myotubes were exposed to 10 µM or 50 µM simvastatin, rosuvastatin or atorvastatin for 24 hours. We demonstrated that myotubes were more susceptible to simvastatin and atorvastatin than to rosuvastatin treatment. Therefore, difference between rosuvastatin and atorvastatin or simvastatin could point to possible mechanisms of toxicity. The cytotoxicity of simvastatin and atorvastatin was associated with a drastic and dose-dependent impairment of AKT signaling cascade that led to inhibition of the protein synthesis, increase of the protein degradation and promotion of apoptosis. Conversely, rosuvastatin blocked AKT signaling only at high concentration and to a lesser extent compared with the other two statins. The reduced effect on cytotoxicity and AKT signaling inhibition in C2C12 myotubes treated with rosuvastatin was accompanied with normal protein synthesis and absence of protein degradation and apoptosis. These results provide evidence that an impairment of AKT signaling pathways might be a causative factor in statin-induced myotoxicity. Our second paper expands on these previous results by showing that the myotoxicity, and with it, the impairment of AKT signaling, can be prevented by the addition of IGF-1. IGF-1 is well known for exerting an anabolic effect on skeletal muscle [9] by activating IGF-1/AKT pathway [10]. Therefore we investigated whether IGF-1 could antagonize the myotoxicity induced by statins. Myotubes were exposed to 10 µM simvastatin and/or 20 ng/ml IGF-1 for 18 hours. Simvastatin-induced myotoxicity was completely antagonized by IGF-1. Moreover, the protective effect of IGF-1 was mediated by the activation of IGF-1/AKT pathway that led to a suppression of atrophic markers and apoptosis, and simultaneously triggered pro-synthetic pathways. These studies provide new insight into the prevention of statin toxicity and may herald new discoveries for the treatment of statin-induced myalgia. The final paper takes the work of the previous two papers and places it into a novel system: the cardiac muscle. Statins are primarily prescribed to cure and prevent cardiovascular disease. Thus, cardiac side-effects may be masked by falsely attributing them to the underlying disease. In this paper, we investigated on the effect of simvastatin in cardiomyocyte in vitro and in vivo. We treated H9c2 rat cardiomyocytes with 10 µM and 100 µM simvastatin for 24 hours. H9c2 cells showed a reduction in the mitochondrial membrane potential and energetic impairment linked to mitochondrial dysfunction. Consequently, the cellular ATP level was decreased. This decrease led to the activation of AMPK, nuclear translocation of FoxO3, upregulation of atrogin-1 and initiation of apoptosis. We confirmed these results in vivo. We demonstrated that the treatment of mice with simvastatin 5 mg/kg/day for 21 days impaired the activity of several enzyme complexes of the electron transport chain in cardiomyocytes and increased mRNA expression of atrogin-1 and markers of apoptosis. This is the first study that shows energetic impairment linked to atrophy and apoptosis induced by statins in the heart, and warrants further investigation to assess statin safety in susceptible patients

Role of cerebrospinal fluid biomarkers to predict conversion to dementia in patients with mild cognitive impairment: a clinical cohort study

Abstract Background: Cerebrospinal fluid (CSF) levels assessment of Aβ1-42 and Tau proteins may be accurate diagnostic biomarkers for the differentiation of preclinical Alzheimer's disease (AD) from age-associated memory impairment, depression and other forms of dementia in patients with mild cognitive impairment (MCI). The aim of our study was to explore the utility of CSF biomarkers in combination with common cognitive markers as predictors for the risk of AD development, and other forms of dementia, and the time to conversion in community patients with MCI. Methods: A group of 71 MCI patients underwent neurological assessment, extended neuropsychological evaluation, routine blood tests, ApoE determination, and lumbar puncture to dose t-tau, p-tau181, Aβ1-42. We investigated baseline CSF and neuropsychological biomarker patterns according to groups stratified with later diagnoses of AD conversion (MCI-AD), other dementia (MCI-NAD) conversion, or clinical stability (sMCI). Results: Baseline Aβ1-42 CSF levels were significantly lower in MCI-AD patients compared to both sMCI and MCI-NAD. Additionally, p-tau181 was higher in the MCI-AD group compared to sMCI. The MCI-AD subgroup analysis confirmed the role of Aβ1-42 in its predictive role of time to conversion: rapid converters had lower Aβ1-42 levels compared to slow converters. Logistic regression and survival analysis further supported the key predictive role of baseline Aβ1-42 for incipient AD and dementia-free survival. Conclusions: Our results confirm the key role of CSF biomarkers in predicting patient conversion from MCI to dementia. The study suggests that CSF biomarkers may also be reliable in a real world clinical setting

Surface-enhanced Raman spectroscopy of the anti-cancer drug irinotecan in presence of human serum albumin

The development of nanotechnological devices and their clinical application in medicine has become increasingly important, especially in the context of targeted and personalized therapy. This is particularly important in cancer therapy, where antitumor drugs are highly cytotoxic and often exert their therapeutic effect at concentrations close to systemic toxicity. In the last years a growing number of studies has started to report the use of plasmonic nanoprobes in the field of theranostics, broadening the application of vibrational spectroscopies like Raman scattering and surface enhanced Raman scattering (SERS) in biomedicine. The present work aims to identify and characterize the vibrational profiles of a widely used anticancer drug, irinotecan (CPT-11). With a rational approach, SERS experiments have been performed on this analyte employing both Au and Ag colloids, starting from simple aqueous solutions up to albumin mixtures. A major step forward for drug detection in albumin solutions has been taken with the adoption of a simple deproteinization strategy, and a two-in-one-step separation and identification by coupling thin layer chromatography, TLC, with SERS (TLC-SERS). The latter has revealed to be a valid system for protein separation and simultaneous analyte detection, showing a potential to become an innovative, sensitive and low cost method for antineoplastic drug profiling in patients' body fluids

The AKT/mTOR signaling pathway plays a key role in statin-induced myotoxicity

Statins are drugs that lower blood cholesterol levels and reduce cardiovascular morbidity and mortality. They are generally well-tolerated, but myopathy is a potentially severe adverse reaction of these compounds. The mechanisms by which statins induce myotoxicity are not completely understood, but may be related to inhibition of the AKT signaling pathway. The current studies were performed to explore the down-stream effects of the statin-associated inhibition of AKT within the AKT signaling pathway and on myocyte biology and morphology in C2C12 myotubes and in mice in vivo. We exposed C2C12 myotubes to 10 μM or 50 μM simvastatin, atorvastatin or rosuvastatin for 24 h. Simvastatin and atorvastatin inhibited AKT phosphorylation and were cytotoxic starting at 10 μM, whereas similar effects were observed for rosuvastatin at 50 μM. Inhibition of AKT phosphorylation was associated with impaired phosphorylation of S6 kinase, ribosomal protein S6, 4E-binding protein 1 and FoxO3a, resulting in reduced protein synthesis, accelerated myofibrillar degradation and atrophy of C2C12 myotubes. Furthermore, impaired AKT phosphorylation was associated with activation of caspases and PARP, reflecting induction of apoptosis. Similar findings were detected in skeletal muscle of mice treated orally with 5 mg/kg/day simvastatin for 3 weeks. In conclusion, this study highlights the importance of the AKT/mTOR signaling pathway in statin-induced myotoxicity and reveals potential drug targets for treatment of patients with statin-associated myopathies

Simvastatin induces mitochondrial dysfunction and increased atrogin-1 expression in H9c2 cardiomyocytes and mice in vivo

Simvastatin is effective and well tolerated, with adverse reactions mainly affecting skeletal muscle. Important mechanisms for skeletal muscle toxicity include mitochondrial impairment and increased expression of atrogin-1. The aim was to study the mechanisms of toxicity of simvastatin on H9c2 cells (a rodent cardiomyocyte cell line) and on the heart of male C57BL/6 mice. After, exposure to 10 μmol/L simvastatin for 24 h, H9c2 cells showed impaired oxygen consumption, a reduction in the mitochondrial membrane potential and a decreased activity of several enzyme complexes of the mitochondrial electron transport chain (ETC). The cellular ATP level was also decreased, which was associated with phosphorylation of AMPK, dephosphorylation and nuclear translocation of FoxO3a as well as increased mRNA expression of atrogin-1. Markers of apoptosis were increased in simvastatin-treated H9c2 cells. Treatment of mice with 5 mg/kg/day simvastatin for 21 days was associated with a 5 % drop in heart weight as well as impaired activity of several enzyme complexes of the ETC and increased mRNA expression of atrogin-1 and of markers of apoptosis in cardiac tissue. Cardiomyocytes exposed to simvastatin in vitro or in vivo sustain mitochondrial damage, which causes AMPK activation, dephosphorylation and nuclear transformation of FoxO3a as well as increased expression of atrogin-1. Mitochondrial damage and increased atrogin-1 expression are associated with apoptosis and increased protein breakdown, which may cause myocardial atrophy

Contractile function and energy metabolism of skeletal muscle in rats with secondary carnitine deficiency

The consequences of carnitine depletion upon metabolic and contractile characteristics of skeletal muscle remain largely unexplored. Therefore, we investigated the effect of N-trimethyl-hydrazine-3-propionate (THP) administration, a carnitine analog inhibiting carnitine biosynthesis and renal reabsorption of carnitine, on skeletal muscle function and energy metabolism. Male Sprague-Dawley rats were fed a standard rat chow in the absence (CON; n = 8) or presence of THP (n = 8) for 3 wk. Following treatment, rats were fasted for 24 h prior to excision of their soleus and EDL muscles for biochemical characterization at rest and following 5 min of contraction in vitro. THP treatment reduced the carnitine pool by ∼80% in both soleus and EDL muscles compared with CON. Carnitine depletion was associated with a 30% decrease soleus muscle weight, whereas contractile function (expressed per gram of muscle), free coenzyme A, and water content remained unaltered from CON. Muscle fiber distribution and fiber area remained unaffected, whereas markers of apoptosis were increased in soleus muscle of THP-treated rats. In EDL muscle, carnitine depletion was associated with reduced free coenzyme A availability (-25%, P < 0.05), impaired peak tension development (-44%, P < 0.05), and increased glycogen hydrolysis (52%, P < 0.05) during muscle contraction, whereas PDC activation, muscle weight, and water content remained unaltered from CON. In conclusion, myopathy associated with carnitine deficiency can have different causes. Although muscle atrophy, most likely due to increased apoptosis, is predominant in muscle composed predominantly of type I fibers (soleus), disturbance of energy metabolism appears to be the major cause in muscle composed of type II fibers (EDL)

IGF-1 prevents simvastatin-induced myotoxicity in C2C12 myotubes

Statins are generally well tolerated, but treatment with these drugs may be associated with myopathy. The mechanisms of statin-associated myopathy are not completely understood. Statins inhibit AKT phosphorylation by an unclear mechanism, whereas insulin-like growth factor (IGF-1) activates the IGF-1/AKT signaling pathway and promotes muscle growth. The aims of the study were to investigate mechanisms of impaired AKT phosphorylation by simvastatin and to assess effects of IGF-1 on simvastatin-induced myotoxicity in C2C12 myotubes. C2C12 mouse myotubes were exposed to 10 μM simvastatin and/or 10 ng/mL IGF-1 for 18 h. Simvastatin inhibited the IGF-1/AKT signaling pathway, resulting in increased breakdown of myofibrillar proteins, impaired protein synthesis and increased apoptosis. Simvastatin inhibited AKT S473 phosphorylation, indicating reduced activity of mTORC2. In addition, simvastatin impaired stimulation of AKT T308 phosphorylation by IGF-1, indicating reduced activation of the IGF-1R/PI3K pathway by IGF-1. Nevertheless, simvastatin-induced myotoxicity could be at least partially prevented by IGF-1. The protective effects of IGF-1 were mediated by activation of the IGF-1R/AKT signaling cascade. Treatment with IGF-1 also suppressed muscle atrophy markers, restored protein synthesis and inhibited apoptosis. These results were confirmed by normalization of myotube morphology and protein content of C2C12 cells exposed to simvastatin and treated with IGF-1. In conclusion, impaired activity of AKT can be explained by reduced function of mTORC2 and of the IGF-1R/PI3K pathway. IGF-1 can prevent simvastatin-associated cytotoxicity and metabolic effects on C2C12 cells. The study gives insight into mechanisms of simvastatin-associated myotoxicity and provides potential targets for therapeutic intervention

Reductive stress impairs myoblasts mitochondrial function and triggers mitochondrial hormesis

Even though oxidative stress damage from excessive production of ROS is a well known phenomenon, the impact of reductive stress remains poorly understood. This study tested the hypothesis that cellular reductive stress could lead to mitochondrial malfunction, triggering a mitochondrial hormesis (mitohormesis) phenomenon able to protect mitochondria from the deleterious effects of statins. We performed several in vitro experiments on L6 myoblasts and studied the effects of N-acetylcysteine (NAC) at different exposure times. Direct NAC exposure (1mM) led to reductive stress, impairing mitochondrial function by decreasing maximal mitochondrial respiration and increasing H₂O₂production. After 24h of incubation, the reactive oxygen species (ROS) production was increased. The resulting mitochondrial oxidation activated mitochondrial biogenesis pathways at the mRNA level. After one week of exposure, mitochondria were well-adapted as shown by the decrease of cellular ROS, the increase of mitochondrial content, as well as of the antioxidant capacities. Atorvastatin (ATO) exposure (100μM) for 24h increased ROS levels, reduced the percentage of live cells, and increased the total percentage of apoptotic cells. NAC exposure during 3days failed to protect cells from the deleterious effects of statins. On the other hand, NAC pretreatment during one week triggered mitochondrial hormesis and reduced the deleterious effect of statins. These results contribute to a better understanding of the redox-dependant pathways linked to mitochondria, showing that reductive stress could trigger mitochondrial hormesis phenomenon

Correlation between In Vitro Neutralization Assay and Serological Tests for Protective Antibodies Detection

Serological assays are useful in investigating the development of humoral immunity against SARS-CoV-2 in the context of epidemiological studies focusing on the spread of protective immunity. The plaque reduction neutralization test (PRNT) is the gold standard method to assess the titer of protective antibodies in serum samples. However, to provide a result, the PRNT requires several days, skilled operators, and biosafety level 3 laboratories. Therefore, alternative methods are being assessed to establish a relationship between their outcomes and PRNT results. In this work, four different immunoassays (Roche Elecsys® Anti SARS-CoV-2 S, Snibe MAGLUMI® SARS-CoV-2 S-RBD IgG, Snibe MAGLUMI® 2019-nCoV IgG, and EUROIMMUN® SARS-CoV-2 NeutraLISA assays, respectively) have been performed on individuals healed after SARS-CoV-2 infection. The correlation between each assay and the reference method has been explored through linear regression modeling, as well as through the calculation of Pearson’s and Spearman’s coefficients. Furthermore, the ability of serological tests to discriminate samples with high titers of neutralizing antibodies (>160) has been assessed by ROC curve analyses, Cohen’s Kappa coefficient, and positive predictive agreement. The EUROIMMUN® NeutraLISA assay displayed the best correlation with PRNT results (Pearson and Spearman coefficients equal to 0.660 and 0.784, respectively), as well as the ROC curve with the highest accuracy, sensitivity, and specificity (0.857, 0.889, and 0.829, respectively)

Statins Trigger Mitochondrial Reactive Oxygen Species-Induced Apoptosis in Glycolytic Skeletal Muscle

Although statins are the most widely used cholesterol-lowering agents, they are associated with a variety of muscle complaints. The goal of this study was to characterize the effects of statins on the mitochondrial apoptosis pathway induced by mitochondrial oxidative stress in skeletal muscle using human muscle biopsies as well as in vivo and in vitro models.; Statins increased mitochondrial H2O2 production, the Bax/Bcl-2 ratio, and TUNEL staining in deltoid biopsies of patients with statin-associated myopathy. Furthermore, atorvastatin treatment for 2 weeks at 10 mg/kg/day in rats increased H2O2 accumulation and mRNA levels and immunostaining of the Bax/Bcl-2 ratio, as well as TUNEL staining and caspase 3 cleavage in glycolytic (plantaris) skeletal muscle, but not in oxidative (soleus) skeletal muscle, which has a high antioxidative capacity. Atorvastatin also decreased the GSH/GSSG ratio, but only in glycolytic skeletal muscle. Cotreatment with the antioxidant, quercetin, at 25 mg/kg/day abolished these effects in plantaris. An in vitro study with L6 myoblasts directly demonstrated the link between mitochondrial oxidative stress following atorvastatin exposure and activation of the mitochondrial apoptosis signaling pathway.; Treatment with atorvastatin is associated with mitochondrial oxidative stress, which activates apoptosis and contributes to myopathy. Glycolytic muscles are more sensitive to atorvastatin than oxidative muscles, which may be due to the higher antioxidative capacity in oxidative muscles.; There is a link between statin-induced mitochondrial oxidative stress and activation of the mitochondrial apoptosis signaling pathway in glycolytic skeletal muscle, which may be associated with statin-associated myopathy