Human Skeletal Muscle Myoblast Cells Research Articles

B-175 Phytochemicals in K. pinnata and the Antioxidant Activity of Combined K. pinnata and Metformin Preparations on Normal and Diabetic Skeletal Muscle Cells

Abstract Background Diabetes Mellitus is associated with oxidative stress, which may play a central role in developing diabetic complications. With the rise in healthcare costs, more people globally are choosing to complement their pharmacological regimen with dietary supplements from natural products. In this study, the compound composition of K. pinnata and the effects of combined preparations of K. pinnata and metformin on antioxidant activity in human skeletal muscle myoblasts (HSMM) and human diabetic skeletal muscle myoblasts (DHSMM) were investigated. Methods K. pinnata leaves were crushed and extracted with hot water, then gravity filtered and freeze-dried. A 1 mL aliquot of the Kalanchoe pinnata extract solution was utilized to determine the chemical composition of the herbal extract. Liquid chromatography-mass spectrometry (LC-MS) was run in negative ion mode, with the K. pinnata extract analyzed in triplicate. A computer program further analyzed the LC-MS data to determine the chemical components in K. pinnata aqueous extract. A database algorithm within Compound Discoverer was used to identify the compounds in K. pinnata aqueous extract. For subsequent cell culture experiments, the K. pinnata leaf extract was weighed and combined with aqueous metformin solutions to prepare various combinatorial treatments. Human skeletal muscle myoblast cells and diabetic human skeletal muscle myoblast cells (Lonza Inc, Walkersville, MD) were cultured and incubated at 37 °C with 5% CO2. A CCK-8 cytotoxicity assay was performed to determine cellular viability, while trypan blue was used to assess the cellular proliferation of in-vitro cells. The effects of combinatorial preparations of K. pinnata and metformin on antioxidant indices were determined in HSMM, DHSMM, and HSMM H2O2 stress-induced cells. Results Liquid chromatography-mass spectrometry (LC-MS) showed that K. pinnata contains biologically active flavanols. The main compounds identified in locally grown K. pinnata were quercetin, kaempferol, apigenin, epigallocatechin gallate (EGCG), and avicularin. Antioxidant results indicated that a combinatorial preparation of K. pinnata with metformin might modulate antioxidant responses by increasing the enzymatic activity of superoxide dismutase and increasing levels of reduced glutathione. A combination of 50 μM and 150 μg/mL of metformin and K. pinnata resulted in a significant increase in reduced glutathione levels in non-diabetic and diabetic human skeletal muscle myoblasts and H2O2 stress-induced human skeletal muscle myoblasts. Additionally, a K. pinnata treatment (400 µg/mL) alone significantly increased catalase (CAT) activity for non-diabetic and diabetic human skeletal muscle myoblasts and an H2O2 stress-induced human skeletal muscle myoblast cell line while significantly lowering malondialdehyde (MDA) levels. The antioxidant activity of the treatment options was more effective at promoting cell viability after 24 h of treatment vs 72 h. Conclusion These treatment options show promise, especially after 24 h for treating oxidative stress-mediated pathophysiological complications associated with type II diabetes.

Bioactive Ingredients in K. pinnata Extract and Synergistic Effects of Combined K. pinnata and Metformin Preparations on Antioxidant Activities in Diabetic and Non-Diabetic Skeletal Muscle Cells.

With healthcare costs rising, many affected by ailments are turning to alternative medicine for treatment. More people are choosing to complement their pharmacological regimen with dietary supplements from natural products. In this study, the compound composition of Kalanchoe Pinnata (K. pinnata) and the effects of combined preparations of K. pinnata and metformin on antioxidant activity in human skeletal muscle myoblasts (HSMMs) and human diabetic skeletal muscle myoblasts (DHSMMs) were investigated. Ultraperformance liquid chromatography fusion orbitrap mass spectrometry (UPLC-OT-FTMS) identified biologically active flavanols in K. pinnata. The main compounds identified in locally grown K. pinnata were quercetin, kaempferol, apigenin, epigallocatechin gallate (EGCG), and avicularin. Antioxidant results indicated that a combinatorial preparation of K. pinnata with metformin may modulate antioxidant responses by increasing the enzymatic activity of superoxide dismutase and increasing levels of reduced glutathione. A combination of 50 μM and 150 μg/mL of metformin and K. pinnata, respectively, resulted in a significant increase in reduced glutathione levels in non-diabetic and diabetic human skeletal muscle myoblasts and H2O2-stress-induced human skeletal muscle myoblasts. Additionally, a K. pinnata treatment (400 µg/mL) alone significantly increased catalase (CAT) activity for non-diabetic and diabetic human skeletal muscle myoblasts and a H2O2-stress-induced human skeletal muscle myoblast cell line, while significantly lowering malondialdehyde (MDA) levels. However, the treatment options were more effective at promoting cell viability after 24 h versus 72 h and did not promote cell viability after 72 h in H2O2-stress-induced HSMM cells. These treatment options show promise for treating oxidative-stress-mediated pathophysiological complications associated with type II diabetes.

Biological effect of dysregulated LBX1 on adolescent idiopathic scoliosis through modulating muscle carbohydrate metabolism

BACKGROUND CONTEXTAbnormal energy metabolism such as lower body weight and body mass index (BMI) and less fat mass is widely reported in patients with adolescent idiopathic scoliosis (AIS) and has been implicated in deformity development. However, the underlying mechanism is largely unclear. LBX1 is one of the promising AIS predisposing genes validated by multicenter studies. PURPOSEThis study aimed to identify differentially expressed proteins (DEPs) relating to energy metabolism in AIS by using proteomic and metabolic analysis and to explore if the expression of these DEPs is associated with clinical parameters and modulated by LBX1. STUDY DESIGNThis is a cross-sectional study using clinical data and biological samples followed by basic study using a cellular model. PATIENT SAMPLEPlasma samples were collected from Chinese girls with nonprogressive and progressive AIS (N=7 and 8, respectively) and age-matched healthy girls (N=50). Paraspinal muscle tissues were collected intraoperatively from concave and convex side of the apex of the major spinal curve in AIS (N=24) and either side from nonscoliosis patients (N=14). OUTCOME MEASURESRadiological Cobb angle and basic anthropometric data of recruited subjects were measured. The DEPs and metabolites were compared in plasma using proteomics and metabolomics technique. The relative expression of selected genes was measured in muscles. METHODSPlasma samples from AIS were collected at first clinical visit and were further divided into nonprogressive or progressive groups according to Cobb angle changes in 6-year follow-up. Age-matched healthy girls were recruited as control. High-performance liquid chromatography-mass spectrometry based proteomic analysis was carried out in three groups to identify DEPs and their annotated metabolic pathways. An independent cohort was used for validation by gas chromatography-mass spectrometry based metabolomic analysis. Paraspinal muscles were subjected to quantitative polymerase chain reaction (qPCR) followed by correlation analysis. Human skeletal muscle myoblast (HSMM) was used as the cellular model. RESULTSThe likelihood of aberrant galactose metabolism and glycolysis was found to be associated with AIS curve progression as evidenced by the thirteen DEPs and seven related metabolites according to proteomic and metabolomic analysis. Some of the DEPs showed significantly altered expression in AIS concave and convex sides paraspinal muscles compared with those in nonscoliosis control. Four DEPs were found significantly and negatively correlated with LBX1 in AIS convex side paraspinal muscles. Overexpressing LBX1 in HSMM cells led to increased expression of three DEPs and decreased expression of three DEPs, respectively. CONCLUSIONSThis is the first integrated proteomic and metabolomic analysis on AIS. Our findings show dysregulated galactose metabolism and glycolysis pathways in progressive group of AIS, suggesting the presence of abnormal energy metabolism at early stage of this disease, and their association with higher risk of progressing into more severe curvature. Evidence from ex vivo study with human muscle biopsies and in vitro study with human myoblast cells propose the possible effect of LBX1 on these two pathways in skeletal muscles. The present study provides new evidence of LBX1 function in AIS via modulating effect on the expression of energy metabolism related genes. This study might provide new insights into etiopathogenesis and development of novel treatment strategy targeting on abnormal body weight and BMI in patients with AIS. Additionally, the plasma proteomic and metabolomic studies suggested new candidates as biomarkers for establishing predictive model for AIS onset/progression.

Plasma exosomal RNAs have potential as both clinical biomarkers and therapeutic targets of dermatomyositis

DM is characterized by skeletal muscle weakness and cutaneous manifestations. Plasma exosomes (EXOs) contain proteins, RNAs, DNA, and lipid cargoes and are transferred among cells. If thoroughly investigated, plasma EXO RNAs could potentially improve our understanding of DM pathogenesis. We aimed to identify potential new biomarkers and therapeutic targets for DM. The RNA (mRNA, miRNA and lncRNA) profiles of plasma EXOs were evaluated by sequencing on the Illumina HiSeq 3000 platform. Differentially expressed (DE) RNAs and bioinformatic analyses were performed. Human skeletal muscle myoblasts cells (HSkMCs) were stimulated with plasma EXOs, rapamycin or IFN-β. Real-time PCR and western blot analysis were used to detect related genes and proteins. A total of 689 DE mRNAs, 53 DE miRNAs and 452 DE lncRNAs were identified in DM plasma EXOs. Bioinformatic analysis inferred that plasma EXOs were secreted mainly by CD8+ T cells, regulatory T cells and natural killer cells. The DE miRNAs participated in the autophagy, TGF-β and Wnt signalling pathways. Three DE miRNAs (hsa-miR-125a-3p, hsa-miR-1246 and hsa-miR-3614-5p) were correlated with serological indices, organ involvement and myositis-specific autoantibodies. The DE lncRNAs participated in autophagy, IFN-β production and mTOR signalling. DM plasma EXOs can induce autophagy in HSkMCs by regulating three miRNAs (hsa-miR-125a-3p, hsa-miR-1246 and hsa-miR-3614-5p) and three lncRNAs (ENST00000584157.1, ENST00000523380.1 and ENST00000560054.1), which formed an autophagy network, playing a role in muscle damage. Our study provides an overview of distinct RNA profiles in DM plasma EXOs, and verified some miRNAs as potential biomarkers and therapeutic targets. The findings provide important clues for more in-depth explorations of plasma EXOs in DM.

Effects of OP2113 on Myocardial Infarct Size and No Reflow in a Rat Myocardial Ischemia/Reperfusion Model.

The present study was to determine whether OP2113 could limit myocardial infarction size and the no-reflow phenomenon in a rat myocardial ischemia/reperfusion model. Rat heart-isolated mitochondria (RHM) were used to investigate mitochondrial respiration and mitochondrial reactive oxygen species (mtROS) generation both in normal conditions and in ischemia/reperfusion-mimicking conditions (using high concentrations of succinate). Human skeletal muscle myoblasts (HSMM) in culture were used to investigate the cellular intermittent deprivation in energy substrates and oxygen as reported in ischemia/reperfusion conditions. In vivo, rats were anesthetized and subjected to 30min of left coronary artery occlusion followed by 3h of reperfusion. Rats were randomized to receive OP2113 as an intravenous infusion starting either 5min prior to coronary artery occlusion (preventive), or 5min prior to reperfusion (curative), or to receive vehicle starting 5min prior to coronary artery occlusion. Infusions continued until the end of the study (3h of reperfusion). RHM treated with OP2113 showed a concentration-dependent reduction of succinate-induced mtROS generation. In HSMM cells, OP2113 treatment (5-10μM) during 48H prevented the reduction in the steady-state level of ATP measured just after reperfusion injuries and decreased the mitochondrial affinity to oxygen. In vivo, myocardial infarct size, expressed as the percentage of the ischemic risk zone, was significantly lower in the OP2113-treated preventive group (44.5 ± 2.9%) versus that in the vehicle group (57.0 ± 3.6%; p < 0.05), with a non-significant trend toward a smaller infarct size in the curative group (50.8 ± 3.9%). The area of no reflow as a percentage of the risk zone was significantly smaller in both the OP2113-treated preventive (28.8 ± 2.4%; p = 0.026 vs vehicle) and curative groups (30.1 ± 2.3%; p = 0.04 vs vehicle) compared with the vehicle group (38.9 ± 3.1%). OP2113 was not associated with any hemodynamic changes. These results suggest that OP2113 is a promising mitochondrial ROS-modulating agent to reduce no-reflow as well as to reduce myocardial infarct size, especially if it is on board early in the course of the infarction. It appears to have benefit on no-reflow even when administered relatively late in the course of ischemia.

Nonthermal atmospheric plasma enhances myoblast differentiation by eliciting STAT3 phosphorylation.

The use of nonthermal atmospheric plasma (NTP) in the biomedical field has recently expanded into cell death induction in cancer, infection prevention, inflammation treatment, and wound-healing enhancement. NTP has been demonstrated to enhance skin and muscle regeneration, but its effects on tissue regeneration, following deep tissue or muscle damage, remains underinvestigated. In this study, we determined the effects of NTP on muscle differentiation and the mechanisms of NTP's contribution to differentiation and regeneration. NTP treatment enhanced cell differentiation in primary normal human skeletal muscle myoblast cells and increased the relative expression of mRNA levels of MyoD which is one of the earliest markers of myogenic commitment, and myogenin, which are important transcription factors required for myogenic differentiation. Furthermore, NTP treatment induced increases in the levels of myosin heavy chain, a differentiated muscle-specific protein, and in myotube formation of myoblasts. We observed that signal transducer and activator of transcription 3 (STAT3) activation induced by NTP treatment affects the myogenic differentiation. In addition, STAT3 phosphorylation was also enhanced by NTP treatment in injured animal muscle. These findings indicate that NTP could enhance musculoskeletal differentiation by acting as an external stimulus for myoblast differentiation, suggesting its treatment potential in promoting regeneration of damaged muscle.-Park, J. K., Kim, Y. S., Kang, S. U., Lee, Y. S., Won, H.-R., Kim, C.-H. Nonthermal atmospheric plasma enhances myoblast differentiation by eliciting STAT3 phosphorylation.

Establishment of a Novel Primary Human Skeletal Myoblast Cellular Model for Chikungunya Virus Infection and Pathogenesis.

Chikungunya virus (CHIKV) is a re-emerging arbovirus known to cause chronic myalgia and arthralgia and is now considered endemic in countries across Asia and Africa. The tissue tropism of CHIKV infection in humans remains, however, ill-defined. Due to the fact that myositis is commonly observed in most patients infected with CHIKV, we sought to develop a clinically relevant cellular model to better understand the pathogenesis of CHIKV infection. In this study, primary human skeletal muscle myoblasts (HSMM) were established as a novel human primary cell line that is highly permissive to CHIKV infection, with maximal amounts of infectious virions observed at 16 hours post infection. Genome-wide microarray profiling analyses were subsequently performed to identify and map genes that are differentially expressed upon CHIKV infection. Infection of HSMM cells with CHIKV resulted in altered expressions of host genes involved in skeletal- and muscular-associated disorders, innate immune responses, cellular growth and death, host metabolism and virus replication. Together, this study has shown the establishment of a clinically relevant primary human cell model that paves the way for the further analysis of host factors and their involvement in the various stages of CHIKV replication cycle and viral pathogenesis.

TIPS to manipulate myogenesis: retention of myoblast differentiation capacity using microsphere culture.

Cell therapy is an emerging option for regenerating skeletal muscle. Improved delivery methods for anchorage-dependent myoblasts are likely to improve integration and function of transplanted muscle cells. Highly porous microspheres, produced using thermally induced phase separation (TIPS), have features ideally suited for minimally invasive cell delivery. The purpose of this study was to investigate, for the first time, the use of TIPS microspheres as highly porous microcarriers for manipulation of human skeletal muscle myoblasts (HSMM) under defined culture conditions. HSMM cells readily attached to the surface of poly (DL-lactide-co-glycolide) (PLGA) TIPS microcarriers, where they were induced to continue proliferating or to be driven towards differentiation whilst under static-dynamic culture conditions for 7 days. Switching from proliferation medium to differentiation medium for 7 days, resulted in increased protein expression of skeletal muscle cell contractile apparatus components, MyoD and skeletal muscle myosin heavy chain, compared with cells cultured on conventional culture plasticware for the same duration (p < 0.001). Growth of myoblasts on the surface of the microcarriers and their migration following simulated delivery, caused no change to the proliferative capacity of cells over 7 days. Results from this study demonstrate that TIPS microspheres provide an ideal vehicle for the expansion and delivery of myoblasts for therapeutic applications. Transplantation of myoblasts anchored to a substrate, rather than in suspension, will reduce the amount of ex vivo manipulation required during preparation of the product and allows cells to be delivered in a more natural state. This will improve the ability to control cell dosage and increase the likelihood of efficacy.

Abstract A57: The secreted Wnt inhibitor SFRP3 is required for PAX3-FOXO1-positive alveolar rhabdomyosarcoma tumorigenesis

Abstract Introduction: This study aims to understand the contribution of the Wnt pathway inhibitor secreted frizzled related protein 3 (SFRP3) to alveolar rhabdomyosarcoma tumorigenesis. Rhabdomyosarcoma is the most common pediatric soft tissue sarcoma and demonstrates features of skeletal muscle. Of the two predominant (pediatric) subtypes, embryonal (eRMS) and alveolar (aRMS), aRMS has the poorer prognosis, with a 5-year survival rate of &amp;lt;30%. The majority of aRMS tumors express the fusion protein PAX3/7-FOXO1. As PAX3/7-FOXO1 is not currently druggable, our goal is to identify proteins that are downstream from or cooperate with PAX3-FOXO1 (PF) to enable tumorigenesis with the hope that these proteins may be more amenable to pharmacological inhibition. Experimental procedures: Microarray analysis using the Affymetrix U133A platform was performed to compare the transcriptomes of human skeletal muscle (HSMM) cells stably expressing either an empty vector or PF. Only genes that were differentially regulated at least three-fold were considered for further study. The microarray was validated and SFRP3 mRNA levels were assessed using RT-PCR. Lentiviral technology was used to stably express shRNAs in human PF-positive aRMS cell lines Rh28 and Rh30. Cell growth, cell proliferation, apoptosis, and cell cycle analysis were measured by MTT assay, BrdU assay, immunoblot for cleaved caspase 3, and by PI staining followed by analysis by flow cytometry, respectively. For the xenograft tumors in mice, doxycycline-inducible SFRP3 shRNAs were stably expressed in aRMS cells. These cells were injected into the flanks of SCID/beige mice to generate the tumors. Induction of the shRNAs by doxycycline administration began once the tumors were palpable. All animal work was performed under institution approved IACUC protocols. Results: In a microarray analysis of the transcriptomes of human skeletal muscle myoblasts (HSMM) with an empty vector or PF, it was noted that the Wnt pathway was preferentially represented in HSMMs with PF. Of these, SFRP3 was selected for further investigation as its mRNA levels were elevated in PF-positive aRMS cells, but not eRMS or HSMM cells. shRNAs against SFRP3 were generated and stably expressed in Rh28 and Rh30 cells, validated to suppress SFRP3, and shown to profoundly decrease cell growth (p&amp;lt;0.001). Associated with this decreased cell growth was a decrease in cell proliferation (p&amp;lt;0.001), an increase in apoptosis, and an increase in the percentage of cells at G1, suggesting a cell cycle arrest. Consistent with a G1 cell cycle arrest, shRNAs against SFRP3 also increased the levels of p21, an inhibitor of cell cycle progression. To assess the effects of SFRP3 shRNAs on tumor growth in a xenograft model of aRMS, a doxycycline-inducible system was used, allowing the shRNAs to be expressed only after the tumors formed. In this model, suppression of SFRP3 reduced tumor growth and tumor weight by more than three-fold. Further, in a pilot study, the combination of SFRP3 suppression with vincristine, a standard chemotherapeutic agent used in the treatment of aRMS, was more effective at reducing cell growth in vitro than with either treatment alone and ablated tumor growth in vivo. Conclusions: In conclusion, SFRP3 is necessary for the growth of aRMS cells both in vitro and in vivo. Inhibition of SFRP3 combined with standard of care chemotherapy should be explored further as SFRP3 is a promising new target in the treatment of PF-positive aRMS. Citation Format: Julie Kephart, Rosanne Jones, Lisa Crose, Jen-Tsan Ashley Chi, Corinne Linardic. The secreted Wnt inhibitor SFRP3 is required for PAX3-FOXO1-positive alveolar rhabdomyosarcoma tumorigenesis. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A57.

Hydrophobized Thermoresponsive Copolymer Brushes for Cell Separation by Multistep Temperature Change

For preparing a thermally modulated biointerface that separates cells without the modification of cell surfaces for regenerative medicine and tissue engineering, poly(N-isopropylacrylamide-co-butyl methacrylate) (P(IPAAm-co-BMA), thermo-responsive hydrophobic copolymer brushes with various BMA composition were formed on glass substrate through a surface-initiated atom transfer radical polymerization (ATRP). Characterization of the prepared surface was performed by X-ray photoelectron spectroscopy (XPS), attenuated total reflection Fourier transform infrared spectroscopy (ATR/FT-IR), and gel-permeation chromatography (GPC) measurement. Prepared copolymer brush surfaces were characterized by observing the adhesion (37 °C) and detachment (20 or 10 °C) of four types of human cells: human umbilical vein endothelial cells (HUVECs), normal human dermal fibroblasts (NHDFs), human aortic smooth muscle cells (SMCs), and human skeletal muscle myoblast cells (HSMMs). HUVECs and NHDFs exhibited their effective detachment temperature at 20 and 10 °C, respectively. Using cells' intrinsic temperature sensitivity for detachment from the copolymer brush, a mixture of green fluorescent protein (GFP)-expressing HUVECs (GFP-HUVECs) and NHDFs was separated.

Dynamically cell separating thermo-functional biointerfaces with densely packed polymer brushes

Poly(N-isopropylacrylamide) (PIPAAm) brush grafted glass surfaces with various brush lengths were prepared as cell separating intelligent interfaces through a surface-initiated atom transfer radical polymerization (ATRP) with a CuCl–Me6TREN catalytic system and α-chloro-p-xylene as a free initiator in 2-propanol at 25 °C for 16 h. Characterization of the prepared surface was performed by X-ray photoelectron spectroscopy (XPS), attenuated total reflection Fourier transform infrared (ATR/FT-IR) spectroscopy, and gel permeation chromatography (GPC) measurement of PIPAAm in ATRP reaction solution for estimating the brush length. Phase transition behavior of PIPAAm in four cell culture media was also investigated by measuring the temperature-dependent turbidities. Prepared PIPAAm brush surfaces as cell separating intelligent interfaces were characterized by observing the adhesion and detachment behavior of four types of human cells: human umbilical vein endothelial cells (HUVECs), normal human dermal fibroblasts (NHDFs), human aortic smooth muscle cells (SMCs), and human skeletal muscle myoblast cells (HSMMs). The PIPAAm brush surface with a moderate brush length exhibited a proper cell adhesion and detachment behavior, while short-brush-surfaces scarcely detached cells and long-brush-surfaces scarcely adhered cells. The PIPAAm brush with a moderate brush length exhibited different cell detachment rates among individual cell types. Utilizing the different cell detachment properties, a mixture of green fluorescent protein (GFP) expressing HUVEC (GFP-HUVEC) and HSMM was separated. After being allowed to adhere on the surfaces at 37 °C for 24 h, the adhered cells on the surfaces were incubated at 20 °C. In the initial period of incubation at 20 °C, the GFP-HUVEC was released from the surface due to its prompt detachment property, and, in the subsequent period of incubation, HSMMs gradually detached themselves from the surface. These results indicated that a precisely designed PIPAAm brush functioned as an intelligent cell separating interface by utilizing the intrinsic cell detachment properties of individual cells.

Human Skeletal Muscle Drug Transporters Determine Local Exposure and Toxicity of Statins

The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, or statins, are important drugs used in the treatment and prevention of cardiovascular disease. Although statins are well tolerated, many patients develop myopathy manifesting as muscle aches and pain. Rhabdomyolysis is a rare but severe toxicity of statins. Interindividual differences in the activities of hepatic membrane drug transporters and metabolic enzymes are known to influence statin plasma pharmacokinetics and risk for myopathy. Interestingly, little is known regarding the molecular determinants of statin distribution into skeletal muscle and its relevance to toxicity. We sought to identify statin transporters in human skeletal muscle and determine their impact on statin toxicity in vitro. We demonstrate that the uptake transporter OATP2B1 (human organic anion transporting polypeptide 2B1) and the efflux transporters, multidrug resistance-associated protein (MRP)1, MRP4, and MRP5 are expressed on the sarcolemmal membrane of human skeletal muscle fibers and that atorvastatin and rosuvastatin are substrates of these transporters when assessed using a heterologous expression system. In an in vitro model of differentiated, primary human skeletal muscle myoblast cells, we demonstrate basal membrane expression and drug efflux activity of MRP1, which contributes to reducing intracellular statin accumulation. Furthermore, we show that expression of human OATP2B1 in human skeletal muscle myoblast cells by adenoviral vectors increases intracellular accumulation and toxicity of statins and such effects were abrogated when cells overexpressed MRP1. These results identify key membrane transporters as modulators of skeletal muscle statin exposure and toxicity.