These cells proliferate as undifferentiated myoblast cells when grown in media containing high concentrations of serum (10%), and terminally differentiate into multinucleated myotubes when cultured in low serum-containing media

These cells proliferate as undifferentiated myoblast cells when grown in media containing high concentrations of serum (10%), and terminally differentiate into multinucleated myotubes when cultured in low serum-containing media. decreased mitochondrial function, skeletal muscle O2 utilization and peak work rate are significantly lower in BTHS patients than control participants [16]. While it is usually widely accepted that skeletal myopathy associated with BTHS stems from mitochondrial dysfunction, the mechanisms linking defective CL remodeling and skeletal myopathy have not been clearly elucidated and likely extend beyond compromised ATP generation. Myogenic differentiation is largely controlled by myogenic transcription factors and is accompanied by major changes in mitochondrial metabolism [17C20], mitochondrial energy Bretylium tosylate production [20, 21], and mitochondria-mediated activation of apoptotic pathways [22C24]. Given the central role of mitochondria in myogenic differentiation, we hypothesized that mitochondrial defects associated with BTHS might contribute to skeletal myopathy by interfering with normal myocyte differentiation. To determine the effect of defective CL remodeling around the myogenic determination, we sought to develop a tafazzin-deficient mammalian skeletal myoblast model. The C2C12 cell line was derived from murine skeletal myoblast cells and represents a widely used model for the study of skeletal muscle development [25], skeletal myopathy [26C28], and skeletal muscle differentiation [29C31]. The cells readily proliferate in high-serum conditions, and differentiate and fuse in low-serum conditions. Tafazzin-deficient C2C12 myocytes would provide a metabolic model for which isogenic cells are available as controls, in Bretylium tosylate contrast to currently used BTHS patient-derived lymphoblast cells. Furthermore, they are experimentally easier and cheaper to manipulate than tafazzin-deficient induced pluripotent stem cells (iPSCs) [32]. In this study, we constructed a CRISPR-generated stable tafazzin knockout (TAZ-KO) C2C12 myocyte cell line. The TAZ-KO cell line exhibits an increased MLCL/CL ratio, decreased mitochondrial respiration, increased mitochondrial ROS production, and defective myocyte differentiation. These results indicate that loss of CL remodeling influences myogenic determination and provide a foundation for future studies to explore potential mechanisms by which CL remodeling affects normal myocyte differentiation. Although BTHS is the only known genetic disorder directly linked to CL, aberrant myocyte differentiation may contribute to the development of skeletal myopathy associated with other mitochondrial diseases. 2. Materials and methods 2. 1 Cell line and growth conditions Wild type C2C12 cell lines were kindly provided by Dr. Steven Cala, Wayne State University. Growth medium consisted of DMEM (Gibco) made up of 10% FBS (Hyclone), 2 mM glutamine (Gibco), penicillin, (100 models/ml) and Bretylium tosylate streptomycin (100 g/ml) (Invitrogen). Cells were produced at 37C in a humidified incubator with 5% CO2. C2C12 myoblast differentiation was induced by shifting cells to DMEM medium containing 2% horse serum (Gibco). 2.2 Construction of TAZ-KO C2C12 cell line using CRISPR A gRNA targeting mouse TAZ exon 3 was identified using the clustered regulatory interspaced short palindromic repeats (CRISPR) design tool at (G2: TCCTAAAACTCCGCCACATC). To express Cas9 and PTPRC guide RNA in the mouse-derived C2C12 myoblast cells, complementary oligonucleotides made up of the gRNA sequence preceded by a G (for expression from the U6 promoter) were cloned into the em Bbs /em I site of plasmid pX330 [33] (a gift from Feng Zhang; Massachusetts Institute of Technology, Cambridge, Massachusetts, USA) [Addgene plasmid # 42230]). The sequence was verified using oligonucleotide primer 330/335 (ACTATCATATGCTTACCGTAAC). The plasmid pPGKpurobpa (a gift from Allan Bradley; Wellcome Trust Sanger Institute, Cambridge, UK) was co-transfected to allow selection under puromycin. Cells were transfected with plasmid pX330-TAZ and pPGKpurobpa using Lipofectamine 2000 (Life Technologies, Inc.). Cells were selected in puromycin-containing DMEM with 10% FBS. Cells were then diluted and put into 96-well plates. Single colonies were picked for screening. To screen for insertions or deletions at the target sites, the following oligonucleotide primers flanking mouse Taz exon 3 were used: FOR: CCAACCACCAGTCTTGCATG; REV: ATCCCTGCCTCCAAGACTTC. Wild type genomic DNA generates a product of 547 bp. Clone No. 3 which generated 3 distinct bands were selected for further analysis. PCR products were inserted into a pGEM?-T Easy Vector (Promega) and 16 individual transformants were analyzed by Sanger sequencing (Applied Genetics Technology Center, Wayne State University School of Medicine). 2.3 Mitochondria extraction Cells were produced to 100% confluency in 150 mm dishes and collected by scraping followed by centrifugation at 800 rpm for 5 min. The cell pellets were washed Bretylium tosylate with cold PBS and suspended in mitochondrial isolation buffer (280 mM sucrose, 0.25 mM EDTA, 20 mM Tris-HCl, pH 7.2). Cells were manually homogenized with a glass homogenizer. Cell debris was removed by centrifugation at 800.