(A-19) Mitochondrial Changes normalized with Transcriptome/Proteomic data in both Endurance and Sedentary subjects

 Transcriptomic and proteomic studies have revealed that mitochondrial genes, proteins and pathways become upregulated in skeletal muscle in response to long-term exercise 1,2. However, it is unclear whether this observed upregulation in mitochondrial-related genes is due to increased mitochondrial number or an increase in gene/protein expression within each mitochondria. RNA sequencing has been performed on skeletal muscle biopsies and results from this study have been published1. Additionally, a proteomic analysis from the muscle of these same individuals was just performed. Both the transcriptomic and proteomic datasets show a significant upregulation of mitochondria-related genes.  By normalizing this transcriptomic and proteomic expression data by mitochondrial volume within trained and untrained human subjects, we will be able to address how each individual mitochondria is changing with long-term exercise. Thus, the goal of this study is to better understand the underlying relationship between individual mitochondrial changes and long-term endurance training. 

The experiment consisted of forty-four total subjects: nine men and nine women in the endurance trained groups (ME and FC), nine men and eight women in the control groups ( MC and FC) as well as nine male strength trained athletes (MS). To quantify mitochondrial volume,  DNA was isolated from vastus lateralis samples from the aforementioned subjects and duplex TaqMan PCR reactions were performed to determine the relative abundance of nuclear DNA and mtDNA. Each reaction was run in triplicate, with the 120-bp Lipolase Protein nuclear gene 3 (LPL) and NADH dehydrogenase 1 mitochondrial encoded gene4(ND1) acting as targets. The resulting mtDNA to nuclear DNA ratio based on the equation: Ct(ND1)- Cr(LPL) indicates the tissue concentration of mitochondria per cell5.  The existing transcriptomic and proteomic differential analysis points will then be adjusted based on mitochondrial volume. We can then cluster these points by comparing the pre-adjustment and post-adjustment analytes. Comparing the different clusters of analytes will reveal which transcriptomic factors and proteomic data points are most affected by mitochondrial volume.

 After performing multiple PCR experiments, the difference in abundance between the LPL gene and the ND1 gene related to the abundance of mitochondria vs nuclei is clear. The Cq average was 26.16 for the LPL gene and 12.7 for the ND1 gene. Normalizing known mitochondrial protein and transcriptomic expression to the relative abundance will reveal which specific upregulated/downregulated pathways are correlated with an increase in mitochondrial volume. Accounting for mitochondrial quantity will, once completed, allow us to determine if changes in mitochondrial volume account for the changes seen in gene expression. Although, differential abundance of these mitochondrial analytes could be explained by a change in mitochondrial composition/function, volume or by a combination of both.

1. 
   

   Chapman MA, Arif M, Emanuelsson EB, Reitzner SM, Lindholm ME, Mardinoglu A, Sundberg CJ. Skeletal Muscle Transcriptomic Comparison between Long-Term Trained and Untrained Men and Women. Cell Rep. 2020 Jun 23;31(12):107808. doi: 10.1016/j.celrep.2020.107808. PMID: 32579934.

   
   

2. 
   

   Chapman, M. A., & Sundberg, C. J. (2019). Exercise, Gene Regulation, and Cardiometabolic Disease. Cardiorespiratory Fitness in Cardiometabolic Diseases, 11–22. https://doi.org/10.1007/978-3-030-04816-7_2

   
   

3. 
   

   Iwona Bogacka, Hui Xie, George A. Bray, Steven R. Smith; Pioglitazone Induces Mitochondrial Biogenesis in Human Subcutaneous Adipose Tissue In Vivo. Diabetes 1 May 2005; 54 (5): 1392–1399. https://doi.org/10.2337/diabetes.54.5.1392

   
   

4. 
   

   He L, Chinnery PF, Durham SE, Blakely EL, Wardell TM, Borthwick GM, Taylor RW, Turnbull DM. Detection and quantification of mitochondrial DNA deletions in individual cells by real-time PCR. Nucleic Acids Res. 2002 Jul 15;30(14):e68. doi: 10.1093/nar/gnf067. PMID: 12136116; PMCID: PMC135769.

   
   

5. 
   

   Nicklas JA, Brooks EM, Hunter TC, Single R, Branda RF. Development of a quantitative PCR (TaqMan) assay for relative mitochondrial DNA copy number and the common mitochondrial DNA deletion in the rat. Environ Mol Mutagen. 2004;44(4):313-20. doi: 10.1002/em.20050. PMID: 15476199.