Mitochondrial Fragmentation in Skeletal Muscle Derived Cells from an Old Male Donor May Relate to Decreased Oxygen Consumption Rates

Abstract

With age, skeletal muscle mitochondria lose their ability to fuse and regenerate following stress and exercise leading to fragmentation. These phenomena can lead to reduced oxygen, muscle atrophy, reduced maximal O2 consumption, loss of metabolic flexibility and risk of developing age‐related diseases such as sarcopenia. Skeletal muscle cells derived from human donors can be used to investigate these physiological capacities in primary culture. Our objective is to investigate mitochondrial morphology and cellular maximal oxygen consumption rates (OCR) of skeletal muscle cells derived from healthy young and old male donors. Primary skeletal muscle cells derived from the Rectus abdominis muscle of healthy active eighteen and sixty‐nine year old men (SKM18M and SKM69M, respectively) were obtained from Cook MyoSite Inc. (Pittsburgh, PA). Cells were stained with MitoTracker Red (Cell Signaling; Danvers, MA) and mitochondrial morphology was observed using a Zeiss LSM 710 AxioObserver confocal inverted scanning microscope (Carl Zeiss; White Plains, NY). The mitochondrial network was analyzed using the Mitochondrial Network Analysis tool in ImageJ (MiNA, FIJI) to estimate mitochondrial footprint from a binarized image. Oxygen consumption rates were measured in intact cells using Seahorse Cell Mito Stress Tests on a XFp extracellular flux analyzer (Agilent Technologies; Santa Clara, CA). Primary cells derived from the young donor (SKM18M) had a larger mitochondrial footprint, longer branch length, and a greater number of network branches compared to the old donor (SKM69M) (Footprint: 34.65 ± 25.30 vs. 11.64 ± 9.53 μm2; Branch Length: 20.59 ± 7.23 vs. 12.10 ± 6.84 μm; Network: 17.25 ± 0.16 vs. 7.67 ± 4.97 counts). SKM18M also showed higher Basal and Maximal OCR compared to SKM69M (Basal: 38.78 ± 8.34 vs. 12.82 ± 2.07; Maximal: 60.09 ±10.84 vs. 20.52 ± 2.36 pmol/min/protein). We observed differences morphologically and metabolically between the primary skeletal muscle cells derived from young and old donors. These preliminary results give us an insight into human skeletal muscle‐derived cellular physiological capacity. Technology to observe human muscle mitochondrial fragmentation in vitro will help us elucidate the effects of aging, illness and injury on skeletal muscle mitochondrial fragmentation, dysfunction, and loss of metabolic flexibility.Support or Funding InformationFunded by the National Institute of Aging of the National Institute of Health; Grant Number R01‐AG059715.