With age, skeletal muscles lose their oxidative capacity and have a reduced mitochondrial fusion leading to fragmentation. These phenomena can lead to a reduction in oxygen consumption, atrophy, and an increased risk of developing age-related diseases such as sarcopenia. Skeletal muscle cells derived from humans can be used to investigate these physiological capacities in primary culture.
PURPOSE: Investigate mitochondrial morphology and maximal oxygen consumption rates (OCR) of skeletal muscle cells derived from healthy young and old men.
METHODS: 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 mitochondria morphology was observed using a Zeiss LSM 710 AxioObserver confocal 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).
RESULTS: 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 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).
CONCLUSIONS: 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 on skeletal muscle mitochondrial fragmentation and loss of metabolic flexibility in aging.