Abstract
Bioenergetics of artery smooth muscle cells is critical in cardiovascular health and disease. An acute rise in metabolic demand causes vasodilation in systemic circulation while a chronic shift in bioenergetic profile may lead to vascular diseases. A decrease in intracellular ATP level may trigger physiological responses while dedifferentiation of contractile smooth muscle cells to a proliferative and migratory phenotype is often observed during pathological processes. Although it is now possible to dissect multiple building blocks of bioenergetic components quantitatively, detailed cellular bioenergetics of artery smooth muscle cells is still largely unknown. Thus, we profiled cellular bioenergetics of human coronary artery smooth muscle cells and effects of metabolic intervention. Mitochondria and glycolysis stress tests utilizing Seahorse technology revealed that mitochondrial oxidative phosphorylation accounted for 54.5% of ATP production at rest with the remaining 45.5% due to glycolysis. Stress tests also showed that oxidative phosphorylation and glycolysis can increase to a maximum of 3.5 fold and 1.25 fold, respectively, indicating that the former has a high reserve capacity. Analysis of bioenergetic profile indicated that aging cells have lower resting oxidative phosphorylation and reduced reserve capacity. Intracellular ATP level of a single cell was estimated to be over 1.1 mM. Application of metabolic modulators caused significant changes in mitochondria membrane potential, intracellular ATP level and ATP:ADP ratio. The detailed breakdown of cellular bioenergetics showed that proliferating human coronary artery smooth muscle cells rely more or less equally on oxidative phosphorylation and glycolysis at rest. These cells have high respiratory reserve capacity and low glycolysis reserve capacity. Metabolic intervention influences both intracellular ATP concentration and ATP:ADP ratio, where subtler changes may be detected by the latter.
Highlights
The energy storing molecule ATP fuels a variety of cell functions including maintenance of transmembrane ionic gradients, muscle contraction, secretion, cell proliferation and migration
Oligomycin is an inhibitor of ATP synthase, and so the difference before and after oligomycin application isolates the oxygen consumption rate (OCR) linked to ATP production
human coronary artery smooth muscle cells (HCASMCs) cultured in high glucose DMEM containing 1 mM sodium pyruvate and 2 mM L-glutamate generate ATP at a rate of 138.93±8.88 pmol/min per 2x104 cells from oxidative phosphorylation (OXPHOS) and 115.87±3.33 pmol/min per 2x104 cells from glycolysis (Fig 2D). These results indicate that HCASMCs produce 54.5% of ATP through OXPHOS at rest with the remaining 45.5% due to glycolysis
Summary
The energy storing molecule ATP fuels a variety of cell functions including maintenance of transmembrane ionic gradients, muscle contraction, secretion, cell proliferation and migration It acts as an intracellular signaling molecule that translates cellular metabolic status to physiological responses. The notion that the Warburg effect is unique to cancer cells has been challenged as aerobic glycolysis is seen among non-cancerous proliferating cells including vascular smooth muscle cells [3]. This may have an intriguing implication in vascular diseases such as atherosclerosis. Though essential in repairing vascular injury, dedifferentiation is the key step at the onset of atherosclerosis where proliferating and migrating vascular smooth muscle cells initiate cap formation [4]
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