Hypoxia and metabolic inhibitors alter the intracellular ATP:ADP ratio and membrane potential in human coronary artery smooth muscle cells.

ATP-sensitive potassium (KATP) channels couple cellular metabolism to excitability, making them ideal candidate sensors for hypoxic vasodilation. However, it is still unknown whether cellular nucleotide levels are affected sufficiently to activate vascular KATP channels during hypoxia. To address this fundamental issue, we measured changes in the intracellular ATP:ADP ratio using the biosensors Perceval/PercevalHR, and membrane potential using the fluorescent probe DiBAC4(3) in human coronary artery smooth muscle cells (HCASMCs). ATP:ADP ratio was significantly reduced by exposure to hypoxia. Application of metabolic inhibitors for oxidative phosphorylation also reduced ATP:ADP ratio. Hyperpolarization caused by inhibiting oxidative phosphorylation was blocked by either 10 µM glibenclamide or 60 mM K+. Hyperpolarization caused by hypoxia was abolished by 60 mM K+ but not by individual K+ channel inhibitors. Taken together, these results suggest hypoxia causes hyperpolarization in part by modulating K+ channels in SMCs

Bioenergetic profile of human coronary artery smooth muscle cells and effect of metabolic intervention

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

Shift in bioenergetic phenotype in response to metabolic modulators.

<p>The change in relative bioenergetic phenotype of HCASMCs after exposure to 10 mM glucose (in glucose-free medium) (A, n = 8), 5 mM 2-DG (B, n = 8), 1 μM rotenone (C, n = 8), 1 μM antimycin (D, n = 7), 1 μM oligomycin (E, n = 8) and 0.75 μM FCCP (F, n = 8). Shift in bioenergetic phenotype can be detected as relative positional change from basal value (filled circle) after drug application (open square) when ECAR is plotted on the X axis and OCR on the Y axis. Panel G and H summarize percent changes of OCR and ECAR against control level.</p

Effects of metabolic inhibitors on cellular ATP:ADP ratio.

<p>Effect of metabolic inhibitors on cellular ATP:ADP ratio measured after 15 min in the absence and presence of, left to right, DMSO (NS), 5 mM 2-DG (p<0.01), 1 μM rotenone (p<0.01), 1 μM antimycin (p<0.05), 6 μM oligomycin (p<0.001) and 1 μM CCCP (p<0.05). Statistical analysis was performed using Student’s un-paired <i>t</i> test (n = 4).</p