Characterization of Intracellular Ca 2+ Handling in Vascular Smooth Muscle Cells Isolated from Amputated Human Limbs

High Ca 2+ scores assessed by computed tomography in the lower limbs of patients with peripheral artery disease are associated with a greater risk of amputation and all‐cause mortality. Direct assessment of intracellular Ca 2+ handling in peripheral vascular smooth muscle cells (VSM) of humans likely to undergo an amputation is unknown due to the invasiveness of such measures. We directly characterized intracellular VSM Ca 2+ handling in arteries from amputated human limbs stratified by the number of diagnosed cardiovascular (CV) risk factors (<2 vs >6 CV risk factors). VSM were enzymatically isolated from peripheral arteries and were digitally imaged using the fluorescent intracellular Ca 2+ indicator, fura‐2. Cells were exposed to 80 mM K + in physiologic Ca 2+ to assess Ca 2+ entry through voltage‐gated Ca 2+ channels (VGCC). Caffeine was used in the presence and absence of extracellular Ca 2+ to release intracellular Ca 2+ from the sarcoplasmic reticulum (SR). VSM from patients with >6 CV risk factors had elevated basal cytosolic Ca 2+ (0.76±0.01 vs 0.72±0.01 F 360 /F 380 ) and greater Ca 2+ entry through VGCC (0.08±0.01 vs 0.05±0.00 ΔF 360 /F 380 ) vs patients with <2 CV risk factors. In the absence of extracellular Ca 2+ , the SR Ca 2+ store release was decreased in VSM in patients with >6 CV risk factors (0.16±0.02 vs 0.22±0.01 F 360 /F 380 ). However, this decrease was absent in the presence of extracellular Ca 2+ , suggesting elevated Ca 2+ ‐activated Ca 2+ influx with more CV risk factors. Taken together, these data suggest that changes in VGCC, SR Ca 2+ stores, and Ca 2+ ‐activated Ca 2+ influx act to elevate cytosolic Ca 2+ in VSM of amputees with more CV risk factors. NIH T32 HL079995, Health‐IUSM Strategic Research Initiative