Arterial calcification is a serious global health issue. It independently increases the risk of CV disease and death, and there are currently no effective treatments available. Factors contributing to arterial calcification include inflammation, endoplasmic reticulum stress, and metabolic imbalances in vascular smooth muscle cells (SMCs). These metabolic imbalances can cause or result from changes in mitochondrial function. Our recent research has demonstrated alterations in mitochondrial function in calcifying SMCs. The five mitochondrial complexes are key to mitochondrial activity and complex I activity was found to be elevated in calcifying SMCs. Therefore, we analyzed the inhibition of mitochondrial complex I, hypothesizing that complex I activity contributes to arterial calcification.
SMCs were calcified in osteogenic media (OM). Inhibiting complex I with rotenone reduced matrix mineralization in calcifying SMCs in a dose-dependent manner (-90%, p<0.001) without impacting cell viability. We assessed mitochondrial respiration using the Seahorse method, which showed an increased basal oxygen consumption rate (OCR) (+25%, p=0.022) in calcifying SMCs, and this was reduced by complex I inhibition with rotenone (-50%) p<0.001). Interestingly, rotenone lowered the extracellular pH (p=0.005) without affecting the intracellular pH. The effect of extracellular acidification on matrix mineralization was supported by showing less calcification in SMCs cultured with OM at a decreased pH (pH 6.6 vs. pH 7.4, -90%, p<0.001). Metabolomic analysis revealed increased lactic acid (cell lysates +100% p=0.031; supernatants +200% p=0.008) and decreased pyruvic acid (cell lysates -50% p=0.010; supernatants -50% p=0.005) in conditioned media and cell lysates of SMCs after rotenone treatment, indicating a metabolic shift. Culturing calcifying SMCs with lactic acid lowered the extracellular pH (p=0.011) and caused a 20% decrease in SMC matrix mineralization (p=0.009), confirming lactic acid as a contributor to extracellular pH decline and mineralization inhibition. Rotenone inhibited tissue non-specific alkaline phosphatase activity, which suggests that the unfavorable, low pH for alkaline phosphatase results in the inhibition of mineralization.
In conclusion, complex I activity may contribute to arterial calcification by altering the metabolism of SMCs, resulting in lactic acid-mediated lowering of the extracellular environment pH. This low extracellular pH inhibits tissue non-specific alkaline phosphatase activity and, consequently, SMC mineralization.
