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Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells
Published on: April 27, 2018
Inhibition of mitochondrial complex I by rotenone reduces arterial calcification in vitro
Andrea Gorgels1, Tabea Köneke1, Setareh Karimian1
1Department of Internal Medicine I, Cardiology, University Hospital Aachen, RWTH Aachen, Aachen, Germany.
Insights
Inhibiting mitochondrial complex I in vascular cells reduces arterial calcification by altering metabolism and increasing lactic acid. This suggests new therapeutic targets for cardiovascular diseases.
Area of Science:
- Biochemistry
- Cell Biology
- Cardiovascular Research
Background:
- Arterial calcification is a risk factor for cardiovascular diseases, particularly in diabetic and kidney disease patients.
- Vascular smooth muscle cell (vSMC) transformation and hydroxyapatite deposition drive arterial calcification.
- Mitochondrial electron transport chain complexes I and IV are implicated in vSMC calcification.
Purpose of the Study:
- To investigate the role of mitochondrial complex I in vSMC calcification.
- To understand the metabolic changes associated with complex I inhibition during calcification.
Main Methods:
- vSMCs were induced to calcify using osteogenic medium.
- Rotenone, a complex I inhibitor, was used to assess its effects.
- Metabolomic profiling, pH measurements, and tissue-nonspecific alkaline phosphatase (TNAP) activity assays were performed.
Main Results:
- Rotenone dose-dependently inhibited matrix mineralization, with near-complete blockage at 50 nM.
- Metabolomic analysis revealed increased lactic acid and decreased pyruvic acid, indicating a shift to glycolysis.
- Rotenone reduced TNAP activity by 59% without altering ALPL transcript levels.
Conclusions:
- Inhibition of mitochondrial complex I induces metabolic reprogramming in vSMCs, leading to lactic acid accumulation.
- Lactic acid and extracellular acidification partially inhibit matrix mineralization via distinct mechanisms.
- Further uncharacterized mechanisms likely contribute to the potent anti-calcific effect of complex I inhibition.
Aim:
Cardiovascular diseases are the leading cause of death worldwide, with arterial calcification being a risk factor, especially in patients with diabetes and kidney disease. Arterial calcification involves hydroxyapatite deposition and the transformation of vascular smooth muscle cells (vSMCs) into osteoblast-like cells, processes mediated in part by tissue-nonspecific alkaline phosphatase (TNAP, encoded by ALPL), a key regulator of mineralization. Previous work showed increased activity of electron transport chain complexes I and IV during vSMC calcification. This study examined the role of mitochondrial complex I in vSMC calcification.
Methods:
vSMCs were calcified using osteogenic medium. Rotenone was used as a complex I inhibitor. Metabolomic profiling, extracellular pH measurements, and TNAP activity assays were performed.
Results:
Rotenone dose-dependently reduced matrix mineralization, with near-complete inhibition at 50 nM, without affecting cell viability. Metabolomic analysis showed that rotenone increased both intracellular and extracellular lactic acid while decreasing pyruvic acid, indicating a shift toward glycolysis. Exogenous lactic acid reduced mineralization by 24%; sodium lactate reduced mineralization without altering extracellular pH; and extracellular acidification independently reduced mineralization by 23%, indicating that both lactate and proton-mediated acidification partially contribute to the anti-calcific mechanism. Additionally, rotenone decreased TNAP activity by 59% without affecting ALPL transcript levels.
Conclusion:
Inhibition of mitochondrial complex I causes metabolic reprogramming in calcifying vSMCs, promoting lactic acid accumulation that partially inhibits matrix mineralization through lactate-mediated and acidification-dependent mechanisms. The near-complete inhibition of calcification by rotenone suggests additional uncharacterized mechanisms beyond the lactic acid-pH axis contribute to its full anti-calcific effect, warranting further investigation.
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