Cholesterol loading in liver mitochondria alters mitochondrial morphology, respiration and respiratory supercomplexes assembly

Trabajo presentado en el Annual Meeting of the American-Association-for-the-Study-of-Liver-Diseases (AASLD) / Liver Meeting, celebrado en San Francisco del 9 al 13 de noviembre de 2018
[Background] Cholesterol accumulation in mitochondrial membranes is a hallmark of alcoholic and non-alcoholic fatty liver diseases and hepatocarcinoma. In physiological conditions mitochondrial cholesterol levels are low, however in pathological conditions cholesterol accumulates within this organelle contributing to the progression of the disease. Cholesterol changes membrane physical properties and impacts negatively in the action of specific solute carriers (e.g. Scl25a11) but its role on mitochondrial function and organization of respiratory chain components has not been deciphered yet. Thus, our aim was to study the effects of cholesterol in the function, morphology and respiration of mitochondria from mice liver.
[Methods] WT C57BL/6J male mice were fed for 2 days with normal chow (Ctrl) or highcholesterol diet (2% Cholesterol and 0,5% Sodium Cholate, HC). A wide range of mitochondrial analysis were subsequently performed to determine: free cholesterol levels by HPLC and immunofluorescence; Membrane fluidity by fluorescence anisotropy; Morphology by Electron Transmission Microscopy; Dynamics by Western Blot; Respirometry by Seahorse XFe 24 Analyzer; and Respiratory supercomplexes assembly by BlueNative Page.
[ Results] HC feeding for two days resulted in a significant increase in the mitochondrial cholesterol levels (2-3 fold), which distributed in both the outer and inner membranes, accompanied by increased fluorescence anisotropy indicative of reduced membrane fluidity. This outcome correlated with increased liver damage assessed by serum ALT levels. Interestingly, mitochondrial cholesterol accumulation resulted in morphological changes of mitochondria that appeared swollen, rounded and with disrupted cristae. In addition, cholesterol decreased expression of mitochondrial fission inducer p-DRP1. High-resolution respirometry analyses using pyruvate+malate revealed that cholesterol loading caused a decreased mitochondrial respiratory profile. Both Respiratory Control Ratio (RCR) (state 3/state 4o respiration) and Uncoupling Control Ratio (UCR) (State 3u/ State 4o respiration) werediminished in mitochondria loaded with cholesterol. In consonance with these observations, a decrease in the respiratory chain complexes assembly into the functional quaternary structures called supercomplexes (III2 +IV, I1 +III2 and I1 +III2 +IV) was observed in cholesterolenriched mitochondria. Conclusion: These data indicate that the accumulation of cholesterol in mitochondrial membranes not only disrupt membrane physical properties but also alters mitochondrial morphology and dynamics, and the functional organization of respiratory supercomplexes assembly.