An Inducible and Vascular Smooth Muscle Cell-Specific Pink1 Knockout Induces Mitochondrial Energetic Dysfunction during Atherogenesis

Abstract

DNA damage and mitochondrial dysfunction are defining characteristics of aged vascular smooth muscle cells (VSMCs) found in atherosclerosis. Pink1 kinase regulates mitochondrial homeostasis and recycles dysfunctional organelles critical for maintaining energetic homeostasis. Here, we generated a new vascular-specific Pink1 knockout and assessed its effect on VSMC-dependent atherogenesis in vivo and VSMC energetic metabolism in vitro. A smooth muscle cell-specific and MHC-Cre-inducible flox’d Pink1f/f kinase knockout was made on a ROSA26+/0 and ApoE−/− C57Blk6/J background. Mice were high fat fed for 10 weeks and vasculature assessed for physiological and pathogical changes. Mitochondrial respiratory activity was then assessed in wild-type and knockout animals vessels and isolated cells for their reliance on oxidative and glycolytic metabolism. During atherogenesis, we find that Pink1 knockout affects development of plaque quality rather than plaque quantity by decreasing VSMC and extracellular matrix components, collagen and elastin. Pink1 protein is important in the wild-type VSMC response to metabolic stress and induced a compensatory increase in hexokinase II, which catalyses the first irreversible step in glycolysis. Pink1 appears to play an important role in VSMC energetics during atherogenesis but may also provide insight into the understanding of mitochondrial energetics in other diseases where the regulation of energetic switching between oxidative and glycolytic metabolism is found to be important.

Inducing Energetic Switching Using Klotho Improves Vascular Smooth Muscle Cell Phenotype

“…XF24 quantifies oxidative phosphorylation by measuring oxygen consumption rate (OCR) as a direct measure of complex 4 activity as the terminal electron acceptor of the mitochondrial respiratory chain, while simultaneously measuring extracellular acidification rate (ECAR) as cytoplasmic milli-pH as in index of glycolysis through glucose to lactate conversion. Drug doses for energetic assays were all 1 µM final concentration for mitochondrial inhibitors oligomycin, FCCP and rotenone, and myxothiazol, and 2-deoxy-glucose (25 mM) to competitively inhibit glucose (2.5 mM), as previously published [ 15 ]. In vivo, soluble Klotho is found to have a wide range of physiological doses (pg–ng) [ 16 ].…”

“…Previously, we identified loss of mitochondrial homeostasis as key to this phenotype in human atherosclerosis [ 1 ]. More recently, we showed that a switch to glycolytic metabolism as a potential survival mechanism could delay these effects [ 15 ]. Here, we undertook a novel drug screen that investigated the role of compounds previously identified as having an energetic switching role through AMPK and are predicted to positively impact energy balance.…”

“…Bright-field images and scratch assay were captured and scored using Olympus IX73 and BX51 microscopes using Adobe Photoshop TM , ImageJ, and Olympus CellSenS software, Southend-on-Sea, United Kingdomas previously described [ 15 , 38 ].…”

“…Building upon our previous work to characterise the role of Pink1-KO in atherosclerotic lesions, this study uses the generation of a vascular specific and inducible Pink1-KO cell line as a novel model of mitochondrial dysfunction [ 15 ]. Here, we investigate the next logical step to investigate if the Pink1 pathway can be augmented by potential drug candidates to mitigate defects in energetic homeostasis found in atherosclerotic development and plaque cap thinning.…”

Mitochondrial Bioenergetics in Different Pathophysiological Conditions 2.0

“…Pink1 kinase regulates mitochondrial homeostasis and recycles dysfunctional organelles critical for maintaining energetic homeostasis. In their study, Docherty et al generated a new vascular-specific Pink1 knockout and assessed its effect on VSMC-dependent atherogenesis in vivo and VSMC energetic metabolism in vitro [ 16 ]. During atherogenesis, the authors found that Pink1 knockout affects the development of plaque quality rather than plaque quantity by decreasing VSMC and extracellular matrix components, collagen and elastin [ 16 ].…”