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Background and Purpose The blood-brain barrier (BBB) is a selectively permeable cerebrovascular endothelial barrier that maintains homeostasis between the periphery and central nervous system (CNS). BBB disruption is a consequence of ischemic stroke and BBB permeability can be altered by infection/inflammation, but the complex cellular and molecular changes that result in this BBB alteration need to be elucidated to determine mechanisms. Methods Infection mimic (LPS) challenge on infarct volume, BBB permeability, infiltrated neutrophils and functional outcomes following murine transient middle cerebral artery occlusion (tMCAO) in vivo; mitochondrial evaluation of cerebrovascular endothelial cells (CVECs) challenged by LPS in vitro; pharmacological inhibition of mitochondria on BBB permeability in vitro and in vivo; the effects of mitochondrial inhibitor on BBB permeability, infarct volume and functional outcomes following tMCAO. Results We report here that LPS worsens ischemic stroke outcome and increases BBB permeability following tMCAO in mice. Further, we elucidate a novel mechanism that compromised mitochondrial function accounts for increased BBB permeability as evidenced by: LPS-induced reductions in oxidative phosphorylation and subunit expression of respiratory chain complexes in CVECs, a compromised BBB permeability induced by pharmacological inhibition of mitochondrial function in CVECs in vitro and in an in vivo animal model, and worsened stroke outcomes in tMCAO mice following inhibition of mitochondrial function. Conclusions We concluded that mitochondria are key players in BBB permeability. These novel findings suggest a potential new therapeutic strategy for ischemic stroke by endothelial cell mitochondrial regulation.

Background Cardiomyocytes are rich in mitochondria which are situated in spatially-distinct subcellular regions including those under the plasma membrane, subsarcolemmal mitochondria; and those between the myofibrils, interfibrillar mitochondria. We previously observed subpopulation-specific differences in mitochondrial proteomes following diabetic insult. The objective of this study was to determine whether mitochondrial genome-encoded proteins are regulated by microRNAs inside the mitochondrion and whether subcellular spatial location or diabetes mellitus influences the dynamics. Methods and Results Using microarray technology coupled with cross-linking immunoprecipitation and next generation sequencing, we identified a pool of mitochondrial microRNAs, termed mitomiRs that are redistributed in spatially-distinct mitochondrial subpopulations in an inverse manner following diabetic insult. Redistributed mitomiRs displayed distinct interactions with the mitochondrial genome requiring specific stoichiometric associations with RISC constituents argonaute-2 (Ago2) and fragile X mental retardation–related protein 1 (FXR1) for translational regulation. In the presence of Ago2 and FXR1, redistribution of mitomiR-378 to the IFM following diabetic insult led to down regulation of mitochondrially-encoded F0 component ATP6. Next generation sequencing analyses identified specific transcriptome and mitomiR sequences associated with ATP6 regulation. Overexpression of mitomiR-378 in HL-1 cells resulted in its accumulation in the mitochondrion and down-regulation of functional ATP6 protein, while antagomir blockade restored functional ATP6 protein and cardiac pump function. Conclusions We propose mitomiRs can translationally regulate mitochondrially-encoded proteins in spatially-distinct mitochondrial subpopulations during diabetes mellitus. The results reveal the requirement of RISC constituents in the mitochondrion for functional mitomiR translational regulation and provide a connecting link between diabetic insult and ATP synthase function.

Mitochondrial dysfunction is a contributor to diabetic cardiomyopathy. Previously, we observed proteomic decrements within the inner mitochondrial membrane (IMM) and matrix of diabetic cardiac interfibrillar mitochondria (IFM) correlating with dysfunctional mitochondrial protein import. The goal of this study was to determine whether overexpression of mitochondria phospholipid hydroperoxide glutathione peroxidase 4 (mPHGPx), an antioxidant enzyme capable of scavenging membrane-associated lipid peroxides in the IMM, could reverse proteomic alterations, dysfunctional protein import, and ultimately, mitochondrial dysfunction associated with the diabetic heart. MPHGPx transgenic mice and controls were made diabetic by multiple low-dose streptozotocin injections and examined after 5 wk of hyperglycemia. Five weeks after hyperglycemia onset, in vivo analysis of cardiac contractile function revealed decreased ejection fraction and fractional shortening in diabetic hearts that was reversed with mPHGPx overexpression. MPHGPx overexpression increased electron transport chain function while attenuating hydrogen peroxide production and lipid peroxidation in diabetic mPHGPx IFM. MPHGPx overexpression lessened proteomic loss observed in diabetic IFM. Posttranslational modifications, including oxidations and deamidations, were attenuated in diabetic IFM with mPHGPx overexpression. Mitochondrial protein import dysfunction in diabetic IFM was reversed with mPHGPx overexpression correlating with protein import constituent preservation. Ingenuity Pathway Analyses indicated that oxidative phosphorylation, tricarboxylic acid cycle, and fatty acid oxidation processes most influenced in diabetic IFM were preserved by mPHGPx overexpression. Specific mitochondrial networks preserved included complex I and II, mitochondrial ultrastructure, and mitochondrial protein import. These results indicate that mPHGPx overexpression can preserve the mitochondrial proteome and provide cardioprotective benefits to the diabetic heart.