Individual electron transport chain complexes have been shown to assemble into the supramolecular structures known as the respiratory chain supercomplexes (RCS). Several studies reported an associative link between RCS disintegration and human diseases, although the physiological role, structural integrity, and mechanisms of RCS formation remain unknown. Our previous studies suggested that the adenine nucleotide translocase (ANT), the most abundant protein of the inner mitochondrial membrane, can be involved in RCS assembly. In this study, we sought to elucidate whether ANT knockdown (KD) affects RCS formation in H9c2 cardiomyoblasts. Results showed that genetic silencing of ANT1, the main ANT isoform in cardiac cells, stimulated proliferation of H9c2 cardiomyoblasts with no effect on cell viability. ANT1 KD reduced the ΔΨm but increased total cellular ATP levels and stimulated the production of total, but not mitochondrial, reactive oxygen species. Importantly, downregulation of ANT1 had no significant effects on the enzymatic activity of individual ETC complexes I–IV; however, RCS disintegration was stimulated in ANT1 KD cells as evidenced by reduced levels of respirasome, the main RCS. The effects of ANT1 KD to induce RCS disassembly was not associated with acetylation of the exchanger. In conclusion, our study demonstrates that ANT is involved in RCS assembly.

Mitochondrial respiratory chain supercomplexes (RCS), particularly, the respirasome, which contains complexes I, III, and IV, have been suggested to participate in facilitating electron transport, reducing the production of reactive oxygen species (ROS), and maintaining the structural integrity of individual electron transport chain (ETC) complexes. Disassembly of the RCS has been observed in Barth syndrome, neurodegenerative and cardiovascular diseases, diabetes mellitus, and aging. However, the physiological role of RCS in high energy-demanding tissues such as the heart remains unknown. This study elucidates the relationship between RCS assembly and cardiac function. Adult male Sprague Dawley rats underwent Langendorff retrograde perfusion in the presence and absence of ethanol, isopropanol, or rotenone (an ETC complex I inhibitor). We found that ethanol had no effects on cardiac function, whereas rotenone reduced heart contractility, which was not recovered when rotenone was excluded from the perfusion medium. Blue native polyacrylamide gel electrophoresis revealed significant reductions of respirasome levels in ethanol- or rotenone-treated groups compared to the control group. In addition, rotenone significantly increased while ethanol had no effect on mitochondrial ROS production. In isolated intact mitochondria in vitro, ethanol did not affect respirasome assembly; however, acetaldehyde, a byproduct of ethanol metabolism, induced dissociation of respirasome. Isopropanol, a secondary alcohol which was used as an alternative compound, had effects similar to ethanol on heart function, respirasome levels, and ROS production. In conclusion, ethanol and isopropanol reduced respirasome levels without any noticeable effect on cardiac parameters, and cardiac function is not susceptible to moderate reductions of RCS.

IntroductionPulmonary arterial hypertension (PAH) is characterized by loss of microvessels. The Wnt pathways control pulmonary angiogenesis, but their role in PAH is incompletely understood. We hypothesized that Wnt activation in pulmonary microvascular endothelial cells (PMVECs) is required for pulmonary angiogenesis, and its loss contributes to PAH.MethodsLung tissue and PMVECs from healthy and PAH patients were screened for Wnt production. Global and endothelial-specific Wnt7a−/–mice were generated and exposed to chronic hypoxia and Sugen-hypoxia (SuHx).ResultsHealthy PMVECs demonstrated >6-fold Wnt7a expression during angiogenesis that was absent in PAH PMVECs and lungs. Wnt7a expression correlated with formation of tip cells, a migratory endothelial phenotype critical for angiogenesis. PAH PMVECs demonstrated reduced VEGF-induced tip cell formation as evidenced by reduced filopodia formation and motility, which was partially rescued by recombinant Wnt7a. We discovered that Wnt7a promotes VEGF signaling by facilitating Y1175 tyrosine phosphorylation in VEGFR2 through ROR2, a Wnt-specific receptor. We found that ROR2 knockdown mimics Wnt7a insufficiency and prevents recovery of tip cell formation with Wnt7a stimulation. While there was no difference between wild-type and endothelial-specific Wnt7a−/–mice under either chronic hypoxia and SuHx, global Wnt7a+/–mice in hypoxia demonstrated higher pulmonary pressures and severe right ventricular and lung vascular remodeling. Similar to PAH, Wnt7a+/–PMVECs exhibited insufficient angiogenic response to VEGF-A that improved with Wnt7a.ConclusionsWnt7a promotes VEGF signaling in lung PMVECs and its loss is associated with insufficient VEGF-A angiogenic response. We propose that Wnt7a deficiency contributes to progressive small vessel loss in PAH.

BackgroundMitochondrial dynamics including fission and fusion, along with mitophagy, play a vital role in the maintenance of the structural and functional integrity and number of mitochondria in the cell. These processes work to sustain the mitochondrial quality in the cell, in order to ensure optimal functioning of the cell. Mitochondrial fission and fusion are mediated by several proteins; including dynamin‐related 1 and fission protein 1 for fission and, optic atrophy type 1 (OPA1) and mitofusins 1 (Mfn1) and 2 (Mfn2) for fusion. Fusion proteins (OPA1, Mfn1 and Mfn2) promote fusion of two mitochondria into one, thereby eliminating dysfunctional mitochondria and thus, maintaining their normal morphology, content, and function, as well as the quality of the mitochondria in different subcellular compartments. The mitochondria in cardiomyocytes can be categorized in subpopulations based on their structural and physiological differences: subsarcolemmal mitochondria (SSM), which are found underneath the sarcolemma; interfibrillar mitochondria (IFM), localized between the myofibrils; and perinuclear mitochondria, which reside near the nuclear poles. Connexin‐43, a transmembrane protein responsible for the formation of gap junctions, can be used to differentiate between the SSM and IFM. In this project, we aim to study the role of mitochondrial fusion proteins between these two populations of mitochondria.HypothesisExpression of fusion proteins are different between the SSM and IFM in intact rat hearts.MethodsMitochondria were isolated from Sprague‐Dawley male rat hearts (n=6). Mitochondrial proteins were visualized by SDS‐PAGE followed by western blot analysis using antibodies against OPA1, Mfn1, and Mfn2.ResultsThe level of connexin‐43 was 95% (P<0.001) lower in IFM compared to SSM. Analysis of fusion proteins revealed that IFM contained 10% (P<0.47), 88% (P<0.001) and 24% (P<0.31) less OPA‐1, Mfn1 and Mfn2, respectively, when compared to SSM. Short and long isoforms of OPA‐1 were 70% (P< 0.002) and 32% (P<0.05) lower in IFM compared to SSM.ConclusionLower expression of fusion proteins in IFM can explain differences in functional parameters between two mitochondrial subpopulations in the heart.Support or Funding InformationSupported by the NHLBI NIH grants SC1HL118669 (S.J.) and 1 R25 HL115473‐01 (R.G‐H.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Background For a long time, mitochondrial electron transport chain (ETC) complexes was thought to function as separate entities. However, studies conducted in the past decade revealed that individual ETC complexes can assemble to form supramolecular structures known as supercomplexes (SCs). The SCs, particularly respirasome are composed of the ETC complexes I, III and IV in various stoichiometry. The respirasome is considered to play an important role in facilitating electron transport, reducing ROS production, and maintaining structural integrity of individual ETC complexes. Despite extensive studies on the molecular identity, the physiological role of SCs in cells, particularly, in high energy‐consuming organs such as the heart and brain remain unknown. These studies seek to determine whether disassembly of SCs affects the cardiac function in rats. Methods Hearts isolated from Sprague Dawley rats (275–325g) were perfused with Krebs Henseleit solution (KHS) via the Langendorff‐perfusion model. Experimental groups were as follows: (i) perfusion for 40 min with KHS, (ii) perfusion for 20 min with KHS and 20 min with rotenone (an inducer of SC dissociation) (n=8), (iii) perfusion for 60 min with KHS (n=4), (iv) perfusion for 20 min with KHS followed by 20 min perfusion with rotenone and 20 min with KHS (washout, no rotenone) (n=6). In control groups (i) and (iii), ethanol was as a vehicle in concentration used for rotenone groups [(ii) and (iv)]. In addition, we compared SC levels in mitochondria isolated from non‐perfused hearts (v), and hearts perfused with KHS (vi) containing no vehicle (ethanol) with those in i–iv groups. Cardiac function was monitored throughout the entire perfusion period. At the end of each protocol, the mitochondria were isolated for analysis of SCs, permeability transition pore opening, respiration rates and ROS production. Results Cardiac function between rats perfused with KHS (vi) and ethanol (i and iii) had no significant difference, though rotenone perfused rats (ii and iv) had a significant reduction (< 40%) in cardiac function. Mitochondrial respiration data shows a significant reduction in complex I oxygen consumption rate in rotenone perfused (ii and iv) rats. Analysis of SCs by blue native PAGE displayed a significant reduction in SC levels in both ethanol (i and iii) and rotenone (ii and iv) perfused rats in comparison to hearts perfused with KHS (vi) alone. No significant differences in SC levels were found between the ethanol (i and iii) and rotenone (ii and iv) perfused groups. Conclusion Our data demonstrate lack of causal relationship link between mitochondrial ETC SCs and cardiac function in rats. Support or Funding Information Supported by the NIGMS NIH grants SC1GM128210 and R25GM061838 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.