Impaired Mitochondrial Bioenergetics Research Articles

Impaired cardiac mitochondrial bioenergetics contribute to loss of cardiac function, in a porcine multiple risk factor model

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Dutch CardioVascular Alliance (an initiative with support of the Dutch Heart Foundation) Grant 2020B008 RECONNEXT, and the German Center for Cardiovascular Research (DZHK; 81Z0600207) Background Heart failure with preserved ejection fraction lacks targeted therapies, due to insufficient understanding of pathogenesis. A multiple comorbidity swine model (MCS) was developed by exposure to three risk factors for six months including diabetes (streptozotocin), chronic kidney disease (renal embolization), and an unhealthy diet (high-fat, high salt diet). An additional risk factor ovariectomy was added on top of the MCS model (MCS+). MCS and MCS+ presented with left ventricular diastolic dysfunction, oxidative stress, coronary microvascular dysfunction and inflammation. Furthermore, myocardial oxygen consumption was increased given the same level of cardiac work (Fig. A), suggesting an impairment in myocardial mitochondrial function. Using a combined-omics approach, the purpose of this study is to unravel alterations in gene expression and proteome abundance regarding mitochondrial function in MCS swine. Methods 15 MCS, 4 MCS+ and 10 healthy control female swine were included in the study. Proteome analysis and single nuclei RNA sequencing, were performed on frozen MCS left ventricle myocardial samples. Cardiac mitochondrial function was measured by an O2k-FlouRespirometer ex vivo in MCS+ swine versus control. Results Proteome analysis showed a reduced abundance of proteins involved in branched-chain amino acid (BCAA) catabolism (Fig. B). Single nuclei RNA sequencing of a subgroup of the same animals demonstrated a downregulation of these genes specifically in cardiomyocyte subpopulations. BCAA can act as a source of ATP via oxidative phosphorylation and can modulate mitochondrial substrate utilisation, and impair mitochondrial function, resulting in increased mitochondrial production of reactive oxygen species (ROS). Mitochondrial function was measured in fresh myocardial tissue, using pyruvate/malate/glutamate as substrate. Low mitochondrial respiratory sensitivity to ADP was observed 26±2.5 in MCS+ vs 43.6±1.5 pmol/(s*mg tissue) in healthy control, reflecting low oxidative capacity. Moreover, the respiratory rate after uncoupling by FCCP was reduced in MCS+ versus healthy control (73.6±11.7 vs 118.4±15.6 pmol/(s*mg tissue). These findings are consistent with our data in the MCS swine and indicate impaired BCAA in the myocardium of animals with comorbidities, which is associated with increased ROS levels (8-isoprostane 12.9±0.8 pg/mg protein in MCS vs 10.3±0.5 in healthy animals) and impaired myocardial efficiency during exercise (Fig. A). Conclusion A combined omics approach suggested an alteration in cardiac mitochondria. Respiratory analyses showed impaired mitochondrial bioenergetics and increased oxidative stress, which can contribute to diastolic dysfunction.

Oxidative stress in the remaining muscle after volumetric muscle loss contributes to impaired mitochondrial bioenergetics and is attenuated by SS-31

Volumetric muscle loss (VML) injury results in the non-recoverable loss of muscle mass and function. Our previous studies indicate mitochondrial dysfunction in the remaining muscle fibers after VML injury, marked by changes in mitochondrial respiration, membrane potential, and enzyme activities. In particular, mitochondrial membrane potential is hyperpolarized during the first 14-days post-injury, a condition that could lead to the production of reactive oxygen species (ROS). When ROS emissions exceed the antioxidant buffering capacity, cellular damage can ensue. The primary objective of this study was to determine ROS production, ROS emission, and antioxidant buffering capacity of remaining muscle fibers after VML injury in the first month after injury. The secondary objective of this study was to determine if a mitochondrial antioxidant (SS-31) could mitigate mitochondrial dysfunction after VML injury. We hypothesized that VML injury will alter the redox status of the remaining muscle. Study 1: Male C57BL/6J mice (n=40) were randomized to Uninjured or VML-injured cohorts. ROS (H2O2) emission/production and endogenous antioxidant buffering capacity were analyzed fluorometrically during live muscle fiber respiration (Oroboros O2k) at 3-, 7-, 14-, 21-, and 30-days post-injury. Study 2: Male C57BL/6J mice (n=32) were randomized into two groups: VML and VML+SS-31. SS-31 is a mitochondrial-targeted peptide with ROS-scavenging properties. VML+SS-31 received SS-31 (8mg/kg/day) for 14 days, and the VML received equal volume saline injections. At 14- and 28-days post-VML, mitochondrial respiration and electron conductance, ROS emission, and antioxidant buffering capacity were assessed in permeabilized gastrocnemius muscle fibers from the muscle remaining after the initial injury. Group differences were detected through ANOVA and unpaired t-test. ROS emission and antioxidant buffering capacity were different between VML-injured and Uninjured mice through 14-days post-injury, specifically at 3-, 7-, and 14-days, mitochondrial ROS emission was significantly higher (+28%, p<0.001), and antioxidant buffering capacity was less (-28%, p=0.002) in VML mice compared to Uninjured mice. Two weeks of SS-31 treatment lead to greater muscle fiber respiration and electron conductance (p≤0.008) at both 14- and 28-days post-VML. Importantly, at 14-days post-injury, mitochondrial ROS emission was 71% lower in VML+SS-31 than VML. At both 14- and 28-days post-injury, antioxidant buffering capacity was 12% greater in VML+SS-31 (p≤0.003) compared to VML. Oxidative stress is a feature of early VML pathophysiology marked by greater ROS emission and less antioxidant buffering capacity. Attenuating oxidative stress during the first two-weeks post-VML injury improves mitochondrial respiration. W81XWH-20-1-0885 and R01AR078903 to SG and JC. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Transketolase drives the development of aortic dissection by impairing mitochondrial bioenergetics.

Aortic dissection (AD) is a disease with rapid onset but with no effective therapeutic drugs yet. Previous studies have suggested that glucose metabolism plays a critical role in the progression of AD. Transketolase (TKT) is an essential bridge between glycolysis and the pentose phosphate pathway. However, its role in the development of AD has not yet been elucidated. In this study, we aimed to explore the role of TKT in AD. We collected AD patients' aortic tissues and used high-throughput proteome sequencing to analyze the main factors influencing AD development. We generated an AD model using BAPN in combination with angiotensin II (Ang II) and pharmacological inhibitors to reduce TKT expression. The effects of TKT and its downstream mediators on AD were elucidated using human aortic vascular smooth muscle cells (HAVSMCs). We found that glucose metabolism plays an important role in the development of AD and that TKT is upregulated in patients with AD. Western blot and immunohistochemistry confirmed that TKT expression was upregulated in mice with AD. Reduced TKT expression attenuated AD incidence and mortality, maintained the structural integrity of the aorta, aligned elastic fibers, and reduced collagen deposition. Mechanistically, TKT was positively associated with impaired mitochondrial bioenergetics by upregulating AKT/MDM2 expression, ultimately contributing to NDUFS1 downregulation. Our results provide new insights into the role of TKT in mitochondrial bioenergetics and AD progression. These findings provide new intervention options for the treatment of AD.

Longevity control by supersulfide-mediated mitochondrial respiration and regulation of protein quality

Supersulfides, which are defined as sulfur species with catenated sulfur atoms, are increasingly being investigated in biology. We recently identified pyridoxal phosphate (PLP)-dependent biosynthesis of cysteine persulfide (CysSSH) and related supersulfides by cysteinyl-tRNA synthetase (CARS). Here, we investigated the physiological role of CysSSH in budding yeast (Saccharomyces cerevisiae) by generating a PLP-binding site mutation K109A in CRS1 (the yeast ortholog of CARS), which decreased the synthesis of CysSSH and related supersulfides and also led to reduced chronological aging, effects that were associated with an increased endoplasmic reticulum stress response and impaired mitochondrial bioenergetics. Reduced chronological aging in the K109A mutant could be rescued by using exogenous supersulfide donors. Our findings indicate important roles for CARS in the production and metabolism of supersulfides—to mediate mitochondrial function and to regulate longevity.

Lipocalin 2 regulates mitochondrial phospholipidome remodeling, dynamics, and function in brown adipose tissue in male mice

Mitochondrial function is vital for energy metabolism in thermogenic adipocytes. Impaired mitochondrial bioenergetics in brown adipocytes are linked to disrupted thermogenesis and energy balance in obesity and aging. Phospholipid cardiolipin (CL) and phosphatidic acid (PA) jointly regulate mitochondrial membrane architecture and dynamics, with mitochondria-associated endoplasmic reticulum membranes (MAMs) serving as the platform for phospholipid biosynthesis and metabolism. However, little is known about the regulators of MAM phospholipid metabolism and their connection to mitochondrial function. We discover that LCN2 is a PA binding protein recruited to the MAM during inflammation and metabolic stimulation. Lcn2 deficiency disrupts mitochondrial fusion-fission balance and alters the acyl-chain composition of mitochondrial phospholipids in brown adipose tissue (BAT) of male mice. Lcn2 KO male mice exhibit an increase in the levels of CLs containing long-chain polyunsaturated fatty acids (LC-PUFA), a decrease in CLs containing monounsaturated fatty acids, resulting in mitochondrial dysfunction. This dysfunction triggers compensatory activation of peroxisomal function and the biosynthesis of LC-PUFA-containing plasmalogens in BAT. Additionally, Lcn2 deficiency alters PA production, correlating with changes in PA-regulated phospholipid-metabolizing enzymes and the mTOR signaling pathway. In conclusion, LCN2 plays a critical role in the acyl-chain remodeling of phospholipids and mitochondrial bioenergetics by regulating PA production and its function in activating signaling pathways.

PHB2 ameliorates Doxorubicin-induced cardiomyopathy through interaction with NDUFV2 and restoration of mitochondrial complex I function

BackgroundDoxorubicin (DOX) is among the most widely employed antitumor agents, although its clinical applications have been largely hindered by severe cardiotoxicity. Earlier studies described an essential role of mitochondrial injury in the pathogenesis of DOX cardiomyopathy. PHB2 (Prohibitin 2) is perceived as an essential regulator for mitochondrial dynamics and oxidative phosphorylation (OXPHOS) although its involvement in DOX cardiomyopathy remains elusive. MethodsTo decipher the possible role of PHB2 in DOX cardiomyopathy, tamoxifen-induced cardiac-specific PHB2 conditional knockout mice were generated and subjected to DOX challenge. Cardiac function and mitochondrial profiles were examined. Screening of downstream mediators of PHB2 was performed using proteomic profiling and bioinformatic analysis, and was further verified using co-immunoprecipitation and pulldown assays. ResultsOur data revealed significantly downregulated PHB2 expression in DOX-challenged mouse hearts. PHB2CKO mice were more susceptible to DOX cardiotoxicity compared with PHB2flox/flox mice, as evidenced by more pronounced cardiac atrophy, interstitial fibrosis and decrease in left ventricular ejection fraction and fractional shortening. Mechanistically, PHB2 deficiency resulted in the impairment of mitochondrial bioenergetics and oxidative phosphorylation in DOX cardiotoxicity. Proteomic profiling and interactome analyses revealed that PHB2 interacted with NDUFV2 (NADH-ubiquinone oxidoreductase core subunit V2), a key subunit of mitochondrial respiratory Complex I to mediate regulatory property of PHB2 on mitochondrial metabolism. PHB2 governed the expression of NDUFV2 by promoting its stabilization, while PHB2 deficiency significantly downregulated NDUFV2 in DOX-challenged hearts. Cardiac overexpression of PHB2 alleviated mitochondrial defects in DOX cardiomyopathy both in vivo and in vitro. ConclusionsOur study defined a novel role for PHB2 in mitochondrial dynamics and energetic metabolism through interacting with NDUFV2 in DOX-challenged hearts. Forced overexpression of PHB2 may be considered a promising therapeutic approach for patients with DOX cardiomyopathy.

Andrographolide protects against atrial fibrillation by alleviating oxidative stress injury and promoting impaired mitochondrial bioenergetics.

Atrial fibrillation (AF) is the most prevalent cardiac arrhythmia seen in clinical settings, which has been associated with substantial rates of mortality and morbidity. However, clinically available drugs have limited efficacy and adverse effects. We aimed to investigate the mechanisms of action of andrographolide (Andr) with respect to AF. We used network pharmacology approaches to investigate the possible therapeutic effect of Andr. To define the role of Andr in AF, HL-1 cells were pro-treated with Andr for 1 h before rapid electronic stimulation (RES) and rabbits were pro-treated for 1 d before rapid atrial pacing (RAP). Apoptosis, myofibril degradation, oxidative stress, and inflammation were determined. RNA sequencing (RNA-seq) was performed to investigate the relevant mechanism. Andr treatment attenuated RAP-induced atrial electrophysiological changes, inflammation, oxidative damage, and apoptosis both in vivo and in vitro. RNA-seq indicated that oxidative phosphorylation played an important role. Transmission electron microscopy and adenosine triphosphate (ATP) content assay respectively validated the morphological and functional changes in mitochondria. The translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) to the nucleus and the molecular docking suggested that Andr might exert a therapeutic effect by influencing the Keap1-Nrf2 complex. In conclusions, this study revealed that Andr is a potential preventive therapeutic drug toward AF via activating the translocation of Nrf2 to the nucleus and the upregulation of heme oxygenase-1 (HO-1) to promote mitochondrial bioenergetics.

IPSC-Based Modeling of Variable Clinical Presentation in Hypertrophic Cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease and a frequent cause of heart failure and sudden cardiac death. Our understanding of the genetic bases and pathogenic mechanisms underlying HCM has improved significantly in the recent past, but the combined effect of various pathogenic gene variants and the influence of genetic modifiers in disease manifestation are very poorly understood. Here, we set out to investigate genotype-phenotype relationships in 2 siblings with an extensive family history of HCM, both carrying a pathogenic truncating variant in the MYBPC3 gene (p.Lys600Asnfs*2), but who exhibited highly divergent clinical manifestations. We used a combination of induced pluripotent stem cell (iPSC)-based disease modeling and CRISPR (clustered regularly interspersed short palindromic repeats)/Cas9 (CRISPR-associated protein 9)-mediated genome editing to generate patient-specific cardiomyocytes (iPSC-CMs) and isogenic controls lacking the pathogenic MYBPC3 variant. Mutant iPSC-CMs developed impaired mitochondrial bioenergetics, which was dependent on the presence of the mutation. Moreover, we could detect altered excitation-contraction coupling in iPSC-CMs from the severely affected individual. The pathogenic MYBPC3 variant was found to be necessary, but not sufficient, to induce iPSC-CM hyperexcitability, suggesting the presence of additional genetic modifiers. Whole-exome sequencing of the mutant carriers identified a variant of unknown significance in the MYH7 gene (p.Ile1927Phe) uniquely present in the individual with severe HCM. We finally assessed the pathogenicity of this variant of unknown significance by functionally evaluating iPSC-CMs after editing the variant. Our results indicate that the p.Ile1927Phe variant of unknown significance in MYH7 can be considered as a modifier of HCM expressivity when found in combination with truncating variants in MYBPC3. Overall, our studies show that iPSC-based modeling of clinically discordant subjects provides a unique platform to functionally assess the effect of genetic modifiers.

The harmful acute effects of clomipramine in the rat liver: Impairments in mitochondrial bioenergetics

Clomipramine, a tricyclic antidepressant used to treat depression and obsessive-compulsive disorder, has been linked to a few cases of acute hepatotoxicity. It is also recognized as a compound that hinders the functioning of mitochondria. Hence, the effects of clomipramine on mitochondria should endanger processes that are somewhat connected to energy metabolism in the liver. For this reason, the primary aim of this study was to examine how the effects of clomipramine on mitochondrial functions manifest in the intact liver. For this purpose, we used the isolated perfused rat liver, but also isolated hepatocytes and isolated mitochondria as experimental systems. According to the findings, clomipramine harmed metabolic processes and the cellular structure of the liver, especially the membrane structure. The considerable decrease in oxygen consumption in perfused livers strongly suggested that the mechanism of clomipramine toxicity involves the disruption of mitochondrial functions. Coherently, it could be observed that clomipramine inhibited both gluconeogenesis and ureagenesis, two processes that rely on ATP production within the mitochondria. Half-maximal inhibitory concentrations for gluconeogenesis and ureagenesis ranged from 36.87 μM to 59.64 μM. The levels of ATP as well as the ATP/ADP and ATP/AMP ratios were reduced, but distinctly, between the livers of fasted and fed rats. The results obtained from experiments conducted on isolated hepatocytes and isolated mitochondria unambiguously confirmed previous propositions about the effects of clomipramine on mitochondrial functions. These findings revealed at least three distinct mechanisms of action, including uncoupling of oxidative phosphorylation, inhibition of the FoF1-ATP synthase complex, and inhibition of mitochondrial electron flow. The elevation in activity of cytosolic and mitochondrial enzymes detected in the effluent perfusate from perfused livers, coupled with the increase in aminotransferase release and trypan blue uptake observed in isolated hepatocytes, provided further evidence of the hepatotoxicity of clomipramine. It can be concluded that impaired mitochondrial bioenergetics and cellular damage are important factors underlying the hepatotoxicity of clomipramine and that taking excessive amounts of clomipramine can lead to several risks including decreased ATP production, severe hypoglycemia, and potentially fatal outcomes.

Lower and higher volumes of physical exercise build up brain reserves against memory deficits triggered by a head injury in mice

Decreasing neurotrophic support and impaired mitochondrial bioenergetics are key mechanisms for long-term neurodegeneration and cognitive decline after traumatic brain injury (TBI). We hypothesize that preconditioning with lower and higher volumes of physical exercise upregulates the CREB-BDNF axis and bioenergetic capability, which might serve as neural reserves against cognitive impairment after severe TBI. Using a running wheel mounted in the home cage, mice were engaged in lower (LV, 48 h free access, and 48 h locked) and higher (HV, daily free access) exercise volumes for thirty days. Subsequently, LV and HV mice remained for additional thirty days in the home cage with the running wheel locked and were euthanized. The sedentary group had the running wheel always locked. For the same type of exercise stimulus in a given time, daily workout presents higher volume than alternate days workout. The total distance ran in the wheel was the reference parameter to confirm distinct exercise volumes. On average, LV exercise ran 27.522 m and HV exercise ran 52.076 m. Primarily, we investigate whether LV and HV protocols increase neurotrophic and bioenergetic support in the hippocampus thirty days after exercise ceased. Regardless of volume, exercise increased hippocampal pCREBSer133-CREB-proBDNF-BDNF signaling and mitochondrial coupling efficiency, excess capacity, and leak control, that may compose the neurobiological basis for neural reserves. Further, we challenge these neural reserves against secondary memory deficits triggered by a severe TBI. After thirty days of exercise LV and HV, and sedentary (SED) mice were submitted to the CCI model. Mice remained for additional thirty days in the home cage with the running wheel locked. The mortality after severe TBI was approximately 20% in LV and HV, while in the SED was 40%. Also, LV and HV exercise sustained hippocampal pCREBSer133-CREB-proBDNF-BDNF signaling, mitochondrial coupling efficiency, excess capacity, and leak control for thirty days after severe TBI. Corroborating these benefits, the mitochondrial H2O2 production linked to complexes I and II was attenuated by exercise regardless of the volume. These adaptations attenuated spatial learning and memory deficits caused by TBI. In summary, preconditioning with LV and HV exercise builds up long-lasting CREB-BDNF and bioenergetic neural reserves that preserve memory fitness after severe TBI.

P019 Host-Enterobacteriaceae interactions and mitochondrial dysfunction in Crohn’s colitis

Abstract Background Crohn’s disease (CD) is characterized by chronic gastrointestinal inflammation and unfavourable changes in the composition of intestinal microbiota (‘dysbiosis’). Members of Enterobacteriaceae such as K. pneumoniae and E. coli are often over-represented in colonic mucosa of CD patients. These bacteria contribute to vicious circle of dysbiosis and inflammation by expressing multiple virulence factors. Further, mitochondrial dysfunction in Paneth cells has recently been reported in ileal CD. In agreement, high-fat diet and repeated use of antibiotics increases susceptibility to colitis by damaging mitochondria. However, it remains unknown whether impaired mitochondrial bioenergetics could enhance mucosal colonization by Enterobacteriaceae and thus, contribute to the pathophysiology of colonic CD. Furthermore, the molecular mechanism leading to mitochondrial damage in colonic CD are incompletely understood. Methods Right colonic biopsies were collected from 74 adult CD patients and 28 healthy participants at St. Paul’s Hospital (Vancouver, BC, Canada). Microbes in colonic biopsies were characterized by 16S rDNA sequencing using Illumina MiSeq platform, followed by analysis of differentially abundant families via ANCOM-BC methodology. Further, high-throughput data-independent acquisition (DIA) proteomics was performed in colonic biopsies (Bruker Daltonics TimsTof Pro2 LC-MS/MS), and later analyzed via DIA-neural network software. Spearman's rank correlation approach was used for host proteomics- Enterobacteriaceae correlational analysis. Microbiome and proteomic data were corrected for false discovery rate (FDR; < 0.5 for statistical significance). For validation studies, human colonic TC7 epithelial cells and patient-derived organoid cultures were infected with CD-patient derived E. coli ± C75, a chemical inhibitor of fatty acid synthase (FASN; de novo fatty acid synthesis enzyme). Results We found an increased abundance of Enterobacteriaceae members in colonic mucosa of CD patients compared to healthy controls (Fig A). Next, our proteomics data revealed a significant downregulation in a group of proteins which were functionally related to mitochondrial health and activity, in CD patients compared to controls (Fig B). Further, Spearman’s analysis showed a positive correlation between Enterobacteriaceae and host FASN enzyme (Fig C), a protein directly related to mitochondrial metabolism. Interestingly, we found that blocking FASN with chemical inhibitor (C75) significantly enhanced colonization by E. coli in TC7 cells (Fig D). Conclusion Mechanistically, our study reports that aberrant activity of host FASN enzyme may cause mitochondrial dysfunction and thus, facilitates intestinal colonization by Enterobacteriaceae in CD patients.

Translational potential of JAX humanized‐APOE mice: sex differences in bioenergetic function in brain

AbstractBackgroundThe preclinical phase of late onset Alzheimer’s disease (LOAD), known as the preclinical phase, can start 10 to 20 years prior to onset of clinical symptoms. Hypometabolism and impaired mitochondrial bioenergetics can emerge during prodromal phase as early indicators of AD risk. APOEe4, the greatest genetic risk factor for LOAD, is well documented to induce lipid metabolic dysregulation. We therefore sought to determine the impact of APOEe4. The following study analyzed body composition and mitochondrial functions to elucidate how the presence of APOEe4 in a mouse model contributes to bioenergetic changes, and whether they affect females and males equally.Method18mo old transgenic mouse models (JAX #029018 and #027894) carrying humanized APOE alleles (hAPOE3/3, hAPOE3/4 or hAPOE4/4) were assessed for fat and lean mass content via EchoMRI. A week later, fasting blood glucose levels were assessed prior to sacrifice, and brain tissues were collected to determine mitochondria function by Complex I and IV activity.ResultBody composition analyses indicated that hAPOEe4 males had significantly lower fat percentage than male APOEe3 mice. Females, irrespective of APOE genotype, exhibited significantly lower lean weight relative to males. No sex or APOE genotype difference was detected in fasting blood glucose plasma levels nor in mitochondrial Complex I activity. In contrast, mitochondrial Complex IV activity was significantly lower in females relative to males. Lastly, Complex IV activity was lower in mice exhibiting dominance aggressive behavior, specifically barbering, irrespective of sex or genotype.ConclusionAPOE mice resembling a human population of approximately 60 years of age displayed a sex difference in body composition parameters, where total and lean weight was higher in males than females. Sex differences also emerged in bioenergetic function, with females exhibiting lower mitochondria Complex IV activity relative to males. Sex differences are consistent with human data, whereas no genotype differences were observed and thus may influence the translational potential of the humanized APOE mouse model in LOAD research. However, the translational potential might be greater when linked with phenotypic characteristics.This work was supported by NIA grants R01AG057931, and RF1AG059093

Activating SIRT3 in peritoneal mesothelial cells alleviates postsurgical peritoneal adhesion formation by decreasing oxidative stress and inhibiting the NLRP3 inflammasome

Peritoneal adhesions (PAs) are a serious complication of abdominal surgery and negatively affect the quality of life of millions of people worldwide. However, a clear molecular mechanism and a standard therapeutic strategy for PAs have not been established. Here, we developed a standardized method to mimic the pathological changes in PAs and found that sirtuin 3 (SIRT3) expression was severely decreased in adhesion tissues, which was consistent with our bioinformatics analysis and patient adhesion tissue analysis. Thus, we hypothesized that activating SIRT3 could alleviate postsurgical PAs. Sirt3-deficient (Sirt3−/−) mice exhibited many more PAs after standardized abdominal surgery. Furthermore, compared with wild-type (Sirt3+/+) mice, Sirt3-deficient (Sirt3−/−) mice showed more prominent reactive oxygen species (ROS) accumulation, increased levels of inflammatory factors, and exacerbated mitochondrial damage and fragmentation. In addition, we observed NLRP3 inflammasome activation in the adhesion tissues of Sirt3−/− but, not Sirt3+/+ mice. Furthermore, mesothelial cells sorted from Sirt3−/− mice exhibited impaired mitochondrial bioenergetics and redox homeostasis. Honokiol (HKL), a natural compound found in several species of the genus Magnolia, could activate SIRT3 in vitro. Then, we demonstrated that treatment with HKL could reduce oxidative stress and the levels of inflammatory factors and suppress NLRP3 activation in vivo, reducing the occurrence of postsurgical PAs. In vitro treatment with HKL also restored mitochondrial bioenergetics and promoted mesothelial cell viability under oxidative stress conditions. Taken together, our findings show that the rescue of SIRT3 by HKL may be a new therapeutic strategy to alleviate and block postsurgical PA formation.

Evidence of impaired mitochondrial cellular bioenergetics in ocular fibroblasts derived from glaucoma patients

Glaucoma is a progressive optic neuropathy characterized by the neurodegeneration of the retinal ganglion cells (RGCs) resulting in irreversible visual impairment and eventual blindness. RGCs are extremely susceptible to mitochondrial compromise due to their marked bioenergetic requirements and morphology. There is increasing interest in therapies targeting mitochondrial health as a method of preventing visual loss in managing glaucoma. The bioenergetic profile of Tenon's ocular fibroblasts from glaucoma patients and controls was investigated using the Seahorse XF24 analyser. Impaired mitochondrial cellular bioenergetics was detected in glaucomatous ocular fibroblasts including basal respiration, maximal respiration and spare capacity. Spare respiratory capacity levels reflect mitochondrial bio-energetic adaptability in response to pathophysiological stress. Basal oxidative stress was elevated in glaucomatous Tenon's ocular fibroblasts and hydrogen peroxide (H2O2) induced reactive oxygen species (ROS) simulated the glaucomatous condition in normal Tenon's ocular fibroblasts. This work supports the role of therapeutic interventions to target oxidative stress or provide mitochondrial energetic support in glaucoma.

309-OR: Lipocalin 2 Deficiency Impairs Mitochondrial Dynamics and Phospholipid Metabolism in Brown Adipocytes

Impaired mitochondrial bioenergetics in brown adipocytes links to the disrupted energy metabolism and thermogenesis in obesity. Phospholipids, particularly cardiolipin and phosphatidic acid (PA) are critical regulators of mitochondrial membrane architecture and dynamics. Mitochondria-associated endoplasmic reticulum membranes (MAMs) serve as the platform for phospholipid biosynthesis. MAM dysfunction disturbs phospholipid metabolism, thereby disrupting mitochondrial function. However, little is known about the participating factors that regulate MAM phospholipid metabolism. We have previously characterized the role of lipocalin 2 (Lcn2) in thermogenesis and mitochondrial function in adipocytes. Herein, we found that Lcn2 was located at MAMs and had a specific PA binding capability. The mitochondrial fusion-fission process induced by the fasting-refeeding cycle was impaired in Lcn2 knockout (KO) brown adipocytes. We also observed that the levels of phosphorylated Drp1 (Ser616) and Opa1 were decreased in brown adipose tissue (BAT) of Lcn2 KO mice compared to WT controls upon acute cold stimulation, whereas Lcn2 overexpression in BAT led to an increase in Drp1 phosphorylation and Opa1 level. Moreover, Lcn2 deficiency significantly enhanced the cold-stimulated accumulation of Opa1 as well as MAM markers SigmaR1, Ip3R1, and Facl4 at MAMs in BAT, suggesting impaired MAM function. CL316,243, a β3-adrenergic receptor agonist has a cold mimic effect on mitochondrial bioenergetics. In brown adipocytes, Lcn2 deficiency significantly attenuated the CL316,243 induction of Drp1 phosphorylation. Intriguingly, lipidomic analysis of BAT mitochondria isolated from mice treated with CL316,243 for 14 days showed that Lcn2 deficiency blunted CL316,243-induced cardiolipin biosynthesis and altered phospholipid metabolism. Together, we discovered a role of Lcn2 as a novel PA transfer protein in mitochondrial bioenergetics via regulating phospholipid metabolism. Disclosure H.Su: None. H.Guo: None. X.Qiu: None. T.Lin: None. X.Chen: None. Funding This work was supported by NIDDK Grant (RDK123042) awarded to Xiaoli Chen.

Characterisation of a novel OPA1 splice variant resulting in cryptic splice site activation and mitochondrial dysfunction

Autosomal dominant optic atrophy (DOA) is an inherited optic neuropathy that results in progressive, bilateral visual acuity loss and field defects. OPA1 is the causative gene in around 60% of cases of DOA. The majority of patients have a pure ocular phenotype, but 20% have extra-ocular features (DOA +). We report on a patient with DOA + manifesting as bilateral optic atrophy, spastic paraparesis, urinary incontinence and white matter changes in the central nervous system associated with a novel heterozygous splice variant NM_015560.2(OPA1):c.2356-1 G > T. Further characterisation, which was performed using fibroblasts obtained from a skin biopsy, demonstrated that this variant altered mRNA splicing of the OPA1 transcript, specifically a 21 base pair deletion at the start of exon 24, NM_015560.2(OPA1):p.Cys786_Lys792del. The majority of variant transcripts were shown to escape nonsense-mediated decay and modelling of the predicted protein structure suggests that the in-frame 7 amino acid deletion may affect OPA1 oligomerisation. Fibroblasts carrying the c.2356-1 G > T variant demonstrated impaired mitochondrial bioenergetics, membrane potential, increased cell death, and disrupted and fragmented mitochondrial networks in comparison to WT cells. This study suggests that the c.2356-1 G > T OPA1 splice site variant leads to a cryptic splice site activation and may manifest in a dominant-negative manner, which could account for the patient’s severe syndromic phenotype.

CEST-2.2 overexpression alters lipid metabolism and extends longevity of mitochondrial mutants.

Mitochondrial dysfunction can either extend or decrease Caenorhabditis elegans lifespan, depending on whether transcriptionally regulated responses can elicit durable stress adaptation to otherwise detrimental lesions. Here, we test the hypothesis that enhanced metabolic flexibility is sufficient to circumvent bioenergetic abnormalities associated with the phenotypic threshold effect, thereby transforming short‐lived mitochondrial mutants into long‐lived ones. We find that CEST‐2.2, a carboxylesterase mainly localizes in the intestine, may stimulate the survival of mitochondrial deficient animals. We report that genetic manipulation of cest‐2.2 expression has a minor lifespan impact on wild‐type nematodes, whereas its overexpression markedly extends the lifespan of complex I‐deficient gas‐1(fc21) mutants. We profile the transcriptome and lipidome of cest‐2.2 overexpressing animals and show that CEST‐2.2 stimulates lipid metabolism and fatty acid beta‐oxidation, thereby enhancing mitochondrial respiratory capacity through complex II and LET‐721/ETFDH, despite the inherited genetic lesion of complex I. Together, our findings unveil a metabolic pathway that, through the tissue‐specific mobilization of lipid deposits, may influence the longevity of mitochondrial mutant C. elegans.

Inhalation of PM2.5 from diesel exhaust promote impairment of mitochondrial bioenergetics and dysregulate mitochondrial quality in rat heart: implications in isoproterenol-induced myocardial infarction model

Aim: Ambient exposure of PM2.5 from diesel exhaust (termed as diesel particulate matter [DPM]) can induce cardiotoxicity that can be manifested into myocardial ischemia/infarction, where the survival depends on mitochondrial function. The mechanism for DPM-induced mitochondrial dysfunction is yet to be elucidated and the consequential impact of impaired mitochondria on the severity of myocardial infarction (MI) has not been established. Materials and methods: Female Wistar rats were exposed to DPM (0.5 mg/ml) for 3 h daily (to achieve a PM2.5 concentration of 250 µg/m3) for 21 d trailed by an induction of MI using isoproterenol (ISO). Conclusion: DPM exposure altered the basal ECG pattern and increased heart weight (HW) to body weight (BW) ratio from control. Loss of mitochondrial quality in the cardiac tissue was observed in DPM exposed animals, measured via declined ETC enzyme activity, reduced ATP levels, high oxidative stress, low mitochondrial copy number, and low expression of the mitochondrial genes involved in mitophagy (PINK and PARKIN) and mitochondrial fusion (MFN-1). Subsequent induction of MI in DPM exposed animals (DPM + ISO) further deteriorated the normal sinus rhythm, accompanied by elevated plasma CK and LDH level, increased myocardial caspase activity, downregulation of Peroxisome proliferator-activated receptor-gamma coactivator (PGC1-α), transcription factor A (TFAM), DNA polymerase subunit gamma (POLG), and other mitochondrial quality control genes. Based on these results, we conclude that DPM alters the electrophysiology and ultrastructure of the heart that aggravates the MI-induced cardiotoxicity, where the diminished mitochondrial quality can be the potential contributor.

DYRK1B-STAT3 Drives Cardiac Hypertrophy and Heart Failure by Impairing Mitochondrial Bioenergetics.

Heart failure is a global public health issue that is associated with increasing morbidity and mortality. Previous studies have suggested that mitochondrial dysfunction plays critical roles in the progression of heart failure; however, the underlying mechanisms remain unclear. Because kinases have been reported to modulate mitochondrial function, we investigated the effects of DYRK1B (dual-specificity tyrosine-regulated kinase 1B) on mitochondrial bioenergetics, cardiac hypertrophy, and heart failure. We engineered DYRK1B transgenic and knockout mice and used transverse aortic constriction to produce an in vivo model of cardiac hypertrophy. The effects of DYRK1B and its downstream mediators were subsequently elucidated using RNA-sequencing analysis and mitochondrial functional analysis. We found that DYRK1B expression was clearly upregulated in failing human myocardium and in hypertrophic murine hearts, as well. Cardiac-specific DYRK1B overexpression resulted in cardiac dysfunction accompanied by a decline in the left ventricular ejection fraction, fraction shortening, and increased cardiac fibrosis. In striking contrast to DYRK1B overexpression, the deletion of DYRK1B mitigated transverse aortic constriction-induced cardiac hypertrophy and heart failure. Mechanistically, DYRK1B was positively associated with impaired mitochondrial bioenergetics by directly binding with STAT3 to increase its phosphorylation and nuclear accumulation, ultimately contributing toward the downregulation of PGC-1α (peroxisome proliferator-activated receptor gamma coactivator-1α). Furthermore, the inhibition of DYRK1B or STAT3 activity using specific inhibitors was able to restore cardiac performance by rejuvenating mitochondrial bioenergetics. Taken together, the findings of this study provide new insights into the previously unrecognized role of DYRK1B in mitochondrial bioenergetics and the progression of cardiac hypertrophy and heart failure. Consequently, these findings may provide new therapeutic options for patients with heart failure.

Myocardial Cytoskeletal Adaptations in Advanced Kidney Disease.

BackgroundThe myocardial cytoskeleton functions as the fundamental framework critical for organelle function, bioenergetics and myocardial remodeling. To date, impairment of the myocardial cytoskeleton occurring in the failing heart in patients with advanced chronic kidney disease has been largely undescribed.Methods and ResultsWe conducted a 3‐arm cross‐sectional cohort study of explanted human heart tissues from patients who are dependent on hemodialysis (n=19), hypertension (n=10) with preserved renal function, and healthy controls (n=21). Left ventricular tissues were subjected to pathologic examination and next‐generation RNA sequencing. Mechanistic and interference RNA studies utilizing in vitro human cardiac fibroblast models were performed. Left ventricular tissues from patients undergoing hemodialysis exhibited increased myocardial wall thickness and significantly greater fibrosis compared with hypertension patients (P<0.05) and control (P<0.01). Transcriptomic analysis revealed that the focal adhesion pathway was significantly enriched in hearts from patients undergoing hemodialysis. Hearts from patients undergoing hemodialysis exhibited dysregulated components of the focal adhesion pathway including reduced β‐actin (P<0.01), β‐tubulin (P<0.01), vimentin (P<0.05), and increased expression of vinculin (P<0.05) compared with controls. Cytoskeletal adaptations in hearts from the hemodialysis group were associated with impaired mitochondrial bioenergetics, including dysregulated mitochondrial dynamics and fusion, and loss of cell survival pathways. Mechanistic studies revealed that cytoskeletal changes can be driven by uremic and metabolic abnormalities of chronic kidney disease, in vitro. Furthermore, focal adhesion kinase silencing via interference RNA suppressed major cytoskeletal proteins synergistically with mineral stressors found in chronic kidney disease in vitro.ConclusionsMyocardial failure in advanced chronic kidney disease is characterized by impairment of the cytoskeleton involving disruption of the focal adhesion pathway, mitochondrial failure, and loss of cell survival pathways.