Myocardial glycophagy flux dysregulation and glycogen accumulation characterize diabetic cardiomyopathy.

Diabetic heart disease morbidity and mortality is escalating. No specific therapeutics exist and mechanistic understanding of diabetic cardiomyopathy etiology is lacking. While lipid accumulation is a recognized cardiomyocyte phenotype of diabetes, less is known about glycolytic fuel handling and storage. Based on in vitro studies, we postulated the operation of an autophagy pathway in the myocardium specific for glycogen homeostasis - glycophagy. Here we visualize occurrence of cardiac glycophagy and show that the diabetic myocardium is characterized by marked glycogen elevation and altered cardiomyocyte glycogen localization. We establish that cardiac glycophagy flux is disturbed in diabetes. Glycophagy may represent a potential therapeutic target for alleviating the myocardial impacts of metabolic disruption in diabetic heart disease.

DIAPH1-MFN2 interaction regulates mitochondria-SR/ER contact and modulates ischemic/hypoxic stress.

Inter-organelle contact and communication between mitochondria and sarco/endoplasmic reticulum (SR/ER) maintain cellular homeostasis and are profoundly disturbed during tissue ischemia. We tested the hypothesis that the formin Diaphanous-1 (DIAPH1), which regulates actin dynamics, signal transduction and metabolic functions, contributes to these processes. We demonstrate that DIAPH1 interacts directly with Mitofusin-2 (MFN2) to shorten mitochondria-SR/ER distance, thereby enhancing mitochondria-ER contact in cells including cardiomyocytes, endothelial cells and macrophages. Solution structure studies affirm the interaction between the Diaphanous Inhibitory Domain and the cytosolic GTPase domain of MFN2. In male rodent and human cardiomyocytes, DIAPH1-MFN2 interaction regulates mitochondrial turnover, mitophagy, and oxidative stress. Introduction of synthetic linker construct, which shorten the mitochondria-SR/ER distance, mitigated the molecular and functional benefits of DIAPH1 silencing in ischemia. This work establishes fundamental roles for DIAPH1-MFN2 interaction in the regulation of mitochondria-SR/ER contact networks. We propose that targeting pathways that regulate DIAPH1-MFN2 interactions may facilitate recovery from tissue ischemia.

TBK1 and GABARAP family members suppress Coxsackievirus B infection by limiting viral production and promoting autophagic degradation of viral extracellular vesicles.

Host-pathogen dynamics are constantly at play during enteroviral infection. Coxsackievirus B (CVB) is a common juvenile enterovirus that infects multiple organs and drives inflammatory diseases including acute pancreatitis and myocarditis. Much like other enteroviruses, CVB is capable of manipulating host machinery to hijack and subvert autophagy for its benefit. We have previously reported that CVB triggers the release of infectious extracellular vesicles (EVs) which originate from autophagosomes. These EVs facilitate efficient dissemination of infectious virus. Here, we report that TBK1 (Tank-binding kinase 1) suppresses release of CVB-induced EVs. TBK1 is a multimeric kinase that directly activates autophagy adaptors for efficient cargo recruitment and induces type-1 interferons during viral-mediated STING recruitment. Positioning itself at the nexus of pathogen elimination, we hypothesized that loss of TBK1 could exacerbate CVB infection due to its specific role in autophagosome trafficking. Here we report that infection with CVB during genetic TBK1 knockdown significantly increases viral load and potentiates the bulk release of viral EVs. Similarly, suppressing TBK1 with small interfering RNA (siRNA) caused a marked increase in intracellular virus and EV release, while treatment in vivo with the TBK1-inhibitor Amlexanox exacerbated viral pancreatitis and EV spread. We further demonstrated that viral EV release is mediated by the autophagy modifier proteins GABARAPL1 and GABARAPL2 which facilitate autophagic flux. We observe that CVB infection stimulates autophagy and increases the release of GABARAPL1/2-positive EVs. We conclude that TBK1 plays additional antiviral roles by inducing autophagic flux during CVB infection independent of interferon signaling, and the loss of TBK1 better allows CVB-laden autophagosomes to circumvent lysosomal degradation, increasing the release of virus-laden EVs. This discovery sheds new light on the mechanisms involved in viral spread and EV propagation during acute enteroviral infection and highlights novel intracellular trafficking protein targets for antiviral therapy.

Calcium, mitochondria and the initiation of acute pancreatitis.

Acute pancreatitis is characterized by necrosis of its parenchymal cells and influx and activation of inflammatory cells that further promote injury and necrosis. This review is intended to discuss the central role of disorders of calcium metabolism and mitochondrial dysfunction in the mechanism of pancreatitis development. The disorders are placed in context of calcium and mitochondria in physiologic function of the pancreas. Moreover, we discuss potential therapeutics for preventing pathologic calcium signals that injure mitochondria and interventions that promote the removal of injured mitochondria and regenerate new and heathy populations of mitochondria.

PACT establishes a posttranscriptional brake on mitochondrial biogenesis by promoting the maturation of miR-181c.

The double-stranded RNA-dependent protein kinase activating protein (PACT), an RNA-binding protein that is part of the RNA-induced silencing complex, plays a key role in miR-mediated translational repression. Previous studies showed that PACT regulates the expression of various miRs, selects the miR strand to be loaded onto RNA-induced silencing complex, and determines proper miR length. Apart from PACT's role in mediating the antiviral response in immune cells, what PACT does in other cell types is unknown. Strikingly, it has also been shown that cold exposure leads to marked downregulation of PACT protein in mouse brown adipose tissue (BAT), where mitochondrial biogenesis and metabolism play a central role. Here, we show that PACT establishes a posttranscriptional brake on mitochondrial biogenesis (mitobiogenesis) by promoting the maturation of miR-181c, a key suppressor of mitobiogenesis that has been shown to target mitochondrial complex IV subunit I (Mtco1) and sirtuin 1 (Sirt1). Consistently, we found that a partial reduction in PACT expression is sufficient to enhance mitobiogenesis in brown adipocytes in culture as well as during BAT activation in mice. In conclusion, we demonstrate an unexpected role for PACT in the regulation of mitochondrial biogenesis and energetics in cells and BAT.

Asporin, an extracellular matrix protein, is a beneficial regulator of cardiac remodeling.

Heart failure is accompanied by adverse cardiac remodeling involving extracellular matrix (ECM). Cardiac ECM acts as a major reservoir for many proteins including growth factors, cytokines, collagens, and proteoglycans. Activated fibroblasts during cardiac injury can alter the composition and activity of these ECM proteins. Through unbiased analysis of a microarray dataset of human heart tissue comparing normal hearts (n = 135) to hearts with ischemic cardiomyopathy (n = 94), we identified Asporin (ASPN) as the top differentially regulated gene (DEG) in ischemic cardiomyopathy; its gene-ontology terms relate closely to fibrosis and cell death. ASPN is a Class I small leucine repeat protein member implicated in cancer, osteoarthritis, and periodontal ligament mineralization. However, its role in cardiac remodeling is still unknown. Here, we initially confirmed our big dataset analysis through cells, mice, and clinical atrial biopsy samples to demonstrate increased Aspn expression after pressure overload or cardiac ischemia/reperfusion injury. We tested the hypothesis that Aspn, being a TGFß1 inhibitor, can attenuate fibrosis in mouse models of cardiac injury. We found that Aspn is released by cardiac fibroblasts and attenuates TGFß signaling. Moreover, Aspn-/- mice displayed increased fibrosis and decreased cardiac function after pressure overload by transverse aortic constriction (TAC) in mice. In addition, Aspn protected cardiomyocytes from hypoxia/reoxygenation-induced cell death and regulated mitochondrial bioenergetics in cardiomyocytes. Increased infarct size after ischemia/reperfusion injury in Aspn-/- mice confirmed Aspn's contribution to cardiomyocyte viability. Echocardiography revealed greater reduction in left ventricular systolic function post-I/R in the Aspn-/- animals compared to wild type. Furthermore, we developed an ASPN-mimic peptide using molecular modeling and docking which when administered to mice prevented TAC-induced fibrosis and preserved heart function. The peptide also reduced infarct size after I/R in mice, demonstrating the translational potential of ASPN-based therapy. Thus, we establish the role of ASPN as a critical ECM molecule that regulates cardiac remodeling to preserve heart function.

Autophagy deficiency abolishes liver mitochondrial DNA segregation.

Mutations in the mitochondrial genome (mtDNA) are ubiquitous in humans and can lead to a broad spectrum of disorders. However, due to the presence of multiple mtDNA molecules in the cell, co-existence of mutant and wild-type mtDNAs (termed heteroplasmy) can mask disease phenotype unless a threshold of mutant molecules is reached. Importantly, the mutant mtDNA level can change across lifespan as mtDNA segregates in an allele- and cell-specific fashion, potentially leading to disease. Segregation of mtDNA is mainly evident in hepatic cells, resulting in an age-dependent increase of mtDNA variants, including non-synonymous potentially deleterious mutations. Here we modeled mtDNA segregation using a well-established heteroplasmic mouse line with mtDNA of NZB/BINJ and C57BL/6N origin on a C57BL/6N nuclear background. This mouse line showed a pronounced age-dependent NZB mtDNA accumulation in the liver, thus leading to enhanced respiration capacity per mtDNA molecule. Remarkably, liver-specific atg7 (autophagy related 7) knockout abolished NZB mtDNA accumulat ion, resulting in close-to-neutral mtDNA segregation through development into adulthood. prkn (parkin RBR E3 ubiquitin protein ligase) knockout also partially prevented NZB mtDNA accumulation in the liver, but to a lesser extent. Hence, we propose that age-related liver mtDNA segregation is a consequence of macroautophagic clearance of the less-fit mtDNA. Considering that NZB/BINJ and C57BL/6N mtDNAs have a level of divergence comparable to that between human Eurasian and African mtDNAs, these findings have potential implications for humans, including the safe use of mitochondrial replacement therapy.Abbreviations: Apob: apolipoprotein B; Atg1: autophagy-related 1; Atg7: autophagy related 7; Atp5a1: ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit 1; BL6: C57BL/6N mouse strain; BNIP3: BCL2/adenovirus E1B interacting protein 3; FCCP: carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; MAP1LC3A: microtubule-associated protein 1 light chain 3 alpha; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; mt-Atp8: mitochondrially encoded ATP synthase 8; MT-CO1: mitochondrially encoded cytochrome c oxidase I; MT-CO2: mitochondrially encoded cytochrome c oxidase II; mt-Co3: mitochondrially encoded cytochrome c oxidase III; mt-Cytb: mitochondrially encoded cytochrome b; mtDNA: mitochondrial DNA; MUL1: mitochondrial ubiquitin ligase activator of NFKB 1; nDNA: nuclear DNA; Ndufa9: NADH:ubiquinone oxireductase subunit A9; NDUFB8: NADH:ubiquinone oxireductase subunit B8; Nnt: nicotinamide nucleotide transhydrogenase; NZB: NZB/BINJ mouse strain; OXPHOS: oxidative phosphorylation; PINK1: PTEN induced putative kinase 1; Polg2: polymerase (DNA directed), gamma 2, accessory subunit; Ppara: peroxisome proliferator activated receptor alpha; Ppia: peptidylprolyl isomerase A; Prkn: parkin RBR E3 ubiquitin protein ligase; P10: post-natal day 10; P21: post-natal day 21; P100: post-natal day 100; qPCR: quantitative polymerase chain reaction; Rpl19: ribosomal protein L19; Rps18: ribosomal protein S18; SD: standard deviation; SEM: standard error of the mean; SDHB: succinate dehydrogenase complex, subunit B, iron sulfur (Ip); SQSTM1: sequestosome 1; Ssbp1: single-stranded DNA binding protein 1; TFAM: transcription factor A, mitochondrial; Tfb1m: transcription factor B1, mitochondrial; Tfb2m: transcription factor B2, mitochondrial; TOMM20: translocase of outer mitochondrial membrane 20; UQCRC2: ubiquinol cytochrome c reductase core protein 2; WT: wild-type.

Myocardial ultrastructure can augment genetic testing for sporadic dilated cardiomyopathy with initial heart failure.

AIMS: The aim of the present study was to consider whether the ultrastructural features of cardiomyocytes in dilated cardiomyopathy can be used to guide genetic testing. METHODS AND RESULTS: Endomyocardial biopsy and whole-exome sequencing were performed in 32 consecutive sporadic dilated cardiomyopathy patients [51.0 (40.0-64.0) years, 75% men] in initial phases of decompensated heart failure. The predicted pathogenicity of ultrarare (minor allele frequency ≤0.0005), non-synonymous variants was determined using the American College of Medical Genetics guidelines. Focusing on 75 cardiomyopathy-susceptibility and 41 arrhythmia-susceptibility genes, we identified 404 gene variants, of which 15 were considered pathogenic or likely pathogenic in 14 patients (44% of 32). There were five sarcomeric gene variants (29% of 17 variants) found in five patients (16% of 32), involving a variant of MYBPC3 and four variants of TTN. A patient with an MYBPC3 variant showed disorganized sarcomeres, three patients with TTN variants located in the region encoding the A-band domain showed sparse sarcomeres, and a patient with a TTN variant in encoding the I-band domain showed disrupted sarcomeres. The distribution of diffuse myofilament lysis depended on the causal genes; three patients with the same TMEM43 variant had diffuse myofilament lysis near nuclei (P = 0.011), while two patients with different DSP variants had lysis in the peripheral areas of cardiomyocytes (P = 0.033). CONCLUSIONS: Derangement patterns of myofilament and subcellular distribution of myofilament lysis might implicate causal genes. Large-scale studies are required to confirm whether these ultrastructural findings are related to the causative genes.

Proteomics of Mouse Heart Ventricles Reveals Mitochondria and Metabolism as Major Targets of a Post-Infarction Short-Acting GLP1Ra-Therapy.

Cardiovascular disease is the main cause of death worldwide, making it crucial to search for new therapies to mitigate major adverse cardiac events (MACEs) after a cardiac ischemic episode. Drugs in the class of the glucagon-like peptide-1 receptor agonists (GLP1Ra) have demonstrated benefits for heart function and reduced the incidence of MACE in patients with diabetes. Previously, we demonstrated that a short-acting GLP1Ra known as DMB (2-quinoxalinamine, 6,7-dichloro-N-[1,1-dimethylethyl]-3-[methylsulfonyl]-,6,7-dichloro-2-methylsulfonyl-3-N-tert-butylaminoquinoxaline or compound 2, Sigma) also mitigates adverse postinfarction left ventricular remodeling and cardiac dysfunction in lean mice through activation of parkin-mediated mitophagy following infarction. Here, we combined proteomics with in silico analysis to characterize the range of effects of DMB in vivo throughout the course of early postinfarction remodeling. We demonstrate that the mitochondrion is a key target of DMB and mitochondrial respiration, oxidative phosphorylation and metabolic processes such as glycolysis and fatty acid beta-oxidation are the main biological processes being regulated by this compound in the heart. Moreover, the overexpression of proteins with hub properties identified by protein-protein interaction networks, such as Atp2a2, may also be important to the mechanism of action of DMB. Data are available via ProteomeXchange with identifier PXD027867.

Autophagy-mitophagy induction attenuates cardiovascular inflammation in a murine model of Kawasaki disease vasculitis.

Kawasaki disease (KD) is the leading cause of acquired heart disease among children. Murine and human data suggest that the NLRP3-IL-1ß pathway is the main driver of KD pathophysiology. NLRP3 can be activated during defective autophagy/mitophagy. We used the Lactobacillus casei cell wall extract (LCWE) murine model of KD vasculitis to examine the role of autophagy/mitophagy on cardiovascular lesion development. LCWE-injected mice had impaired autophagy/mitophagy and increased levels of ROS in cardiovascular lesions, together with increased systemic 8-OHdG release. Enhanced autophagic flux significantly reduced cardiovascular lesions in LCWE-injected mice, whereas autophagy blockade increased inflammation. Vascular smooth muscle cell-specific deletion of Atg16l1 and global Parkin-/- significantly increased disease formation, supporting the importance of autophagy/mitophagy in this model. Ogg1-/- mice had significantly increased lesions with increased NLRP3 activity, whereas treatment with MitoQ reduced vascular tissue inflammation, ROS production, and systemic 8-OHdG release. Treatment with MN58b or Metformin (increasing AMPK and reducing ROS) resulted in decreased cardiovascular lesions. Our results demonstrate that impaired autophagy/mitophagy and ROS-dependent damage exacerbate the development of murine KD vasculitis. This pathway can be efficiently targeted to reduce disease severity. These findings enhance our understanding of KD pathogenesis and identify potentially novel therapeutic avenues for KD treatment.

Hypothermia promotes mitochondrial elongation In cardiac cells via inhibition of Drp1.

Hypothermia is a valuable clinical tool in mitigating against the consequences of ischemia in surgery, stroke, cardiac arrest and organ preservation. Protection is afforded principally by a reduction of metabolism, manifesting as reduced rates of oxygen uptake, preservation of ATP levels, and a curtailing of ischemic calcium overload. The effects of non-ischemic hypothermic stress are relatively unknown. We sought to investigate the effects of clinically mild-to-severe hypothermia on mitochondrial morphology, oxygen consumption and protein expression in normoxic hearts and cardiac cells. Normoxic perfusion of rat hearts at 28-32 °C was associated with inhibition of mitochondrial fission, evidenced by a reduced abundance of the active phosphorylated form of the fission receptor Drp1 (pDrp1S616). Abundance of the same residue was reduced in H9c2 cells subjected to hypothermic culture (25-32 °C), in addition to a reduced abundance of the Drp1 receptor MFF. Hypothermia-treated H9c2 cardiomyocytes exhibited elongated mitochondria and depressed rates of mitochondrial-associated oxygen consumption, which persisted upon rewarming. Hypothermia also promoted a reduction in mRNA expression of the capsaicin receptor TRPV1 in H9c2 cells. When normothermic H9c2 cells were transfected with TRPV1 siRNA we observed reduced pDrp1S616 and MFF abundance, elongated mitochondria, and reduced rates of mitochondrial-associated oxygen consumption, mimicking the effects of hypothermic culture. In conclusion hypothermia promoted elongation of cardiac mitochondria via reduced pDrp1S616 abundance which was also associated with suppression of cellular oxygen consumption. Silencing of TRPV1 in H9c2 cardiomyocytes reproduced the morphological and respirometric phenotype of hypothermia. This report demonstrates a novel mechanism of cold-induced inhibition of mitochondrial fission.

Elevated Asparagine Biosynthesis Drives Brain Tumor Stem Cell Metabolic Plasticity and Resistance to Oxidative Stress.

Asparagine synthetase (ASNS) is a gene on the long arm of chromosome 7 that is copy-number amplified in the majority of glioblastomas. ASNS copy-number amplification is associated with a significantly decreased survival. Using patient-derived glioma stem cells (GSC), we showed that significant metabolic alterations occur in gliomas when perturbing the expression of ASNS, which is not merely restricted to amino acid homeostasis. ASNS-high GSCs maintained a slower basal metabolic profile yet readily shifted to a greatly increased capacity for glycolysis and oxidative phosphorylation when needed. This led ASNS-high cells to a greater ability to proliferate and spread into brain tissue. Finally, we demonstrate that these changes confer resistance to cellular stress, notably oxidative stress, through adaptive redox homeostasis that led to radiotherapy resistance. Furthermore, ASNS overexpression led to modifications of the one-carbon metabolism to promote a more antioxidant tumor environment revealing a metabolic vulnerability that may be therapeutically exploited. IMPLICATIONS: This study reveals a new role for ASNS in metabolic control and redox homeostasis in glioma stem cells and proposes a new treatment strategy that attempts to exploit one vulnerable metabolic node within the larger multilayered tumor network.

At the heart of mitochondrial quality control: many roads to the top.

Mitochondrial quality control depends upon selective elimination of damaged mitochondria, replacement by mitochondrial biogenesis, redistribution of mitochondrial components across the network by fusion, and segregation of damaged mitochondria by fission prior to mitophagy. In this review, we focus on mitochondrial dynamics (fusion/fission), mitophagy, and other mechanisms supporting mitochondrial quality control including maintenance of mtDNA and the mitochondrial unfolded protein response, particularly in the context of the heart.

Attenuation of Adverse Postinfarction Left Ventricular Remodeling with Empagliflozin Enhances Mitochondria-Linked Cellular Energetics and Mitochondrial Biogenesis.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors such as empagliflozin are known to reduce the risk of hospitalizations related to heart failure irrespective of diabetic state. Meanwhile, adverse cardiac remodeling remains the leading cause of heart failure and death in the USA. Thus, understanding the mechanisms that are responsible for the beneficial effects of SGLT2 inhibitors is of the utmost relevance and importance. Our previous work illustrated a connection between adverse cardiac remodeling and the regulation of mitochondrial turnover and cellular energetics using a short-acting glucagon-like peptide-1 receptor agonist (GLP1Ra). Here, we sought to determine if the mechanism of the SGLT2 inhibitor empagliflozin (EMPA) in ameliorating adverse remodeling was similar and/or to identify what differences exist, if any. To this end, we administered permanent coronary artery ligation to induce adverse remodeling in wild-type and Parkin knockout mice and examined the progression of adverse cardiac remodeling with or without EMPA treatment over time. Like GLP1Ra, we found that EMPA affords a robust attenuation of PCAL-induced adverse remodeling. Interestingly, unlike the GLP1Ra, EMPA does not require Parkin to improve/maintain mitochondria-related cellular energetics and afford its benefits against developing adverse remodeling. These findings suggests that further investigation of EMPA is warranted as a potential path for developing therapy against adverse cardiac remodeling for patients that may have Parkin and/or mitophagy-related deficiencies.

Recruitment of pro-IL-1α to mitochondrial cardiolipin, via shared LC3 binding domain, inhibits mitophagy and drives maximal NLRP3 activation.

The balance between NLRP3 inflammasome activation and mitophagy is essential for homeostasis and cellular health, but this relationship remains poorly understood. Here we found that interleukin-1α (IL-1α)-deficient macrophages have reduced caspase-1 activity and diminished IL-1ß release, concurrent with reduced mitochondrial damage, suggesting a role for IL-1α in regulating this balance. LPS priming of macrophages induced pro-IL-1α translocation to mitochondria, where it directly interacted with mitochondrial cardiolipin (CL). Computational modeling revealed a likely CL binding motif in pro-IL-1α, similar to that found in LC3b. Thus, binding of pro-IL-1α to CL in activated macrophages may interrupt CL-LC3b-dependent mitophagy, leading to enhanced Nlrp3 inflammasome activation and more robust IL-1ß production. Mutation of pro-IL-1α residues predicted to be involved in CL binding resulted in reduced pro-IL-1α-CL interaction, a reduction in NLRP3 inflammasome activity, and increased mitophagy. These data identify a function for pro-IL-1α in regulating mitophagy and the potency of NLRP3 inflammasome activation.

Neurotropin Inhibits Lipid Accumulation by Maintaining Mitochondrial Function in Hepatocytes via AMPK Activation.

The accumulation of lipid droplets in the cytoplasm of hepatocytes, known as hepatic steatosis, is a hallmark of non-alcoholic fatty liver disease (NAFLD). Inhibiting hepatic steatosis is suggested to be a therapeutic strategy for NAFLD. The present study investigated the actions of Neurotropin (NTP), a drug used for chronic pain in Japan and China, on lipid accumulation in hepatocytes as a possible treatment for NAFLD. NTP inhibited lipid accumulation induced by palmitate and linoleate, the two major hepatotoxic free fatty acids found in NAFLD livers. An RNA sequencing analysis revealed that NTP altered the expression of mitochondrial genes. NTP ameliorated palmitate-and linoleate-induced mitochondrial dysfunction by reversing mitochondrial membrane potential, respiration, and ß-oxidation, suppressing mitochondrial oxidative stress, and enhancing mitochondrial turnover. Moreover, NTP increased the phosphorylation of AMPK, a critical factor in the regulation of mitochondrial function, and induced PGC-1ß expression. Inhibition of AMPK activity and PGC-1ß expression diminished the anti-steatotic effect of NTP in hepatocytes. JNK inhibition could also be associated with NTP-mediated inhibition of lipid accumulation, but we did not find the association between AMPK and JNK. These results suggest that NTP inhibits lipid accumulation by maintaining mitochondrial function in hepatocytes via AMPK activation, or by inhibiting JNK.

Parkin, an E3 ubiquitin ligase, enhances airway mitochondrial DNA release and inflammation.

INTRODUCTION: Parkin (Park2), an E3 ubiquitin ligase, is critical to maintain mitochondrial function by regulating mitochondrial biogenesis and degradation (mitophagy), but recent evidence suggests the involvement of Parkin in promoting inflammation. In the present study, we determined if Parkin regulates airway mitochondrial DNA (mtDNA) release and inflammatory responses to type 2 cytokine interleukin (IL)-13 and allergens. METHODS: We measured Parkin mRNA expression in brushed bronchial epithelial cells and mtDNA release in the paired bronchoalveolar lavage fluid (BALF) from normal subjects and asthmatics. Parkin-deficient primary human tracheobronchial epithelial (HTBE) cells generated using the CRISPR-Cas9 system were stimulated with IL-13. To determine the in vivo function of Parkin, Parkin knockout (PKO) and wild-type (WT) mice were treated with IL-13 or allergen (house dust mite, HDM) in the presence or absence of mtDNA isolated from normal mouse lungs. RESULTS: Parkin mRNA expression in asthmatic airway epithelium was upregulated, which positively correlated with the levels of released mtDNA in BALF. IL-13-stimulated HTBE cells increased Parkin expression. Moreover, IL-13 induced mtDNA release in Parkin-sufficient, but not in Parkin-deficient HTBE cells. PKO (vs WT) mice attenuated airway mtDNA release and inflammation following IL-13 or HDM treatments. mtDNA amplified airway inflammation in mice treated with IL-13 or HDM. Notably, Parkin also mediated mtDNA-induced exacerbation of airway inflammation. CONCLUSION: Our research findings suggest that Parkin promotes mtDNA release and inflammation in airways, thus improving our understanding of the complex role of Parkin and mitochondrial dysfunction in asthma pathogenesis.

Minimal Invasive Pericardial Perfusion Model in Swine: A Translational Model for Cardiac Remodeling After Ischemia/Reperfusion Injury.

RATIONALE: Adverse remodeling leads to heart failure after myocardial infarction (MI), with important impact on morbidity and mortality. New therapeutic approaches are needed to further improve and broaden heart failure therapy. We established a minimally invasive, reproducible pericardial irrigation model in swine, as a translational model to study the impact of temperature on adverse cardiac remodeling and its molecular mechanisms after MI. OBJECTIVE: Chronic heart failure remains a leading cause of death in western industrialized countries, with a tremendous economic impact on the health care system. Previously, many studies have investigated mechanisms to reduce infarct size after ischemia/reperfusion injury, including therapeutic hypothermia. Nonetheless, the molecular mechanisms of adverse remodeling after MI remain poorly understood. By deciphering the latter, new therapeutic strategies can be developed to not only reduce rehospitalization of heart failure patients but also reduce or prevent adverse remodeling in the first place. METHODS AND RESULTS: After 90 min of MI, a 12Fr dual lumen dialysis catheter was place into the pericardium via minimal invasive, sub-xiphoidal percutaneous puncture. We performed pericardial irrigation with cold or warm saline for 60 min in 25 female farm pigs after ischemia and reperfusion. After one week of survival the heart was harvested for further studies. After cold pericardial irrigation we observed a significant decrease of systemic body temperature measured with a rectal probe in the cold group, reflecting that the heart was chilled throughout its entire thickness. The temperature remained stable in the control group during the procedure. We did not see any difference in arrhythmia or hemodynamic stability between both groups. CONCLUSION: We established a minimally invasive, reproducible and translational model of pericardial irrigation in swine. This method enables the investigation of mechanisms involved in myocardial adverse remodeling after ischemia/reperfusion injury in the future.

Intermittent Use of a Short-Course Glucagon-like Peptide-1 Receptor Agonist Therapy Limits Adverse Cardiac Remodeling via Parkin-dependent Mitochondrial Turnover.

Given that adverse remodeling is the leading cause of heart failure and death in the USA, there is an urgent unmet need to develop new methods in dealing with this devastating disease. Here we evaluated the efficacy of a short-course glucagon-like peptide-1 receptor agonist therapy-specifically 2-quinoxalinamine, 6,7-dichloro-N-(1,1-dimethylethyl)-3-(methylsulfonyl)-,6,7-dichloro-2-methylsulfonyl-3-N-tert-butylaminoquinoxaline (DMB; aka Compound 2) - in attenuating adverse LV remodeling. We also examined the role, if any, of mitochondrial turnover in this process. Wild-type, Parkin knockout and MitoTimer-expressing mice were subjected to permanent coronary artery ligation, then treated briefly with DMB. LV remodeling and cardiac function were assessed by histology and echocardiography. Autophagy and mitophagy markers were examined by western blot and mitochondrial biogenesis was inferred from MitoTimer protein fluorescence and qPCR. We found that DMB given post-infarction significantly reduced adverse LV remodeling and the decline of cardiac function. This paralleled an increase in autophagy, mitophagy and mitochondrial biogenesis. The salutary effects of the drug were lost in Parkin knockout mice, implicating Parkin-mediated mitophagy as part of its mechanism of action. Our findings suggest that enhancing Parkin-associated mitophagy and mitochondrial biogenesis after infarction is a viable target for therapeutic mitigation of adverse remodeling.