Adenoviral Expression Research Articles

Polymeric immunoglobulin receptor promotes Th2 immune response in the liver by increasing cholangiocytes derived IL-33: a diagnostic and therapeutic biomarker of biliary atresia

Biliary atresia (BA) is a devastating neonatal cholangiopathy with an unclear pathogenesis, and prompt diagnosis of BA is currently challenging. Proteomic and immunoassay analyses were performed with serum samples from 250 patients to find potential BA biomarkers. The expression features of polymeric immunoglobulin receptor (PIGR) were investigated using human biopsy samples, three different experimental mouse models, and cultured human biliary epithelial cells (BECs). Chemically modified small interfering RNA and adenovirus expression vector were applied for invivo silencing and overexpressing PIGR in a rotavirus-induced BA mouse model. Luminex-based multiplex cytokine assays and RNA sequencing were used to explore the molecular mechanism of PIGR involvement in the BA pathogenesis. Serum levels of PIGR, poliovirus receptor (PVR), and aldolase B (ALDOB) were increased in BA patients and accurately distinguished BA from infantile hepatitis syndrome (IHS). Combined PIGR and PVR analysis distinguished BA from IHS with an area under the receiver operating characteristic curve of 0.968 and an accuracy of 0.935. PIGR expression was upregulated in the biliary epithelium of BA patients; Th1 cytokines TNF-α and IFN-γ induced PIGR expression in BECs via activating NF-κB pathway. Silencing PIGR alleviated symptoms, reduced IL-33 expression, and restrained hepatic Th2 inflammation in BA mouse model; while overexpressing PIGR increased liver fibrosis and IL-33 expression, and boosted hepatic Th2 inflammation in BA mouse model. PIGR expression promotes the proliferation and epithelial-mesenchymal transition, and reduced the apoptosis of BECs. PIGR participated in BA pathogenesis by promoting hepatic Th2 inflammation via increasing cholangiocytes derived IL-33; PIGR has the value as a diagnostic and therapeutic biomarker of BA. This study was financially supported by the National Natural Science Foundation of China (82170529), the National Key R&D Program (2021YFC2701003), and the National Natural Science Foundation of China (82272022).

Folate as a potential treatment for lethal ventricular arrhythmias in TANGO2-deficiency disorder.

TANGO2-deficiency disorder (TDD) is an autosomal-recessive genetic disease caused by biallelic loss-of-function variants in the TANGO2 gene. TDD-associated cardiac arrhythmias are recalcitrant to standard antiarrhythmic medications and constitute the leading cause of death. Disease modeling for TDD has been primarily carried out using human dermal fibroblast and, more recently, in Drosophila by multiple research groups. No human cardiomyocyte system has been reported, which greatly hinders the investigation and understanding of TDD-associated arrhythmias. Here, we established potentially novel patient-derived induced pluripotent stem cell differentiated cardiomyocyte (iPSC-CM) models that recapitulate key electrophysiological abnormalities in TDD. These electrophysiological abnormalities were rescued in iPSC-CMs with either adenoviral expression of WT-TANGO2 or correction of the pathogenic variant using CRISPR editing. Our natural history study in patients with TDD suggests that the intake of multivitamin/B complex greatly diminished the risk of cardiac crises in patients with TDD. In agreement with the clinical findings, we demonstrated that high-dose folate (vitamin B9) virtually abolishes arrhythmias in TDD iPSC-CMs and that folate's effect was blocked by the dihydrofolate reductase inhibitor methotrexate, supporting the need for intracellular folate to mediate antiarrhythmic effects. In summary, data from TDD iPSC-CM models together with clinical observations support the use of B vitamins to mitigate cardiac crises in patients with TDD, providing potentially life-saving treatment strategies during life-threatening events.

A face-to-face comparison of the BBB cell models hCMEC/D3 and hBMEC for their applicability to adenoviral expression of transporters.

The blood-brain barrier (BBB) is a structure mainly formed by brain capillary endothelial cells (BCEC) whose role is to regulate the exchange of compounds between the blood and the brain. In this process efflux and uptake transporters play a key role. Aim of this study was to compare the two previously established cell lines hCMEC/D3 and hBMEC as BBB cell models for the application of an adenoviral system to transiently express OATP2B1 and Pgp. Comparison of hCMEC/D3 and hBMEC mRNA and protein levels of BBB markers showed a unique expression pattern for each cell line. While showing similar expression of the efflux transporter BCRP, transferrin receptor (TFRC) and of the tight junctions proteins Occludin and ZO-1, hCMEC/D3 displayed higher levels of the endothelial marker PECAM1, VE-cadherin, Von Willebrand Factor (VWF) and of the efflux transporter Pgp. Moreover, measuring integrity of the monolayer by determining the Trans-Endothelial Electrical Resistance (TEER), electrical capacitance (CCl), and inulin apparent permeability coefficient (Papp) revealed higher TEER and lower CCl for hBMEC but comparable Papp in the two cell lines. Following adenoviral infection, enhanced OATP2B1 and Pgp expression and functionality could be observed only in hBMEC. Importantly, the adenoviral expression system did not affect expression of BBB markers and permeability in both cell lines. Taken together, our results provide first evidence that hBMEC is an applicable human BBB cell model in which adenoviral infection can be used to transiently express and investigate transporters of interest.

Peak transgene expression after intramuscular immunization of mice with adenovirus 26-based vector vaccines correlates with transgene-specific adaptive immune responses.

Non-replicating adenovirus-based vectors have been broadly used for the development of prophylactic vaccines in humans and are licensed for COVID-19 and Ebola virus disease prevention. Adenovirus-based vectored vaccines encode for one or more disease specific transgenes with the aim to induce protective immunity against the target disease. The magnitude and duration of transgene expression of adenovirus 5- based vectors (human type C) in the host are key factors influencing antigen presentation and adaptive immune responses. Here we characterize the magnitude, duration, and organ biodistribution of transgene expression after single intramuscular administration of adenovirus 26-based vector vaccines in mice and evaluate the differences with adenovirus 5-based vector vaccine to understand if this is universally applicable across serotypes. We demonstrate a correlation between peak transgene expression early after adenovirus 26-based vaccination and transgene-specific cellular and humoral immune responses for a model antigen and SARS-CoV-2 spike protein, independent of innate immune activation. Notably, the memory immune response was similar in mice immunized with adenovirus 26-based vaccine and adenovirus 5-based vaccine, despite the latter inducing a higher peak of transgene expression early after immunization and a longer duration of transgene expression. Together these results provide further insights into the mode of action of adenovirus 26-based vector vaccines.

Overexpression of Slc22a18 facilitates fat accumulation in mice

We previously reported that solute carrier family 22 member 18 (Slc22a18) regulates lipid accumulation in 3T3-L1 adipocytes. Here, we provide additional evidence derived from experiments with adenoviral vector expression and genetic manipulation of mice. In primary cultured rat hepatocytes, adenoviral overexpression of mouse Slc22a18 increased triglyceride accumulation and triglyceride synthetic activity, which was decreased in an adenoviral knockdown experiment. Adenoviral overexpression of mouse Slc22a18 in vivo caused massive fatty liver in mice, even under normal dietary conditions. Conversely, adenoviral knockdown of mouse Slc22a18 reduced hepatic lipid accumulation induced by a high-glucose and high-sucrose diet. We created Slc22a18 knockout mice, which grew normally and showed no obvious spontaneous phenotypes. However, compared with control littermates, the knockout mice exhibited decreased hepatic triglyceride content under refeeding conditions, significantly reduced epididymal fat mass, and tended to have lower liver weight in conjunction with leptin deficiency. Finally, we created transgenic mice overexpressing rat Slc22a18 in an adipose-specific manner, which had increased body weight and epididymal fat mass primarily because of increased adipocyte cell volume. In these transgenic mice, a positive correlation was observed between adiposity and the expression levels of the rat Slc22a18 transgene. Taken together, these results indicate that Slc22a18 has positive effects on lipid accumulation in vivo.

Antiviral activity of adenoviral vector expressing human interferon lambda-4 against influenza virus.

Interferon lambda (IFNλ), classified as a type III IFN, is a representative cytokine that plays an important role in innate immunity along with type I IFN. IFNλ can elicit antiviral states by inducing peculiar sets of IFN-stimulated genes (ISGs). In this study, an adenoviral vector expression system with a tetracycline operator system was used to express human IFNλ4 in cells and mice. The formation of recombinant adenovirus (rAd-huIFNλ4) was confirmed using immunohistochemistry assays and transmission electron microscopy. Its purity was verified by quantifying host cell DNA and host cell proteins, as well as by confirming the absence of the replication-competent adenovirus. The transduction of rAd-huIFNλ4 induced ISGs and inhibited four subtypes of the influenza virus in both mouse-derived (LA-4) and human-derived cells (A549). The antiviral state was confirmed in BALB/c mice following intranasal inoculation with 109 PFU of rAd-huIFNλ4, which led to the inhibition of four subtypes of the influenza virus in mouse lungs, with reduced inflammatory lesions. These results imply that human IFNλ4 could induce antiviral status by modulating ISG expression in mice.

Abstract 3070: CDK12 is a novel therapeutic target in glioblastoma

Abstract Glioblastoma (GBM), the most common primary brain tumor in adults carries a highly unfavorable prognosis of only one to two years despite treatment with the standard of care, involving surgery, temozolomide and radiation. Patient-derived xenograft (PDX) GBM cultures were assessed for their susceptibility to loss of CDK12 function, which was either mediated genetically (shRNA) or by the novel inhibitor, SR-4835. GBM metabolism was measured by utilizing extracellular flux analysis as well as LC/MS based carbon tracing analyses. In vivo efficacy was determined by orthotopic GBM models in mice. Based on Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) and shRNA library screens in multiple GBM cell cultures we identified CDK12 as essential for the growth of GBM. Consistently, loss of function of CDK12 (genetically and pharmacologically) led to a reduction of growth of GBM patient-derived xenograft (PDX) lines in vitro. Notably, SR-4835 reduced GBM growth in the low nanomolar range and synergistically enhanced the potency of temozolomide. Extracellular flux analysis and metabolite screening analyses, including carbon tracing (U13C-Glucose, U13C-Glutamine) suggested that loss of function of CDK12 elicited a substantial inhibition of cellular respiration, causing energy deprivation with subsequent cell death with apoptotic features. On the molecular level, loss of function of CDK12 increased ATF4 along with pro-apoptotic Noxa, whereas anti-apoptotic Mcl-1 decreased. In turn, adenoviral mediated ectopic expression of Mcl-1 inhibited cell death mediated by loss of function of CDK12 in GBM cells. In an orthotopic murine PDX model of GBM animals receiving treatment with SR-4835 had a significantly longer overall survival without induction of toxicity as compared to mice treated with vehicle. In summary, these findings suggest that CDK12 is a novel treatment target for GBM and that CDK12 is an important driver of energy metabolism in GBM to partially mediate apoptotic resistance. Moreover, SR-4835 enhanced the potency of temozolomide in GBM models. Therefore, the novel CDK12 inhibitor, SR-4835 should be tested in a clinical trial in patients suffering from GBM. Citation Format: Jeong-Yeon Mun, Chang Shu, Qiuqiang Gao, Mike-Andrew Westhoff, Georg Karpel-Massler, Markus D. Siegelin. CDK12 is a novel therapeutic target in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3070.

Novel Relationship between Mitofusin 2-Mediated Mitochondrial Hyperfusion, Metabolic Remodeling, and Glycolysis in Pulmonary Arterial Endothelial Cells.

The disruption of mitochondrial dynamics has been identified in cardiovascular diseases, including pulmonary hypertension (PH), ischemia-reperfusion injury, heart failure, and cardiomyopathy. Mitofusin 2 (Mfn2) is abundantly expressed in heart and pulmonary vasculature cells at the outer mitochondrial membrane to modulate fusion. Previously, we have reported reduced levels of Mfn2 and fragmented mitochondria in pulmonary arterial endothelial cells (PAECs) isolated from a sheep model of PH induced by pulmonary over-circulation and restoring Mfn2 normalized mitochondrial function. In this study, we assessed the effect of increased expression of Mfn2 on mitochondrial metabolism, bioenergetics, reactive oxygen species production, and mitochondrial membrane potential in control PAECs. Using an adenoviral expression system to overexpress Mfn2 in PAECs and utilizing 13C labeled substrates, we assessed the levels of TCA cycle metabolites. We identified increased pyruvate and lactate production in cells, revealing a glycolytic phenotype (Warburg phenotype). Mfn2 overexpression decreased the mitochondrial ATP production rate, increased the rate of glycolytic ATP production, and disrupted mitochondrial bioenergetics. The increase in glycolysis was linked to increased hypoxia-inducible factor 1α (HIF-1α) protein levels, elevated mitochondrial reactive oxygen species (mt-ROS), and decreased mitochondrial membrane potential. Our data suggest that disrupting the mitochondrial fusion/fission balance to favor hyperfusion leads to a metabolic shift that promotes aerobic glycolysis. Thus, therapies designed to increase mitochondrial fusion should be approached with caution.

AAV production in stable packaging cells requires expression of adenovirus 22/33K protein to allow episomal amplification of integrated rep/cap genes

Efficient manufacture of recombinant adeno-associated virus (rAAV) vectors for gene therapy remains challenging. Packaging cell lines containing stable integration of the AAV rep/cap genes have been explored, however rAAV production needs to be induced using wild-type adenoviruses to promote episomal amplification of the integrated rep/cap genes by mobilizing a cis-acting replication element (CARE). The adenovirus proteins responsible are not fully defined, and using adenovirus during rAAV manufacture leads to contamination of the rAAV preparation. ‘TESSA’ is a helper adenovirus with a self-repressing Major Late Promoter (MLP). Its helper functions enable efficient rAAV manufacture when the rep and cap genes are provided in trans but is unable to support rAAV production from stable packaging cells. Using rAAV-packaging cell line HeLaRC32, we show that expression of the adenovirus L4 22/33K unit is essential for rep/cap amplification but the proteins are titrated away by binding to replicating adenovirus genomes. siRNA-knockdown of the adenovirus DNA polymerase or the use of a thermosensitive TESSA mutant decreased adenovirus genome replication whilst maintaining MLP repression, thereby recovering rep/cap amplification and efficient rAAV manufacture. Our findings have direct implications for engineering more efficient adenovirus helpers and superior rAAV packaging/producer cells.

Investigating the role of mitochondrial calcium uniporter beta in Alzheimer’s disease

AbstractBackgroundWe have recently shown that mitochondrial calcium (mCa2+) overload significantly contributes to the development of Alzheimer’s disease (AD). We found that either genetic inhibition of the neuronal mitochondrial calcium uniporter (MCU, the primary route for mCa2+ influx)1 or enhancement of mitochondrial Na+/Ca2+ exchanger (NCLX, the primary route for mCa2+ efflux)2 in the AD mouse model reduces AD‐associated phenotype and mitochondrial permeability transition pore (MPTP)‐induced cell death. However, blocking either the mCa2+ influx or enhancing mCa2+ efflux can also shut down the bioenergetics. Thus, it is vital to find a regulator of mCa2+ homeostasis that will positively impact Ca2+‐dependent physiological functions.MethodsTo define the new molecular regulatory mechanism to limit mCa2+ overload in AD that will maintain bioenergetics and prevent cell death, we have examined the role of Mitochondrial Calcium Uniporter Beta (MCUB), a paralog of MCU that acts as a negative regulator of mCa2+ uptake3. This study used AD patient samples, mice, and cell lines to examine MCUB expression. A neuroblastoma cell line stably expressing the human Swedish mutant amyloid precursor protein (N2a/APPswe) with adenovirus encoding MCUB was examined for mitochondrial alterations in: Ca2+ handling, ROS generation, permeability transition pore activation, oxidative phosphorylation (OxPHOS) and cell death.Results5xFAD mice and human AD brain samples displayed significant reductions in the expression of MCUB. Studies performed in N2a/APPswe neurons discovered that mCa2 handling, redox, and bioenergetics was severely impaired in AD cells. Rescue of MCUB via adenoviral expression enhanced the clearance of pathogenic mCa2+, enhanced OxPHOS, and prevented ROS and cell death.ConclusionsOur data suggest that MCUB modulation has the therapeutic potential to preserve bioenergetics and limit cell death in AD. This project will further utilize neuronal‐specific knockouts and overexpression of MCUB to increase or decrease mCa2+ uptake, respectively, in 5xFAD mouse models and will explore whether and how MCUB‐mediated changes in uptake are able to preserve metabolism, mCa2+ dynamics, and AD‐phenotype in vivo.

Mitochondrial hyperfusion induces metabolic remodeling in lung endothelial cells by modifying the activities of electron transport chain complexes I and III

ObjectivePulmonary hypertension (PH) is a progressive disease with vascular remodeling as a critical structural alteration. We have previously shown that metabolic reprogramming is an early initiating mechanism in animal models of PH. This metabolic dysregulation has been linked to remodeling the mitochondrial network to favor fission. However, whether the mitochondrial fission/fusion balance underlies the metabolic reprogramming found early in PH development is unknown. MethodsUtilizing a rat early model of PH, in conjunction with cultured pulmonary endothelial cells (PECs), we utilized metabolic flux assays, Seahorse Bioassays, measurements of electron transport chain (ETC) complex activity, fluorescent microscopy, and molecular approaches to investigate the link between the disruption of mitochondrial dynamics and the early metabolic changes that occur in PH. ResultsWe observed increased fusion mediators, including Mfn1, Mfn2, and Opa1, and unchanged fission mediators, including Drp1 and Fis1, in a two-week monocrotaline-induced PH animal model (early-stage PH). We were able to establish a connection between increases in fusion mediator Mfn1 and metabolic reprogramming. Using an adenoviral expression system to enhance Mfn1 levels in pulmonary endothelial cells and utilizing 13C-glucose labeled substrate, we found increased production of 13C lactate and decreased TCA cycle metabolites, revealing a Warburg phenotype. The use of a 13C5-glutamine substrate showed evidence that hyperfusion also induces oxidative carboxylation. The increase in glycolysis was linked to increased hypoxia-inducible factor 1α (HIF-1α) protein levels secondary to the disruption of cellular bioenergetics and higher levels of mitochondrial reactive oxygen species (mt-ROS). The elevation in mt-ROS correlated with attenuated ETC complexes I and III activities. Utilizing a mitochondrial-targeted antioxidant to suppress mt-ROS, limited HIF-1α protein levels, which reduced cellular glycolysis and reestablished mitochondrial membrane potential. ConclusionsOur data connects mitochondrial fusion-mediated mt-ROS to the Warburg phenotype in early-stage PH development.

Engineered Human Adenoviruses of Species B and C Report Early, Intermediate Early, and Late Viral Gene Expression.

Adenoviruses (AdVs) are being developed for oncolytic or vaccination therapy against existing and emerging conditions. Well-characterized replication-competent human and human primate AdVs expressing multiple payloads are desirable, but their replication in rodent models is limited. To score the timing of adenoviral gene expression in cell cultures, we developed fully replication-competent transcriptional reporter viruses for HAdV-C5, -B3, and -B35. The picornavirus-derived 2A sequence, which induces cotranslational peptide splitting and reinitiation (skipping), was linked to GFP and the fused sequence was inserted C-terminal of the early gene E1A, the intermediate early gene protein IX and the late fiber gene. The 2A peptide induced ribosomal skipping during translation of the messenger RNA (mRNA) and gave rise to GFP from the corresponding viral promoters, as shown by immunoblotting and flow cytometry analyses of human and rodent cells. In human cells, both species B and C AdV exhibited highest reporter expression for fiber, followed by protein IX and lowest for E1A. Inoculation with either HAdV-C5 or -B3/35 viruses encoding protein IX- or fiber-GFP gave rise to higher GFP levels in hamster than mouse cells. Remarkably, despite rather low 2A ribosomal skipping efficiency of ∼50% for E1A-2A-GFP, protein IX-2A-GFP, and fiber-2A-GFP, unprocessed protein IX-2A-GFP and fiber-2A-GFP fusion proteins were efficiently incorporated into HAdV-B3 virions, respectively. These data indicate that the B3 C-termini of protein IX and fiber can be considered for retargeting engineered oncolytic or vaccination vectors, or for antigen display. The variable expression levels of transgenes from different subviral promoters may be used to improve oncolytic AdV vectors expressing therapeutic genes.

Insights from the Construction of Adenovirus-Based Vaccine Candidates against SARS-CoV-2: Expecting the Unexpected.

To contain the spread of the SARS-CoV-2 pandemic, rapid development of vaccines was required in 2020. Rational design, international efforts, and a lot of hard work yielded the market approval of novel SARS-CoV-2 vaccines based on diverse platforms such as mRNA or adenovirus vectors. The great success of these technologies, in fact, contributed significantly to control the pandemic. Consequently, most scientific literature available in the public domain discloses the results of clinical trials and reveals data of efficaciousness. However, a description of processes and rationales that led to specific vaccine design is only partially available, in particular for adenovirus vectors, even though it could prove helpful for future developments. Here, we disclose our insights from the endeavors to design compatible functional adenoviral vector platform expression cassettes for the SARS-CoV-2 spike protein. We observed that contextualizing genes from an ssRNA virus into a DNA virus provides significant challenges. Besides affecting physical titers, expression cassette design of adenoviral vaccine candidates can affect viral propagation and spike protein expression. Splicing of mRNAs was affected, and fusogenicity of the spike protein in ACE2-overexpressing cells was enhanced when the ER retention signal was deleted.

Abstract 023: Prior Sflt1 Induced Preeclampsia Exacerbates Post-partum Hypertension-mediated Aortic Stiffness And Hypercholesterolemia-induced Atherosclerotic Inflammation

Preeclampsia (PE) is new high blood pressure (BP) and organ damage occurring late in pregnancy in association with increased soluble VEGF receptor (sFlt1). PE survivors have increased risk of future hypertension and myocardial infarction/stroke. We hypothesized that exposure to high sFlt1 during pregnancy exacerbates hypertension and atherosclerosis post-partum. PE was induced by transient adenoviral expression of sFlt1 compared to control virus in pregnant mice. PE mice had elevated sFlt1, BP and kidney damage during pregnancy which resolve post partum. Two months post-partum, prior PE mice had significantly increased BP response to high salt diet. After high BP stimuli, we studied large and small artery function. Prior PE mice had diminished nitric oxide-mediated vasodilation and stiffer aortas with more fibrosis as measured by pulse wave velocity (Con:3.7 vs sFlt:5.9 mm/ms, p<0.0001) and trichrome staining (Con:13.7% vs sFlt:33.9%, p=0.0009). Atherosclerosis studies exposed LDLR knockout mice to PE followed by high fat diet feeding for 8 weeks. Body weight, fasting glucose and cholesterol were not altered by prior PE. Aortic roots were histologically analyzed for plaque size and composition. Aortic root plaque size (Con:73.3 vs sFlt:78.0 AU, p=0.60) and necrotic core were not different between groups. Aortic arch plaque inflammation was quantified via flow cytometry showing significantly increased plaque CD45+ leukocytes (sFlt:1.6 fold, p=0.036) and T cells (sFlt:1.8 fold, p=0.031) from mice with prior PE. Atheroprone mice with prior PE had significantly increased aortic expression of CCL22 (sFlt:1.5 fold, p=0.047), providing a potential mechanism for increased T cell infiltration into atherosclerotic plaques. Experimental PE produces a state of enhanced sensitivity to hypertensive stimuli and increased vascular inflammation in atherosclerotic plaques supporting the concept that PE directly exacerbates hypertension, vascular stiffness, and atherosclerotic inflammation independent of pre-existing risk factors.

Abstract P3180: Cardiomyocyte Pgc-1α Mediates The Adaptive Response To Endurance Exercise Training In Mice

Exercise has myriad systemic benefits in reducing the incidence from and morbidity of cardio-metabolic diseases. In healthy individuals, aerobic exercise promotes physiologic hypertrophy of the heart, which requires increased mitochondrial numbers and function. In skeletal muscle and heart, a key orchestrator of the adaptation to increased energetic needs is the transcriptional regulator PGC-1α. PGC-1α may mediate cardiac adaptation to physiological stress through its function in mitochondrial biogenesis but also through other mechanisms. The role of cardiac PGC-1α to the systemic training response is poorly understood. We generated PGC-1α cardiomyocyte-specific knockout (cKO) mice to study its role in endurance exercise training. Sedentary cKO mice demonstrated normal cardiac function and no significant differences in acute treadmill exercise tolerance. However, cKO mice demonstrated a lack of ability to augment exercise capacity after 6 weeks of wheel running compared to WT (work achieved 32.9 J in WT [39% increase] vs 23.1 J in cKOs [no change vs baseline], p<0.001, unpaired t-test). This was despite comparable voluntary wheel distance run during training (2.93-3.67 km/day for both over 5 weeks, p=0.58). Unexpectedly, after 6 weeks of training, cKO mice developed a dilated cardiomyopathy at rest and exercise-related dysfunction (contractile reserve [ΔFS stress-rest ] +6.0 in WT vs -6.4% in KO, p<0.05, unpaired t-test). They also demonstrated features of cardiac cachexia as measured by 30% and 21% reductions in inguinal adipose and gastrocnemius mass, respectively (P<0.05, unpaired t-test). Gene expression profiling demonstrated activation of pathological ( Nppa , Nppb , Myh7/Myh6 ) and atrophic pathways ( Gdf11 , Gdf15 , Fstl3 ) in the trained cKOs. In vitro, gain of function studies using adenoviral expression in neonatal rat ventricular myocytes demonstrated that PGC-1α overexpression could abrogate pathologic hypertrophy and atrophy gene expression pathways stimulated by phenylephrine. Our preliminary studies support cardiomyocyte PGC-1α as a key mediator of the adaptive response to exercise training and unveil a key role of this transcriptional activator in protecting from exercise-related cardiac dysfunction in mice.

Abstract P2068: β Iv -spectrin/stat3 Are Responsible For Concentric Versus Eccentric Cardiac Hypertrophy

Hypertrophy is critical to development of heart failure following cardiac stress. In pressure overload, hypertrophy is divided by early concentric and chronic eccentric orientations, however, the mechanisms regulating this transition and the impact on heart function are largely unknown. Previous work identified the structural protein β IV -spectrin and its interaction with the transcription factor, STAT3, in coordinating hypertrophic profiles. Specifically, mice expressing truncated β IV -spectrin lacking interaction with the kinase CaMKII ( qv 3J mice) resisted β IV -spectrin degradation and maintained concentric hypertrophy and systolic function compared to WT following 6 weeks transaortic constriction (TAC). Alternatively, mutant β IV -spectrin mice lacking the β IV -spectrin/STAT3 binding domain ( qv 4J ) expressed reduced EF and LV dilation, consistent with eccentric remodeling, suggesting β IV -spectrin/STAT3 is a regulator of cardiac profiles. This relationship was examined by adenoviral expression of the hypertrophic driver MEK1/2 on isolated myocytes, where MEK increased the width of WT cells but length in qv 4J . Notably, application of STAT3 inhibitor S3I-201 on qv 4J myocytes restored growth in width. Additionally, in vivo delivery of S3I-201 to WT mice after 6 weeks TAC reduced LV dilation and recovered systolic function. To enable hypertrophy, recent work identified the need for microtubules to traffic and locally translate sarcomeric transcripts. Therefore, we tested the hypothesis that β IV -spectrin/STAT3 direct orientation by altering microtubule stability and sarcomeric transcript trafficking. Isolated myocytes from WT and qv 4J mice were treated with the microtubule depolymerizing drug Nocodazole and then washed to evaluate microtubule repolymerization. Microtubules restored faster in qv 4J myocytes but could be reduced to WT levels by treating with S3I-201. Furthermore, localization of the sarcomeric gene, actc1 (α-actinin) by RNAScope showed more peripheral localization in qv 4J myocytes, compared to WT, and was normalized by S3I-201. Together, these data identify β IV -spectrin/STAT3 as modulators of microtubule dynamics, sarcomeric transcript distribution, hypertrophic orientation, and systolic function.

Mitochondrial rupture after photodamage initiates parkin-independent mitophagy in mouse hepatocytes.

Involvement of parkin in mitophagy is well studied. Translocation of parkin from cytosol to depolarized mitochondria triggers ubiquitination-dependent mitophagy. In this study, we used Airyscan super-resolution microscopy and correlative light-electron microscopy (CLEM) to characterize parkin association during photodamage-induced mitophagy, a form of phosphoinositide 3-kinase-independent or Type 2 mitophagy. After photodamage with 488-nm light to small groups of mitochondria within GFP-LC3 transgenic mouse hepatocytes, U-shaped crescents of GFP-LC3 fluorescence formed around damaged, depolarized mitochondria beginning after 31 ± 18 min. Fluorescence of crescents progressively intensified, but their length remained unchanged. Subsequently, LC3-containing puncta merged with damaged mitochondria containing crescents to form a continuous ring. Adenovirally expressed mCherry-TOMM20 (outer membrane marker) labeled the surface of all mitochondria. After photodamage, gaps of mCherry-TOMM20 surface labeling appeared as a result of mitochondrial swelling and outer membrane rupture. Crescents of GFP-LC3 fluorescence formed over these gaps not containing mCherry-TOMM20. Brightfield microscopy confirmed mitochondrial swelling after photodamage, leading to outer membrane rupture and herniation of the inner membrane. CLEM then revealed that GFP-LC3 labeling occurred at the bare surfaces of herniated inner membranes at early stages of photodamage-induced mitophagy. Adenoviral expression of mCherry-parkin showed that parkin did not associate with photodamaged mitochondria at any stage after up to 2 h. In parkin KO mouse hepatocytes adenovirally expressing GFP-LC3 and mCherry-TOMM20, the same mitophagy pattern was observed. Thus, mitochondrial swelling, outer membrane rupture, and inner membrane herniation after photodamage exteriorizes bare surfaces of mitochondrial inner membranes that become the proximate site for LC3 but not parkin association and the initiation of mitophagy (AA021191, AA025379, DK073336).

Ryanodine receptor hyperactivity promotes mitochondrial dysfunction via calpain-dependent OPA1 proteolysis.

Hereditary RyR2 gain-of function mutations are linked to the highly malignant arrhythmia syndrome, catecholaminergic polymorphic tachycardia (CPVT). A mutation-mediated increase in RyR2 activity not only results in unstable sarcoplasmic reticulum Ca2+ release, but also alters mitochondrial structure and function, leading to excessive emission of damaging reactive oxygen species (ROS) by the organelle. However, the exact mechanisms underlying RyR2 hyperactivity-dependent mitochondrial damage remain elusive. We hypothesized that RyR2 hyperactivity activates the intermembrane space (IMS)-residing mitochondrial Ca2+-dependent protease calpain, driving proteolysis of OPA1, a protein responsible for tight cristae arrangement. Concomitant changes in cristae architecture promotes mito-ROS production, thereby aggravating the CPVT phenotype. To test this, we generated a unique CPVT RyR2-S2222L(+/-) rat model. Electron microscopy demonstrated significantly increased mitochondrial cristae diameter in tissue slices from CPVT rat hearts. Biochemical analysis showed reduced OPA1 expression in isolated CPVT myocytes, suggesting that mitochondrial ultrastructural changes in CPVT could be driven by Ca2+-dependent proteolysis of OPA1 by calpain. Importantly, specific inhibition of calpain via adeno-associated virus mediated expression of IMS-targeted calpastatin reduced the incidence of ventricular tachycardia in CPVT rats. At the cellular level, adenoviral expression of IMS-CAST restored OPA1 expression levels and significantly improved intracellular Ca2+ homeostasis in CPVT, indicative of normalized RyR2 function. Furthermore, IMS-calpain inhibition attenuated the reduction in mitochondrial matrix Ca2+ and increase in mito-ROS emission observed in CPVT myocytes using genetic probes mtRCamp1h and MLS-HyPer7. All protective effects were lost in myocytes with additional shRNA-mediated knockdown of OPA1. We conclude that RyR2 hyperactivity promotes mitochondrial structural damage via IMS-calpain-mediated degradation of OPA1 and contributes to proarrhythmic remodeling in CPVT.

Sirtuin 3 relieves inflammatory responses elicited by lipopolysaccharide via the PGC1α-NFκB pathway in bovine mammary epithelial cells

Excessive inflammation in bovine mammary endothelial cells (BMEC) due to mastitis leads to disease progression and eventual culling of cattle. Sirtuin 3 (SIRT3), a mitochondrial deacetylase, downregulates pro-inflammatory cytokines in BMEC exposed to high concentrations of nonesterified fatty acids by blunting nuclear factor-κB (NFκB) signaling. In nonruminants, SIRT3 is under the control of PGC1α, a transcriptional cofactor. Specific aims were to study (1) the effect of SIRT3 on inflammatory responses of lipopolysaccharide (LPS)-challenged bovine mammary epithelial cells (bovine mammary alveolar cells-T, MAC-T) models, and (2) the role of PGC1α in the attenuation of NFκB signaling via SIRT3. To address these objectives, first, MAC-T cells were incubated in triplicate with 0, 50, 100, 150, or 200 μg/mL LPS (derived from Escherichia coli O55:B5) for 12 h with or without a 2-h incubation of the NFκB inhibitor ammonium pyrrolidine dithiocarbamate (APDC, 10 μM). Second, SIRT3 was overexpressed using adenoviral expression (Ad-SIRT3) at different multiplicity of infection (MOI) for 6 h followed by a 12 h incubation with 150 μg/mL LPS. Third, cells were treated with the PGC1α agonist ZLN005 (10 μg/mL) for 24 h and then challenged with 150 μg/mL LPS for 12 h. Fourth, cells were initially treated with the PGC1α inhibitor SR-18292 (100 μM) for 6 h followed by a 6-h culture with or without 50 MOI Ad-SIRT3 and a challenge with 150 μg/mL LPS for 12 h. Data were analyzed using one-way ANOVA with subsequent Bonferroni correction. Linear and quadratic contrasts were used to determine dose-responses to LPS. There were linear and quadratic effects of LPS dosage on cell viability. Incubation with 150 and 200 μg/mL LPS for 12 h decreased cell viability to 78.6 and 34.9%, respectively. Compared with controls, expression of IL1B, IL6, and TNFA was upregulated by 5.2-, 5.9-, and 2.7-fold with 150 μg/mL LPS; concentrations of IL-1β, IL-6, and tumor necrosis factor-α (TNF-α) in cell medium also increased. Compared with the LPS group, LPS+APDC increased cell viability and reversed the upregulation of IL1B, IL6, and TNFA expression. However, mRNA and protein abundance of SIRT3 decreased linearly with increasing LPS dose. Ad-SIRT3 infection (50 MOI) reduced IL1B, IL6, and TNFA expression and also their concentrations in cell medium, and decreased pNFκB P65/NFκB P65 ratio and nuclear abundance of NFκB P65. The PGC1α agonist increased SIRT3 expression, whereas it decreased cytokine expression, pNFκB P65/NFκB P65 ratio, and prevented NFκB P65 nuclear translocation. Contrary to the agonist, the PGC1α inhibitor had opposite effects, and elevated the concentrations of IL-1β, IL-6, and TNF-α in cell medium. Overall, data suggested that SIRT3 activity could attenuate LPS-induced inflammatory responses in mammary cells via alterations in the PGC1α-NFκB pathway. As such, there may be potential benefits for targeting SIRT3 in vivo to help prevent or alleviate negative effects of mastitis.

Abstract 15113: C-cbl E3 Ubiquitin Ligase Regulates Cardiomyocyte Proliferation and Cardiac Regeneration

Background: Neonatal mammalian hearts display considerable regenerative capacity following injury through cardiomyocyte proliferation. However, this regenerative capacity is lost by post-natal day 7. Previous studies have shown the role of receptor tyrosine kinases (RTKs) on cardiomyocyte proliferation and cardiac regeneration. However, the mechanisms that control RTK turnover and signaling during and beyond the regenerative window of the heart remain largely unknown. Hypothesis: This study investigates the role of the proto-oncogene Casitas b-lineage lymphoma (c-Cbl), an adaptor protein with an intrinsic E3 ubiquitin ligase activity that targets RTKs, on cardiomyocyte proliferation and cardiac regeneration following myocardial infarction (MI). Methods and Results: c-Cbl expression was downregulated during post-natal development and re-expressed in the adult heart following MI. RNA-sequencing studies reveal a distinct transcriptional profile in c-Cbl-deficient mouse hearts, of which c-Cbl deletion substantially delayed cardiomyocyte maturation and enhanced cardiomyocyte proliferation. c-Cbl deletion also extended the regenerative window for post-natal mouse hearts following MI and enhanced the ability to recover following MI with improved myocardial function, reduced scar formation, and increased cardiomyocyte cell-cycle activity. Mechanistically, c-Cbl E3 ubiquitin ligase activity was markedly enhanced in the adult than neonatal heart following MI, along with increased ubiquitination and downregulation of key RTKs involved in cardiomyocyte proliferation. Intriguingly, adenoviral expression of a c-Cbl mutant deficient in E3 ligase enhanced RTK signaling and promoted cardiomyocyte proliferation in vitro. Conclusions: These results reveal c-Cbl as a critical regulator of cardiomyocyte proliferation and a potential therapeutic target for the maintenance of cardiac regeneration and function following MI.