CaMKIIδ is the dominant isozyme of Ca2+/calmodulin-dependent protein kinase II in the heart. Under certain pathological conditions, it can be oxidized, causing a constitutive activation that can lead to cardiac failure. We recently showed that, in purified CaMKIIδ exposed to oxidative conditions, a disulfide link formed between Cys273 and Cys290 causes this autonomous activation. Cys273 has a low pKa that facilitates the oxidation of its thiol to a sulfenic acid at physiological pH. Does this matter in vivo? To answer that question, we created a transgenic mouse with Cys273 mutated to serine (CaMKIIδC273S) to prevent disulfide formation. We conducted a detailed assessment of cardiac function at rest and in a dobutamine stress test. We found that the CaMKIIδ Cys273Ser mutation does not have deleterious effects on cardiac physiology. Then, we assessed whether the mutation would protect the heart from ischemia-reperfusion in the Langendorff model. The CaMKIIδC273S mouse had improved cardiac function and decreased infarct size compared to the wild-type mouse. We conclude that blocking disulfide formation at Cys273 protects the heart against ischemia-reperfusion injury. Drugs that specifically target Cys 273 may be therapeutic in human cardiac disease.

Catecholamine-induced polymorphic ventricular tachycardia (CPVT) is associated typically with mutations in Ryanodine Receptor type 2 (RYR2) and cardiac Calsequestrin (CASQ2) genes. RyR2 and Casq2 along with membrane proteins, junctin and triadin, constitute the Ca2+ release unit in the junctional sarcoplasmic reticulum in mammalian cardiomyocytes. Mouse models lacking Casq2 gene phenocopy CPVT and have been used to study CPVT pathophysiology and to identify novel therapies. Here, we analyze roles of normal aging for the development of cardiac dysfunctions of Casq2-null mice. We show that aging intensifies the disease phenotype. Age-dependent changes in triadin but not in RyR2 or junctin correlate with a more severe CPVT phenotype. We also examine transcriptomes at 1 month and 2 years of age. We identify 583 genes whose expression is dependent on Casq2 genotype with enrichment for genes associated with calcium handling and transport, sarcomere structures, extracellular matrix, and stress signaling associated with cardiac failure. 37% of the differentially expressed genes are specifically dysregulated in older mice. These findings establish aging as a powerful modifier of CPVT and suggest that targeting structural protein dynamics and compensatory transcriptional pathways may offer new therapeutic strategies.

Free sialic acid storage disorder (FSASD) is a rare, neurodegenerative, lysosomal storage disease caused by biallelic variants in SLC17A5, which encodes the lysosomal sialic acid exporter, sialin. Although clinical severity varies, all affected individuals exhibit progressive neurological decline and characteristic lysosomal and urinary accumulation of free sialic acid. No US Food and Drug Administration-approved therapies currently exist, and the molecular underpinnings of central nervous system (CNS) dysfunction remain poorly defined. To model the most prevalent FSASD-associated allele, we generated a Slc17a5R39C mouse model carrying the pathogenic p.Arg39Cys variant using CRISPR/Cpf1. Homozygous Slc17a5R39C/R39C mice recapitulated key features of FSASD, including biochemical accumulation of free sialic acid, ataxia, tremors, seizures and shortened lifespan. Neuropathological analyses revealed widespread CNS hypomyelination, with relatively preserved peripheral nervous system myelination, while transcriptomic profiling showed broad suppression of myelin-associated genes and a cerebellum-specific inflammatory signature. This was accompanied by pronounced astrogliosis, progressive Purkinje cell degeneration and elevated neurofilament proteins, including serum neurofilament light polypeptide. Altogether, the Slc17a5R39C/R39C model of FSASD provides critical insights into disease pathogenesis, highlights region-specific neurodegenerative vulnerabilities, and establishes a robust preclinical platform for mechanistic investigation and therapeutic development.

The autosomal dominant p.Ala165Val mutation in LIM Domain Binding Protein 3 (LDB3) causes myofibrillar myopathy marked by Z-disc disruption, accumulation of filamin-C (FLNc) and chaperone proteins, and progressive muscle weakness. We previously showed that this mutation interferes with the LDB3-protein kinase C alpha (PKCα)-FLNc mechanosensing axis and impairs chaperone-assisted selective autophagy (CASA), establishing a gain-of-function mechanism. In this study, we examined whether mutant allele-specific knockdown could reverse the disease or mitigate disease progression in-vivo. A single intramuscular-injection of an AAV9-delivered microRNA-based shRNA produced substantial knockdown of mutant Ldb3 transcripts and protein in Ldb3 Ala165Val/+ knock-in mice treated either before or after the onset of pathology. Treatment after disease onset reduced filamin-C and CASA protein aggregates and improved muscle strength, whereas early intervention prevented development of molecular and histological features of myopathy. Phosphoproteomic profiling further showed broad remodeling of dysregulated phosphorylation networks, including restoration of PKCα-responsive sites and normalization of altered sarcomeric and cytoskeletal signaling observed in Ldb3 Ala165Val/+ mice. These findings identify disruption of the LDB3-PKCα-FLNc mechanosensing pathway as a central disease driver and suggest that restoring this signaling axis may complement mutant allele-specific RNA interference (RNAi). Overall, our results support RNAi as a promising therapeutic strategy for dominant LDB3-related myofibrillar myopathy.

In USA, six million individuals with Sub-Saharan ancestry carry two APOL1 high-risk variants, which increase the risk for kidney diseases. Whether APOL1 high-risk variants increase other diseases under dyslipidemia remains unclear and requires further investigation.We characterized a mouse model to investigate the role of APOL1 in dyslipidemia and cardiovascular diseases. Transgenic mice carrying APOL1 (G0 and G1 variants)on bacterial artificial chromosomes (BAC/APOL1 mice) were crossed with the ApoE knock-out (ApoE-KO) dyslipidemia and atherosclerosis mouse model. The compound transgenic mice were evaluated for the impact of APOL1 on systemic phenotypes. ApoE-KO mice carrying APOL1-G0 and APOL1-G1 did not show differences in the extent of atherosclerotic lesions or aortic calcification, as evaluated by Sudan IV staining and radiographic examination, respectively. However, ~20% of ApoE-KO; BAC/APOL1-G1 mice developed hydrocephalus and required euthanasia. The hydrocephalus was communicating and likely was due to excess cerebrospinal fluid produced by the choroid plexus, where epithelial cells expressed APOL1. Single-nuclear RNA-seq of choroid plexus identified solute transporter upregulation and mTORC2 pathway activation in APOL1-G1-expressing epithelial cells. Further, in the All of Us cohort, we found higher hydrocephalus prevalence among individuals with the APOL1-G1 variant in both recessive and dominant models, supporting the mouse findings. While APOL1-G1 expression in ApoE-KO mice did not worsen cardiovascular disease phenotypes, we uncovered hydrocephalus as a novel APOL1 risk allele-mediated phenotype. These findings extend the spectrum of APOL1-associated pathologies.

Sandhoff disease, a lysosomal storage disorder, is caused by pathogenic variants in the HEXB gene, resulting in the loss of β-hexosaminidase activity and accumulation of sphingolipids including GM2 ganglioside. This accumulation occurs primarily in neurons, and leads to progressive neurodegeneration through a largely unknown process. Lysosomal storage diseases often exhibit dysfunctional mTOR signaling, a pathway crucial for proper neuronal development and function. In this study, Sandhoff disease model mice exhibited reduced mTOR signaling in the brain. To test if restoring mTOR signaling could improve the disease phenotype, we genetically reduced expression of the mTOR inhibitor Tsc2 in these mice. Sandhoff disease mice with reactivated mTOR signaling displayed increased survival rates and motor function, especially in females, increased dendritic-spine density, and reduced neurodegeneration. Tsc2 reduction also partially rescued aberrant synaptic function–related gene expression. These findings imply that enhancing mTOR signaling could be a potential therapeutic strategy for lysosomal-based neurodegenerative diseases.

Introduction: Erythropoietin (EPO) acts primarily in regulating red blood cell production mediated by high EPO receptor (EPOR) expression in erythroid progenitor cells. EPO activity in non-erythroid tissue is evident in mice with EPOR restricted to erythroid tissues (ΔEPORE) that become obese, glucose-intolerant, and insulin-resistant. In animal models, nitric oxide synthase (NOS) contributes to EPO activities including erythropoiesis, neuroprotection, and cardioprotection against ischemia-reperfusion injury. However, we found that extended EPO treatment to increase hematocrit compromised heart function, while the loss of neuronal NOS (nNOS) was protective against the deleterious activity of EPO to promote heart failure.Methods: Wild-type (WT) mice, ΔEPORE mice, and nNOS-knockout mice (nNOS−/−) were placed on a high-fat diet to match the ΔEPORE obese phenotype and were treated with EPO for 3 weeks. Hematocrit and metabolic response to EPO treatment were monitored. Cardiac function was assessed by echocardiography and ultrasonography.Results: ΔEPORE mice showed a decrease in the left ventricular outflow tract (LVOT) peak velocity, ejection fraction, and fractional shortening, showing that endogenous non-erythroid EPO response is protective for heart function. EPO treatment increased hematocrit in all mice and decreased fat mass in male WT, demonstrating that EPO regulation of fat mass requires non-erythroid EPOR. EPO treatment also compromised heart function in WT mice, and decreased the pulmonary artery peak velocity (PA peak velocity), LVOT peak velocity, ejection fraction, and fractional shortening, but it had minimal effect in further reducing the heart function in ΔEPORE mice, indicating that the adverse effect of EPO on heart function is not related to EPO-stimulated erythropoiesis. ΔEPORE mice had increased expression of heart failure-associated genes, hypertrophic cardiomyopathy-related genes, and sarcomeric genes that were also elevated with EPO treatment in WT mice. Male and female nNOS−/− mice were protected against diet-induced obesity. EPO treatment in nNOS−/− mice increased the hematocrit that tended to be lower than WT mice and decreased the PA peak velocity but did not affect the LVOT peak velocity, ejection fraction, and fractional shortening, suggesting that nNOS is required for the adverse effect of EPO treatment on heart function in WT mice. EPO treatment did not change expression of heart failure-associated gene expression in nNOS−/− mice.Discussion: Endogenous EPO has a protective effect on heart function. With EPO administration, in contrast to the protective effect to the cardiac injury of acute EPO treatment, extended EPO treatment to increase hematocrit in WT mice adversely affected the heart function with a corresponding increase in expression of heart failure-associated genes. This EPO activity was independent of EPO-stimulated erythropoiesis and required EPOR in non-erythroid tissue and nNOS activity, while nNOS−/− mice were protected from the EPO-associated adverse effect on heart function. These data provide evidence that nNOS contributes to the negative impact on the heart function of high-dose EPO treatment for anemia.

Mitochondrial calcium concentration ([Ca2+](m)) plays an essential role in bioenergetics, and loss of [Ca2+](m) homeostasis can trigger diseases and cell death in numerous cell types. Ca2+ uptake into mitochondria occurs via the mitochondrial Ca2+ uniporter (MCU), which is regulated by three mitochondrial Ca2+ uptake (MICU) proteins localized in the intermembrane space, MICU1, 2, and 3. We generated a mouse model of systemic MICU3 ablation and examined its physiological role in skeletal muscle. We found that loss of MICU3 led to impaired exercise capacity. When the muscles were directly stimulated there was a decrease in time to fatigue. MICU3 ablation significantly increased the maximal force of the KO muscle and altered fibre type composition with an increase in the ratio of type IIb (low oxidative capacity) to type IIa (high oxidative capacity) fibres. Furthermore, MICU3-KO mitochondria have reduced uptake of Ca2+ and increased phosphorylation of pyruvate dehydrogenase, indicating that KO animals contain less Ca2+ in their mitochondria. Skeletal muscle from MICU3-KO mice exhibited lower net oxidation of NADH during electrically stimulated muscle contraction compared with wild-type. These data demonstrate that MICU3 plays a role in skeletal muscle physiology by setting the proper threshold for mitochondrial Ca2+ uptake, which is important for matching energy demand and supply in muscle.

Model organisms such as Drosophila are powerful tools to study the genetic basis of sleep. Previously, we identified the genes pointed and Arginine kinase 1 using selective breeding for long and short sleep duration in an outbred population of Drosophila. pointed is a transcription factor that is part of the epidermal growth factor receptor signaling pathway, while Arginine kinase 1 is involved in proline and arginine metabolism. Conserved orthologs of these genes exist in mice, leading us to hypothesize that they would also impact sleep in a murine model. We generated mutations in the murine orthologs Ets1 and Ckm using CRISPR in a C57BL/6N background and used video analysis to measure sleep in the mice. Both mutations affected sleep parameters, and the effects were observed predominantly in female mice, with males showing fewer differences from littermate controls. The study of natural populations in flies therefore leads to candidate genes with functional conservation on sleep in mammals.

BACKGROUND:Calcium (Ca2+) uptake by mitochondria occurs via the mitochondrial Ca2+ uniporter. Mitochondrial Ca2+ uniporter exists as a complex, regulated by 3 MICU (mitochondrial Ca2+ uptake) proteins localized in the intermembrane space: MICU1, MICU2, and MICU3. Although MICU3 is present in the heart, its role is largely unknown. METHODS:We used CRISPR-Cas9 to generate a mouse with global deletion of MICU3 and an adeno-associated virus (AAV9) to overexpress MICU3 in wild-type mice. We examined the role of MICU3 in regulating mitochondrial calcium ([Ca2+]m) in ex vivo hearts using an optical method following adrenergic stimulation in perfused hearts loaded with a Ca2+-sensitive fluorophore. Additionally, we studied how deletion and overexpression of MICU3, respectively, impact cardiac function in vivo by echocardiography and the molecular composition of the mitochondrial Ca2+ uniporter complex via Western blot, immunoprecipitation, and Blue native-PAGE analysis. Finally, we measured MICU3 expression in failing human hearts. RESULTS:MICU3 knock out hearts and cardiomyocytes exhibited a significantly smaller increase in [Ca2+]m than wild-type hearts following acute isoproterenol infusion. In contrast, heart with overexpression of MICU3 exhibited an enhanced increase in [Ca2+]m compared with control hearts. Echocardiography analysis showed no significant difference in cardiac function in knock out MICU3 mice relative to wild-type mice at baseline. However, mice with overexpression of MICU3 exhibited significantly reduced ejection fraction and fractional shortening compared with control mice. We observed a significant increase in the ratio of heart weight to tibia length in hearts with overexpression of MICU3 compared with controls, consistent with hypertrophy. We also found a significant decrease in MICU3 protein and expression in failing human hearts. CONCLUSIONS:Our results indicate that increased and decreased expression of MICU3 enhances and reduces, respectively, the uptake of [Ca2+]m in the heart. We conclude that MICU3 plays an important role in regulating [Ca2+]m physiologically, and overexpression of MICU3 is sufficient to induce cardiac hypertrophy, making MICU3 a possible therapeutic target.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes injury to multiple organ systems, including the brain. SARS-CoV-2's neuropathological mechanisms may include systemic inflammation and hypoxia, as well as direct cell damage resulting from viral infections of neurons and glia. How the virus directly causes injury to brain cells, acutely and over the long term, is not well understood. In order to gain insight into this process, we studied the neuropathological effects of open reading frame 3a (ORF3a), a SARS-CoV-2 accessory protein that is a key pathological factor of the virus. Forced ORF3a brain expression in mice caused the rapid onset of neurological impairment, neurodegeneration, and neuroinflammation-key neuropathological features found in coronavirus disease (COVID-19, which is caused by SARS-CoV-2 infection). Furthermore, ORF3a expression blocked autophagy progression in the brain and caused the neuronal accumulation of α-synuclein and glycosphingolipids, all of which are linked to neurodegenerative disease. Studies with ORF3-expressing HeLa cells confirmed that ORF3a disrupted the autophagy-lysosomal pathway and blocked glycosphingolipid degradation, resulting in their accumulation. These findings indicate that, in the event of neuroinvasion by SARS-CoV-2, ORF3a expression in brain cells may drive neuropathogenesis and be an important mediator of both short- and long-term neurological manifestations of COVID-19.

Background Liver cancer remains the third leading cause of worldwide cancer-related deaths.1 Despite clinical advances, established immunotherapies largely fail patients due to poor immune responses.1 2 Peripheral nerves influence tumors, but brain-liver interactions remain largely unstudied.3 A recent study reported a decreased risk of liver cancers in patients that underwent truncal vagotomy (vagus snip) compared to simple suture procedure.4 Here, we identify a vagal-immune arc regulating hepatic cancer. Methods C57BL/6 or BALB/c mice (9–12 weeks old) underwent a surgical hepatic vagotomy (HV) or sham procedure (SV). Following established protocols,5 HV and SV mice received orthotopic tumors via intrahepatic injection (2.5x105 RIL-175 or B16-F10 cells) or tail vein/flank injection (1.0x106 A20, RIL-175 cells) to model primary and metastatic liver cancer. Growth of luciferase-labeled cells was measured via in vivo imaging and immune profiling was conducted via flow cytometry and scRNASEQ. Open field test, Y maze, and phenotyper cages assessed mouse behavior. Highly-multiplexed immunofluorescence (CODEX platform) revealed peripheral nerves in clinical resection samples. Results Precise liver denervation reduced tumor growth in three models of primary (RIL-175) and metastatic (B16-F10, A20) tumors (figure 1A). Outcomes remained organ specific as HV mice exhibited reduced tumor burden of intrahepatic, but not subcutaneous, models. HV livers exhibited decreased levels of vagal neurotransmitter acetylcholine (ACh). As immunofluorescent analyses revealed colocalization of peripheral nerves and lymphocytes in clinical liver cancer, we profiled HV immunity. HV livers exhibited broad anti-tumor immunity, notably increased CD8+ T cells and higher expression of intracellular cytokines (IFNγ, TNFα) (figure 1B). We then examined whether immune alterations were a cause or consequence of HV tumor burden. ACh exposure reduced intracellular cytokine levels in ex vivo CD8+ T cells following anti-CD3/CD8 activation. Treatment with bethanechol (ACh receptor agonist, 400 μg/mL drinking water) increased tumor burden and reduced CD8+TNFα+ subsets. Bethanechol failed to promote tumor growth in Rag1KO mice lacking mature B and T cells, and targeted depletion of CD8+ T cells abrogated the effects of vagotomy (figure 1C). Finally, as the vagus nerve is largely comprised of afferent fibers, we assessed murine behavior and ambulation. Tumor-bearing HV mice displayed decreased anxiety-life features and fatigue compared to sham controls. Conclusions Our findings highlight a vagal-CD8+ T cell axis modulating hepatic tumor burden and behavior. This work furthers the emerging field of cancer neuroscience and identifies ACh signaling targets to alter hepatic immunosuppression and cancer outcomes. References Kim E, Viatour P. Hepatocellular carcinoma: old friends and new tricks. Exp Mol Med. 2020;52:1898–1907. Greten T, et al. Society for Immunotherapy of Cancer (SITC) clinical practice guideline on immunotherapy for the treatment of hepatocellular carcinoma. 2021. JITC. 2021;9:e002794. Monje M, et al. Roadmap for the Emerging Field of Cancer Neuroscience. Cell. 2020;181:219–222. Wu S, et al. Decreased risk of liver and intrahepatic cancer in non-H. pylori-infected perforated peptic ulcer patients with truncal vagotomy: a nationwide study. Sci Rep. 2021;11:e15594. Brown Z, Heinrich B, Greten T. Establishment of orthotopic liver tumors by surgical intrahepatic tumor injection in mice with underlying non-alcoholic fatty liver disease. Methods protoc, 2018;2:e21. Ethics Approval This research was approved by the NCI Division of Intramural Research Animal Care and Use Committee, proposal numbers: MOB-028 and TGOB-015. Patients provided informed consent for clinical tissue acquisition: Institutional Review Board protocol #2017–0365.

Mammalian brains are larger and more densely packed with neurons than reptiles, but the genetic mechanisms underlying the increased connection complexity amongst neurons are unclear. The expression diversity of clustered protocadherins (Pcdhs), which is controlled by CTCF and cohesin, is crucial for proper dendritic arborization and cortical connectivity in vertebrates. Here, we identify a highly-conserved and mammalian-restricted protein, ZFP661, that binds antagonistically at CTCF barriers at the Pcdh locus, preventing CTCF from trapping cohesin. ZFP661 balances the usage of Pcdh isoforms and increases Pcdh expression diversity. Loss of Zfp661 causes cortical dendritic arborization defects and autism-like social deficits in mice. Our study reveals both a novel mechanism that regulates the trapping of cohesin by CTCF and a mammalian adaptation that promoted Pcdh expression diversity to accompany the expanded mammalian brain.

Previous studies of natural variants in Drosophila melanogaster implicated the Wnt signaling receptor frizzled in sleep. Given that the Wnt signaling pathway is highly conserved across species, we hypothesized that frizzled class receptor 1 (Fzd1), the murine homolog of frizzled, would also have a role in sleep. Using a CRISPR transgenic approach, we removed most of the Fzd1 coding region from C57BL/6N mice. We used a video assay to measure sleep characteristics in Fzd1-deficient mice. As Wnt signaling is known to affect visuospatial memory, we also examined the impact of the deletion on learning and memory using the novel object recognition (NOR) paradigm. Fzd1-deficient mice had altered sleep compared to littermate controls. The mice did not respond differently to the NOR paradigm compared to controls but did display anxiety-like behavior. Our strategy demonstrates that the study of natural variation in Drosophila sleep translates into candidate genes for sleep in vertebrate species such as the mouse.

Arginine-specific mono-ADP-ribosylation is a reversible post-translational modification; arginine-specific, cholera toxin-like mono-ADP-ribosyltransferases (ARTCs) transfer ADP-ribose from NAD + to arginine, followed by cleavage of ADP-ribose-(arginine)protein bond by ADP-ribosylarginine hydrolase 1 (ARH1), generating unmodified (arginine)protein. ARTC1 has been shown to enhance tumorigenicity as does Arh1 deficiency. In this study, Artc1 -KO and Artc1/Arh1 -double-KO mice showed decreased spontaneous tumorigenesis and increased age-dependent, multi-organ inflammation with upregulation of pro-inflammatory cytokine TNF- α . In a xenograft model using tumorigenic Arh1 -KO mouse embryonic fibroblasts (MEFs), tumorigenicity was decreased in Artc1 -KO and heterozygous recipient mice, with tumor infiltration by CD8 + T cells and macrophages, leading to necroptosis, suggesting that ARTC1 promotes the tumor microenvironment. Furthermore, Artc1/Arh1 -double-KO MEFs showed decreased tumorigenesis in nude mice, showing that tumor cells as well as tumor microenvironment require ARTC1. By echocardiography and MRI, Artc1 -KO and heterozygous mice showed male-specific, reduced myocardial contractility. Furthermore, Artc1 -KO male hearts exhibited enhanced susceptibility to myocardial ischemia-reperfusion-induced injury with increased receptor-interacting protein kinase 3 (RIP3) protein levels compared to WT mice, suggesting that ARTC1 suppresses necroptosis. Overall survival rate of Artc1 -KO was less than their Artc1 -WT counterparts, primarily due to enhanced immune response and inflammation. Thus, anti-ARTC1 agents may reduce tumorigenesis but may increase multi-organ inflammation and decrease cardiac contractility.

The effect of cytokines on non‐traditional immunological targets under conditions of chronic inflammation is an ongoing subject of study. Fatigue is a symptom often associated with autoimmune diseases. Chronic inflammatory response and activated cell‐mediated immunity are associated with cardiovascular myopathies which can be driven by muscle weakness and fatigue. Thus, we hypothesize that immune dysfunction‐driven changes in myocyte mitochondria may play a critical role in fatigue‐related pathogenesis. We show that persistent low‐level expression of IFN‐γ in designated IFN‐γ AU‐Rich Element deletion mice (ARE mice) under androgen exposure resulted in mitochondrial and metabolic deficiencies in myocytes from male or castrated ARE mice. Most notably, echocardiography unveiled that low ejection fraction in the left ventricle post‐stress correlated with mitochondrial deficiencies, explaining how heart function decreases under stress. We report that inefficiencies and structural changes in mitochondria, with changes to expression of mitochondrial genes, are linked to male‐biased fatigue and acute cardiomyopathy under stress. Our work highlights how male androgen hormone backgrounds and active autoimmunity reduce mitochondrial function and the ability to cope with stress and how pharmacological blockade of stress signal protects heart function. These studies provide new insight into the diverse actions of IFN‐γ in fatigue, energy metabolism, and autoimmunity. © 2023 The Pathological Society of Great Britain and Ireland. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Background: During the first 1 to 3 weeks of LHRH-agonist therapy for recurrent or metastatic prostate cancer, an initial increase in testosterone is associated with a condition known as "flare." Flare often is accompanied by fatigue, increases in immune infiltrate in prostate cancer and cardiovascular distress. Cardiovascular myopathies and fatigue have been associated with chronic inflammatory responses and activated cell-mediated immunity. Methods and Results: Using a model of autoimmune mice with persistent low-level expression of proinflammatory interferon gamma (IFN-g), we found fatigue and aberrant cardiovascular activity in the form of reduced ejection fraction responding to exercise in male but not female mice. This finding suggests that male hormone has a potential role for impacting cardiovascular fatigue with a more activated immune environment. Furthermore, analysis of cardiovascular tissues revealed an increase in factors associated with fatigue, such as lactic acid production and changes in expression of genes associated with an increase in anaerobic respiration and poor mitochondrial performance in cardiac musculature. Conclusion: These results indicate that the chronic expression of IFN-g may be synergetic with androgen to produce a male-biased fatigue and muscle dysfunction. Considering that the most frequent side effects to activated immune therapies to cancer in males undergoing CAR-T therapies are fatigue and cardiomyopathies we believe this insight may be beneficial to patients of other therapies as well. Understanding the biological effects of therapy driven flare may offer opportunities for physiological interventions in patients undergoing persistent or intermittent LHRHa therapy. Citation Format: John M. Fenimore, Danielle Springer, Elijah Edmonston, Kunio Nagashima, Adam Sowalsky, Howard Young. Male-biased metabolic and structural muscular changes driven by testosterone flare-like conditions [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A080.

Aims:Patients with ADP-ribose-acceptor hydrolase 3 ( ARH3 ) deficiency exhibit stress-induced childhood-onset neurodegeneration with ataxia and seizures (CONDSIAS). ARH3 degrades protein-linked poly(ADP- ribose) (PAR) synthesized by poly(ADP-ribose)polymerase (PARP)-1 during oxidative stress, leading to cleavage of the ADP-ribose linked to protein. ARH3 deficiency leads to excess accumulation of PAR, resulting in PAR-dependent cell death or parthanatos. Approximately one-third of patients with homozygous mutant ARH3 die from cardiac arrest, which has been described as neurogenic, suggesting that ARH3 may play an important role in maintaining myocardial function. To address this question, cardiac function was monitored in Arh3 -knockout (KO) and - heterozygous (HT) mice.Methods and results:Arh3 -KO male mice displayed cardiac hypertrophy by histopathology and decreased cardiac contractility assessed by MRI. In addition, both genders of Arh3 -KO and -HT mice showed decreased cardiac contractility by dobutamine stress test assessed by echocardiography. A direct role of ARH3 on myocardial function was seen with a Langendorff-perfused isolated heart model . Arh3 -KO male mouse hearts showed decreased post-ischemic rate pressure products, increased size of ischemia-reperfusion (IR) infarcts, and elevated PAR levels. Consistently, in vivo IR injury showed enhanced infarct size in Arh3 -KO mice in both genders. In addition, Arh3 -HT male mice showed increased size of in vivo IR infarcts. Treatment with an FDA-approved PARP inhibitor, rucaparib, improved cardiac contractility during dobutamine-induced stress and exhibited reduced size of in vivo IR infarcts. To understand better the role of ARH3, CRISPR-Cas9 was used to generate different Arh3 genotypes of myoblasts and myotubes. Incubation with H2O2 decreased viability of Arh3 -KO and -HT myoblasts and myotubes, resulting in PAR-dependent cell death that was reduced by PARP inhibitors or by transfection with the Arh3 gene.Conclusion:ARH3 regulates PAR homeostasis in myocardium to preserve function and protect against oxidative stress; PARP inhibitors reduce the myocardial dysfunction seen with Arh3 mutations.

GM1 gangliosidosis is a rare lysosomal storage disorder affecting multiple organ systems, primarily the central nervous system, and is caused by functional deficiency of β-galactosidase (GLB1). Using CRISPR/Cas9 genome editing, we generated a mouse model to evaluate characteristics of the disease in comparison to GM1 gangliosidosis patients. Our Glb1−/− mice contain small deletions in exons 2 and 6, producing a null allele. Longevity is approximately 50 weeks and studies demonstrated that female Glb1−/− mice die six weeks earlier than male Glb1−/− mice. Gait analyses showed progressive abnormalities including abnormal foot placement, decreased stride length and increased stance width, comparable with what is observed in type II GM1 gangliosidosis patients. Furthermore, Glb1−/− mice show loss of motor skills by 20 weeks assessed by adhesive dot, hanging wire, and inverted grid tests, and deterioration of motor coordination by 32 weeks of age when evaluated by rotarod testing. Brain MRI showed progressive cerebellar atrophy in Glb1−/− mice as seen in some patients. In addition, Glb1−/− mice also show significantly increased levels of a novel pentasaccharide biomarker in urine and plasma which we also observed in GM1 gangliosidosis patients. Glb1−/− mice also exhibit accumulation of glycosphingolipids in the brain with increases in GM1 and GA1 beginning by 8 weeks. Surprisingly, despite being a null variant, this Glb1−/− mouse most closely models the less severe type II disease and will guide the development of new therapies for patients with the disorder.