Failure In Mice Research Articles

Time course changes in in vivo muscle mechanical function and Ca2+ regulation of force following experimentally induced gradual ovarian failure in mice.

The abrupt cessation of ovarian hormone release is associated with declines in muscle contractile function, yet the impact of gradual ovarian failure on muscle contractility across peri-, early- and late-stage menopause remains unclear. In this study, a 4-vinylcyclohexene diepoxide (VCD)-induced ovarian failure mouse model was used to examine time course changes in muscle mechanical function. Plantar flexors of female mice (VCD: n=10; CON: n=8) were assessed at 40 (early perimenopause), 80 (late perimenopause), 120 (menopause onset) and 176 (late menopause) days post-initial VCD injection. A torque-frequency relationship was established across a range of frequencies (10-200Hz). Isotonic dynamic contractions were elicited against relative loads (10-80% maximal isometric torque) to determine the torque-velocity-power relationship. Mice then performed a fatigue task using intermittent 100Hz isometric contractions until torque dropped by 60%. Recovery of twitch, 10Hz and 100Hz torque were tracked for 10min post-task failure. Additionally, intact muscle fibres from the flexor digitorum brevis underwent a fatigue task (50 repetitions at 70Hz), and 10 and 100Hz tetanic [Ca2+ ] were monitored for 10min afterward. VCD mice exhibited 16% lower twitch torque than controls across all time points. Apart from twitch torque, 10Hz torque and 10Hz tetanic [Ca2+ ], where VCD showed greater values relative to pre-fatigue during recovery, no significant differences were observed between control and VCD mice during recovery. These results indicate that gradual ovarian failure has minimal detriments to in vivo muscle mechanical function, with minor alterations observed primarily for low-frequency stimulation during recovery from fatigue.

Inhibition of 7-dehydrocholesterol reductase prevents hepatic ferroptosis under an active state of sterol synthesis

Recent evidence indicates ferroptosis is implicated in the pathophysiology of various liver diseases; however, the organ-specific regulation mechanism is poorly understood. Here, we demonstrate 7-dehydrocholesterol reductase (DHCR7), the terminal enzyme of cholesterol biosynthesis, as a regulator of ferroptosis in hepatocytes. Genetic and pharmacological inhibition (with AY9944) of DHCR7 suppress ferroptosis in human hepatocellular carcinoma Huh-7 cells. DHCR7 inhibition increases its substrate, 7-dehydrocholesterol (7-DHC). Furthermore, exogenous 7-DHC supplementation using hydroxypropyl β-cyclodextrin suppresses ferroptosis. A 7-DHC-derived oxysterol metabolite, 3β,5α-dihydroxycholest-7-en-6-one (DHCEO), is increased by the ferroptosis-inducer RSL-3 in DHCR7-deficient cells, suggesting that the ferroptosis-suppressive effect of DHCR7 inhibition is associated with the oxidation of 7-DHC. Electron spin resonance analysis reveals that 7-DHC functions as a radical trapping agent, thus protecting cells from ferroptosis. We further show that AY9944 inhibits hepatic ischemia-reperfusion injury, and genetic ablation of Dhcr7 prevents acetaminophen-induced acute liver failure in mice. These findings provide new insights into the regulatory mechanism of liver ferroptosis and suggest a potential therapeutic option for ferroptosis-related liver diseases.

Mechanosensitive channels TMEM63A and TMEM63B mediate lung inflation-induced surfactant secretion.

Pulmonary surfactant is a lipoprotein complex lining the alveolar surface to decrease the surface tension and facilitate inspiration. Surfactant deficiency is often seen in premature infants and in children and adults with respiratory distress syndrome. Mechanical stretch of alveolar type 2 epithelial (AT2) cells during lung expansion is the primary physiological factor that stimulates surfactant secretion; however, it is unclear whether there is a mechanosensor dedicated to this process. Here, we show that loss of the mechanosensitive channels TMEM63A and TMEM63B (TMEM63A/B) resulted in atelectasis and respiratory failure in mice due to a deficit of surfactant secretion. TMEM63A/B were predominantly localized at the limiting membrane of the lamellar body (LB), a lysosome-related organelle that stores pulmonary surfactant and ATP in AT2 cells. Activation of TMEM63A/B channels during cell stretch facilitated the release of surfactant and ATP from LBs fused with the plasma membrane. The released ATP evoked Ca2+ signaling in AT2 cells and potentiated exocytic fusion of more LBs. Our study uncovered a vital physiological function of TMEM63 mechanosensitive channels in preparing the lungs for the first breath at birth and maintaining respiration throughout life.

Modulation of the crosstalk between Keap1/Nrf2/HO-1 and NF-κB signaling pathways by Tomatidine protects against inflammation/oxidative stress-driven fulminant hepatic failure in mice

Fulminant hepatic failure (FHF) is the terminal phase of acute liver injury, which is characterized by massive hepatocyte necrosis and rapid hepatic dysfunction in patients without preexisting liver disease. There are currently no therapeutic options for such a life-threatening hepatic failure except liver transplantation; therefore, the terminal phase of the underlying acute liver injury should be avoided. Tomatidine (TOM), asteroidal alkaloid, may have different biological activities, including antioxidant and anti-inflammatory effects. Herein, the lipopolysaccharide (LPS)/D-galactosamine (D-GalN)-induced FHF mouse model was established to explore the protective potential of TOM and the underlying mechanisms of action. TOM pretreatment significantly inhibited hepatocyte necrosis and decreased serum aminotransferase activities in LPS/D-GalN-stimulated mice. TOM further increased the level of different antioxidant enzymes while reducing lipid peroxidation biomarkers in the liver. These beneficial effects of TOM were shown to be associated with targeting of NF-κB signaling pathways, where TOM repressed NF-κB activation and decreased LPS/D-GalN-induced TNF-α, IL-6, IL-1β, and iNOS production. Moreover, TOM prevented LPS/D-GalN-induced upregulation of Keap1 expression and downregulation of Nrf2 and HO-1 expression, leading to increased Nrf2-binding activity and HO-1 levels. Besides, TOM pretreatment repressed LPS/D-GalN-induced upregulation of proliferating cell nuclear antigen (PCNA) expression, which spared the hepatocytes from damage and subsequent repair following the LPS/D-GalN challenge. Collectively, our findings revealed that TOM has a protective effect on LPS/D-GalN-induced FHF in mice, showing powerful antioxidant and anti-inflammatory effects, primarily mediated via modulating Keap1/Nrf2/HO-1 and NF-κB/TNF-α/IL-6/IL-1β/iNOS signaling pathways.

P53 Acetylation Exerts Critical Roles in Pressure Overload-Induced Coronary Microvascular Dysfunction and Heart Failure in Mice.

Coronary microvascular dysfunction (CMD) has been shown to contribute to cardiac hypertrophy and heart failure (HF) with preserved ejection fraction. At this point, there are no proven treatments for CMD. We have shown that histone acetylation may play a critical role in the regulation of CMD. By using a mouse model that replaces lysine with arginine at residues K98, K117, K161, and K162R of p53 (p534KR), preventing acetylation at these sites, we test the hypothesis that acetylation-deficient p534KR could improve CMD and prevent the progression of hypertensive cardiac hypertrophy and HF. Wild-type and p534KR mice were subjected to pressure overload by transverse aortic constriction to induce cardiac hypertrophy and HF. Echocardiography measurements revealed improved cardiac function together with a reduction of apoptosis and fibrosis in p534KR mice. Importantly, myocardial capillary density and coronary flow reserve were significantly improved in p534KR mice. Moreover, p534KR upregulated the expression of cardiac glycolytic enzymes and Gluts (glucose transporters), as well as the level of fructose-2,6-biphosphate; increased PFK-1 (phosphofructokinase 1) activity; and attenuated cardiac hypertrophy. These changes were accompanied by increased expression of HIF-1α (hypoxia-inducible factor-1α) and proangiogenic growth factors. Additionally, the levels of SERCA-2 were significantly upregulated in sham p534KR mice, as well as in p534KR mice after transverse aortic constriction. In vitro, p534KR significantly improved endothelial cell glycolytic function and mitochondrial respiration and enhanced endothelial cell proliferation and angiogenesis. Similarly, acetylation-deficient p534KR significantly improved coronary flow reserve and rescued cardiac dysfunction in SIRT3 (sirtuin 3) knockout mice. Our data reveal the importance of p53 acetylation in coronary microvascular function, cardiac function, and remodeling and may provide a promising approach to improve hypertension-induced CMD and to prevent the transition of cardiac hypertrophy to HF.

Mycn ameliorates cardiac hypertrophy-induced heart failure in mice by mediating the USP2/JUP/Akt/β-catenin cascade

BackgroundPathological cardiac hypertrophy is associated with cardiac dysfunction and is a key risk factor for heart failure and even sudden death. This study investigates the function of Mycn in cardiac hypertrophy and explores the interacting molecules.MethodsA mouse model of cardiac hypertrophy was induced by isoproterenol (ISO). The cardiac dysfunction was assessed by the heart weight-to-body weight ratio (HW/BW), echocardiography assessment, pathological staining, biomarker detection, and cell apoptosis. Transcriptome alteration in cardiac hypertrophy was analyzed by bioinformatics analysis. Gain- or loss-of-function studies of MYCN proto-oncogene (Mycn), ubiquitin specific peptidase 2 (USP2), and junction plakoglobin (JUP) were performed. The biological functions of Mycn were further examined in ISO-treated cardiomyocytes. The molecular interactions were verified by luciferase assay or immunoprecipitation assays.ResultsMycn was poorly expressed in ISO-treated mice, and its upregulation reduced HW/BW, cell surface area, oxidative stress, and inflammation while improving cardiac function of mice. It also reduced apoptosis of cardiomyocytes in mice and those in vitro induced by ISO. Mycn bound to the USP2 promoter to activate its transcription. USP2 overexpression exerted similar myocardial protective functions. It stabilized JUP protein by deubiquitination modification, which blocked the Akt/β-catenin pathway. Knockdown of JUP restored phosphorylation of Akt and β-catenin protein level, which negated the protective effects of USP2.ConclusionThis study demonstrates that Mycn activates USP2 transcription, which mediates ubiquitination and protein stabilization of JUP, thus inactivating the Akt/β-catenin axis and alleviating cardiac hypertrophy-induced heart failure.

Larixyl acetate, a TRPC6 inhibitor, attenuates pressure overload‑induced heart failure in mice.

Heart failure is a primary cause of global mortality. In the present study, whether larixyl acetate, an inhibitor of transient receptor potential cation channel subfamily C member 6, attenuates pressure overload‑induced heart failure in mice was investigated. To test this hypothesis, a transverse aortic constriction (TAC) animal model and an angiotensin II (Ang II)‑treated H9c2 cell model were used. Cardiac and cellular structure, function and the expression levels of hypertrophy, endoplasmic reticulum (ER) stress, apoptosis, autophagy and pmTOR/mTOR related mRNAs or proteins were assessed to explore the underlying molecular mechanisms. The results indicated that treatment with TAC or Ang II leads to significant hypertrophy and dysfunction of the heart or H9c2 cells, accompanied by an increase in ER stress, apoptosis and activation of the mTOR signaling pathway, and a decrease in autophagy. The administration of larixyl acetate attenuated these impairments, which can be reversed by inhibiting autophagy through the activation of the mTOR signaling pathway. These findings suggested that larixyl acetate can effectively protect against pressure overload‑induced heart failure by enhancing autophagy and limiting ER stress and apoptosis through inhibition of the mTOR pathway.

ZBP1 activation triggers hematopoietic stem and progenitor cell death resulting in bone marrow failure in mice

Human bone marrow failure (BMF) syndromes result from the loss of hematopoietic stem and progenitor cells (HSPC), and this loss has been attributed to cell death; however, the cell death triggers, and mechanisms remain unknown. During BMF, tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ) increase. These ligands are known to induce necroptosis, an inflammatory form of cell death mediated by RIPK1, RIPK3, and MLKL. We previously discovered that mice with a hematopoietic RIPK1 deficiency (Ripk1HEM KO) exhibit inflammation, HSPC loss, and BMF, which is partially ameliorated by a RIPK3 deficiency; however, whether RIPK3 exerts its effects through its function in mediating necroptosis or other forms of cell death remains unclear. Here, we demonstrate that similar to a RIPK3 deficiency, an MLKL deficiency significantly extends survival and like Ripk3 deficiency partially restores hematopoiesis in Ripk1HEM KO mice revealing that both necroptosis and apoptosis contribute to BMF in these mice. Using mouse models, we show that the nucleic acid sensor Z-DNA binding protein 1 (ZBP1) is up-regulated in mouse RIPK1-deficient bone marrow cells and that ZBP1's function in endogenous nucleic acid sensing is necessary for HSPC death and contributes to BMF. We also provide evidence that IFNγ mediates HSPC death in Ripk1HEM KO mice, as ablation of IFNγ but not TNFα receptor signaling significantly extends survival of these mice. Together, these data suggest that RIPK1 maintains hematopoietic homeostasis by preventing ZBP1 activation and induction of HSPC death.

Retracted: The Anti-inflammatory Effect of Chitosan Oligosaccharide on Heart Failure in Mice.

[This retracts the article DOI: 10.1155/2022/8746530.].

Improved integration of single-cell transcriptome data demonstrates common and unique signatures of heart failure in mice and humans.

Cardiovascular research heavily relies on mouse (Mus musculus) models to study disease mechanisms and to test novel biomarkers and medications. Yet, applying these results to patients remains a major challenge and often results in noneffective drugs. Therefore, it is an open challenge of translational science to develop models with high similarities and predictive value. This requires a comparison of disease models in mice with diseased tissue derived from humans. To compare the transcriptional signatures at single-cell resolution, we implemented an integration pipeline called OrthoIntegrate, which uniquely assigns orthologs and therewith merges single-cell RNA sequencing (scRNA-seq) RNA of different species. The pipeline has been designed to be as easy to use and is fully integrable in the standard Seurat workflow.We applied OrthoIntegrate on scRNA-seq from cardiac tissue of heart failure patients with reduced ejection fraction (HFrEF) and scRNA-seq from the mice after chronic infarction, which is a commonly used mouse model to mimic HFrEF. We discovered shared and distinct regulatory pathways between human HFrEF patients and the corresponding mouse model. Overall, 54% of genes were commonly regulated, including major changes in cardiomyocyte energy metabolism. However, several regulatory pathways (e.g., angiogenesis) were specifically regulated in humans. The demonstration of unique pathways occurring in humans indicates limitations on the comparability between mice models and human HFrEF and shows that results from the mice model should be validated carefully. OrthoIntegrate is publicly accessible (https://github.com/MarianoRuzJurado/OrthoIntegrate) and can be used to integrate other large datasets to provide a general comparison of models with patient data.

Effects of ivabradine on myocardial autophagia and apoptosis in isoprenaline-induced heart failure in mice.

To investigate the effects and mechanisms of ivabradine (IVA) on isoprenaline-induced cardiac injury. Forty male C57BL/6 mice were randomly divided into control group, model group, high-dose IVA group, and low-dose IVA group. The control group was given saline, other groups were given subcutaneous injections of isoproterenol (ISO) 5 mg/kg/d to make the myocardial remodeling model. A corresponding dose of IVA (high dose 50 mg/kg/d, low dose 10 mg/kg/d) was given by gavage (30 days). A transthoracic echocardiogram was obtained to detect the structure and function of the heart. An electron microscope was used to explore the cardiomyocytes' apoptosis and autophagy. HE staining and Masson's trichrome staining were performed to explore myocardial hypertrophy and fibrosis. Western blot was used to detect Bax, Bcl-2, cleaved caspase-3, Becline-1, LC3, phosphorylated p38 mitogen-activated protein kinase (p-p38MAPK), phosphorylated extracellular regulated protein kinases1/2 (p-ERK1/2), phosphorylated c-Jun N-terminal kinase (p-JNK), and α-smooth muscle actin (α-SMA) in the myocardium. Heart rate in the IVA groups was reduced, and the trend of heart rate reduction was more obvious in the high-dose group. Echocardiography showed that IVA improved the cardiac structure and function compared to the model group. IVA attenuated cardiac fibrosis, decreased cardiomyocyte apoptosis, and increased autophagy. The phosphorylated MAPK in the ISO-induced groups was increased. IVA treatment decreased the p-p38MAPK level. There were no differences in p-ERK and p-JNK levels. The beneficial effects of IVA on myocardial injury are related to blocking the p38MAPK signal pathway, decreasing cardiomyocyte apoptosis, and increasing cardiomyocyte autophagy.

DIA-Based Proteomics Reveals the Protective Effect of Quercetin against Cyclophosphamide-Induced Testicular Toxicity in Mice

Cyclophosphamide (CP) is a common employed immunosuppressive and anti-neoplastic alkylating antineoplastic drug with documented male reproductive harm. Quercetin, a widely distributed anti-oxidant flavonoid, has been demonstrated to have a variety of biological roles, including tissue-protective properties. Here, administration of quercetin alleviated the CP-induced damage to the testicles, as evidenced by improvements in body weight, testis and epididymis index, serum testosterone concentration, the pathomorphological of testis, and reduced apoptosis in testicular cells. Moreover, Proteomic data indicated that all DEPs were classified into five categories, Cluster 1 (C1) and C4 were regulated by cyclophosphamide and quercetin. KEGG pathway analyses showed that mTOR signaling pathways and Proteasome were the most abundant pathways in C1 and C4, respectively. In the protein-protein interaction (PPI) network analysis, quercetin inhibits the expression of GSK3B, EIF4E, and RPS6KA2 proteins in CP-induced spermatogenesis failure in mice via the mTOR signaling pathway, protecting against spermatogonia injury. On the other hand, quercetin alleviates spermatogonia injury by stimulating the production of Rps23, Rpl8, Rps11, and Mrps17 proteins in CP-induced testis injury in mice via the pathways of Proteasome. Our results have showed that quercetin has a putative protective effect via modulating the pathways of mTOR and Proteasome in CP induced testis injury

BNC1 deficiency induces mitochondrial dysfunction-triggered spermatogonia apoptosis through the CREB/SIRT1/FOXO3 pathway: the therapeutic potential of nicotinamide riboside and metformin†.

Male infertility is a global health problem that disturbs numerous couples worldwide. Basonuclin 1 (BNC1) is a transcription factor mainly expressed in proliferative keratinocytes and germ cells. A frameshift mutation of BNC1 was identified in a large Chinese primary ovarian insufficiency pedigree. The expression of BNC1 was significantly decreased in the testis biopsies of infertile patients with nonobstructive azoospermia. Previous studies have revealed that mice with BNC1 deficiency are generally subfertile and undergo gradual spermatogenic failure. We observed that apoptosis of spermatogonia is tightly related to spermatogenic failure in mice with a Bnc1 truncation mutation. Such impairment is related to mitochondrial dysfunction causing lower mitochondrial membrane potential and higher reactive oxygen species. We showed that downregulation of CREB/SIRT1/FOXO3 signaling participates in the above impairment. Administration of nicotinamide riboside or metformin reversed mitochondrial dysfunction and inhibited apoptosis in Bnc1-knockdown spermatogonia by stimulating CREB/SIRT1/FOXO3 signaling. Dietary supplementation with nicotinamide riboside or metformin in mutated mice increased SIRT1 signaling, improved the architecture of spermatogenic tubules, inhibited apoptosis of the testis, and improved the fertility of mice with a Bnc1 truncation mutation. Our data establish that oral nicotinamide riboside or metformin can be useful for the treatment of spermatogenic failure induced by Bnc1 mutation.

Dapagliflozin protects against chronic heart failure in mice by inhibiting macrophage-mediated inflammation, independent of SGLT2

The specific mechanism of sodium-glucose cotransporter 2 (SGLT2) inhibitor in heart failure (HF) needs to be elucidated. In this study, we use SGLT2-global-knockout (KO) mice to assess the mechanism of SGLT2 inhibitor on HF. Dapagliflozin ameliorates both myocardial infarction (MI)- and transverse aortic constriction (TAC)-induced HF. Global SGLT2 deficiency does not exert protection against adverse remodeling in both MI- and TAC-induced HF models. Dapagliflozin blurs MI- and TAC-induced HF phenotypes in SGLT2-KO mice. Dapagliflozin causes major changes in cardiac fibrosis and inflammation. Based on single-cell RNA sequencing, dapagliflozin causes significant differences in the gene expression profile of macrophages and fibroblasts. Moreover, dapagliflozin directly inhibits macrophage inflammation, thereby suppressing cardiac fibroblasts activation. The cardio-protection of dapagliflozin is blurred in mice treated with a C-C chemokine receptor type 2 antagonist. Taken together, the protective effects of dapagliflozin against HF are independent of SGLT2, and macrophage inhibition is the main target of dapagliflozin against HF.

Caspase 8 Deletion Causes Infection/Inflammation-Induced MDS-like Bone Marrow Failure in Mice

Myelodysplastic syndromes (MDS) are a heterogeneous group of pre-leukemic hematopoietic disorders characterized by persistent cytopenia in peripheral blood (PB) and normo- or hypercellular and morphologic dysplasia in bone marrow (BM) due to ineffective hematopoiesis, as well as resulting in an elevated risk for leukemic transformation. Bacterial or viral infections increase the severity of MDS patients' symptoms. An inflammatory BM micro-environment and programmed cell death of hematopoietic stem/progenitor cells (HSPCs) have been thought to be the major causes leading to ineffective hematopoiesis. Three types of programed cell death, apoptosis, necroptosis and pyroptosis, were observed in BM tissues of MDS patients, suggesting PANoptosis might play a critical role in the pathogenesis of MDS. Caspase 8 (Casp8) is a master regulator of PANoptosis. Casp8 is downregulated in HSPCs from patients with MDS, and is associated with increased Ripk1 protein, which is another master regulator of PANoptosis. To study the role of PANoptosis in normal hematopoiesis and the pathogenesis of MDS, we generated Mx1-Cre and Rosa-ERT 2Cre-mediated inducible C asp8 knockout mice ( Casp8 -/-). Mx1-CreCasp8 -/- mice died of BM failure within 10 days of three polyI:C injections (administered every other day) due to the massive depletion of HSPCs. This could be completely rescued by Ripk3 deletion, suggesting that HSPCs were eliminated by necroptosis. Rosa-ERT 2Cre- Casp8 -/- mice are completely normal without any significant changes in BM hematopoiesis after induction by five doses of tamoxifen (TAM) when maintained in specific pathogen-free facilities. However, such mice developed a spontaneous BM failure phenotype when maintained in quarantine facilities. This BM failure can be attenuated by antibiotic treatment, suggesting that the phenotype is infection-induced. In addition, low dose polyI:C injections or treatment with LPS also induces BM failure in Rosa-ERT 2Cre - Casp8 -/- mice living in germ-free conditions. Detailed analysis suggested that Casp8 -/- HSPCs are hypersensitive to polyI:C, LPS, TNFα and IFNα/γ-induced cell death. Most importantly, all these phenotypic and functional abnormalities observed in Rosa-ERT 2Cre- Casp8 -/- mice can be completely prevented by Ripk3 deletion. In addition, serial transplantationstudy demonstrated that Casp8 -/- HSCs have impaired self-renewal capacity. Interestingly, the self-renewal of Casp8 -/-Ripk3 -/- HSCs is significantly enhanced compared to Wild-type HSCs. Our study suggests an essential role for a balance in Casp8 and Ripk3 activities in the regulation of normal hematopoiesis and the self-renewal of HSCs, the disruption of which might induce inflammation and BM failure, resulting in MDS-like disease.

Carfilzomib Mitigates Lipopolysaccharide/D-Galactosamine/Dimethylsulfoxide-Induced Acute Liver Failure in Mice.

Acute liver failure (ALF) is a disease accompanied by severe liver inflammation. No effective therapy is available yet apart from liver transplantation; therefore, developing novel treatments for ALF is urgently required. Inflammatory mediators released by NF-кB activation play an essential role in ALF. Proteasome inhibitors have many medical uses, such as reducing inflammation and NF-кB inhibition, which are believed to account for most of their repurposing effects. This study was undertaken to explore the possible protective effects and the underlying mechanisms of carfilzomib, a proteasome inhibitor, in a mouse model of ALF induced by lipopolysaccharide/D-galactosamine/dimethylsulfoxide (LPS/GalN/DMSO). Carfilzomib dose-dependently protected mice from LPS/GalN/DMSO-induced liver injury, as indicated by the decrease in serum alanine aminotransferase and aspartate aminotransferase levels. LPS/GalN/DMSO increased TNF-α, NF-кB, lipid peroxidation, NO, iNOS, cyclooxygenase-II, myeloperoxidase, and caspase-3 levels. Carfilzomib administration mitigated LPS/GalN/DMSO-induced liver damage by decreasing the elevated levels of TNF-α, NF-кB, lipid peroxidation, nitric oxide, iNOS, cyclooxygenase-II, myeloperoxidase, caspase-3, and histopathological changes. A restored glutathione level was also observed in the carfilzomib-treated LPS/GalN/DMSO mice. Our results demonstrate that carfilzomib protects against LPS/GalN/DMSO-induced ALF by inhibiting NF-кB, decreasing inflammatory mediators, oxidative/nitrosative stress, neutrophil recruitment, and apoptosis, suggesting that carfilzomib may be a potential therapeutic agent for ALF.

Microfluidic Encapsulation of Exosomes Derived from Lipopolysaccharide-Treated Mesenchymal Stem Cells in Hyaluronic Acid Methacryloyl to Restore Ovarian Function in Mice.

Premature ovarian failure (POF) features an upward incidence nowadays, and the human umbilical cord mesenchymal stem cells (hUC-MSCs)-derived exosomes (MSC-Exos) have shown applied values in the recovery of ovarian function. Here, a novel exosome-encapsulated microcarrier prepared by microfluidic technology for ovarian repair after chemotherapy damage is presented. The exosomes derived from lipopolysaccharide (LPS)-preconditioned hUC-MSCs are encapsulated with hyaluronic acid methacryloyl (HAMA) via microfluidic electrospray, which is named HAMA/MSC-Exos. Attributing to the biocompatibility and semipermeable property of HAMA, the encapsulated exosomes show great viability and controllable release behavior from HAMA. It is demonstrated that in situ transplantation of HAMA/MSC-Exos can rescue ovarian functions of cyclophosphamide-induced ovarian failure in mice by increasing ovarian volume, improving the number of antral follicles and restoring fertility. It is believed that the transplantation of HAMA/MSC-Exos will provide a new concept for the treatment of POF in clinical practice.

Abstract 16532: Mosaic Loss of Y Chromosome in Leukocytes Predicts Death in Patients With Chronic Heart Failure Across the Whole Spectrum of Ejection Fraction

Aims: Mosaic loss of Y chromosome (LOY) in men increases with age and correlates with clonal expansion of myeloid cells. Experimental modeling demonstrated that LOY in hematopoietic cells leads to diffuse cardiac fibrosis and subsequent development of heart failure in mice. Cardiac fibrosis is an important pathogenic feature in heart failure irrespective of etiology. Therefore, we investigated the prognostic significance of LOY in men with chronic heart failure. Methods and Results: In a total of 705 male patients with chronic heart failure with reduced and preserved ejection fraction, LOY was determined by digital PCR of peripheral blood cells by assessing the 6bp sequence difference between the AMELX and AMELY genes. ROC analysis relating the extent of LOY to mortality revealed a LOY of 17% as the optimal cut-off value to predict death during follow-up. LOY >17% was observed in 18.6% of all patients, increased with age and was associated with increased mortality after three years (13,7% in patients without LOY compared to 29.0% in patients with LOY, p<0.001, Figure 1). LOY remained an independent predictor of death in multivariate analysis including, age, LVEF and nt-pro BNP serum levels (HR 1,67; 95% CI 1.08-2.57; p=0.017). Importantly, LOY was associated with increased mortality in patients with heart failure with reduced ejection fraction defined as LVEF < 50% (p<0.001) as well as in patients with preserved LVEF ≥ 50% (p=0.01). Conclusion: Among patients with heart failure, LOY in blood cells is associated with increased risk of death across the complete range of ejection fraction. Further studies are warranted to discern if LOY associates with cardiac fibrosis in men with heart failure and triggers increased profibrotic signaling. The results of this study may inform trials evaluating precision-targeted anti-fibrotic therapies in patients with chronic heart failure.

Elimination of CaMKIIδ Autophosphorylation by CRISPR-Cas9 Base Editing Improves Survival and Cardiac Function in Heart Failure in Mice.

Cardiovascular diseases are the main cause of worldwide morbidity and mortality, highlighting the need for new therapeutic strategies. Autophosphorylation and subsequent overactivation of the cardiac stress-responsive enzyme CaMKIIδ (Ca2+/calmodulin-dependent protein kinase IIδ) serves as a central driver of multiple cardiac disorders. To develop a comprehensive therapy for heart failure, we used CRISPR-Cas9 adenine base editing to ablate the autophosphorylation site of CaMKIIδ. We generated mice harboring a phospho-resistant CaMKIIδ mutation in the germline and subjected these mice to severe transverse aortic constriction, a model for heart failure. Cardiac function, transcriptional changes, apoptosis, and fibrosis were assessed by echocardiography, RNA sequencing, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, and standard histology, respectively. Specificity toward CaMKIIδ gene editing was assessed using deep amplicon sequencing. Cellular Ca2+ homeostasis was analyzed using epifluorescence microscopy in Fura-2-loaded cardiomyocytes. Within 2 weeks after severe transverse aortic constriction surgery, 65% of all wild-type mice died, and the surviving mice showed dramatically impaired cardiac function. In contrast to wild-type mice, CaMKIIδ phospho-resistant gene-edited mice showed a mortality rate of only 11% and exhibited substantially improved cardiac function after severe transverse aortic constriction. Moreover, CaMKIIδ phospho-resistant mice were protected from heart failure-related aberrant changes in cardiac gene expression, myocardial apoptosis, and subsequent fibrosis, which were observed in wild-type mice after severe transverse aortic constriction. On the basis of identical mouse and human genome sequences encoding the autophosphorylation site of CaMKIIδ, we deployed the same editing strategy to modify this pathogenic site in human induced pluripotent stem cells. It is notable that we detected a >2000-fold increased specificity for editing of CaMKIIδ compared with other CaMKII isoforms, which is an important safety feature. While wild-type cardiomyocytes showed impaired Ca2+ transients and an increased frequency of arrhythmias after chronic β-adrenergic stress, CaMKIIδ-edited cardiomyocytes were protected from these adverse responses. Ablation of CaMKIIδ autophosphorylation by adenine base editing may offer a potential broad-based therapeutic concept for human cardiac disease.

S14G-humanin confers cardioprotective effects against chronic adrenergic and pressure overload-induced heart failure in mice

S14G-humanin (HNG), an analog of the mitochondria-derived peptide humanin, has demonstrated protective effects against various cardiovascular diseases. However, the specific pharmacological effects of HNG in heart failure (HF) have not been previously reported. Therefore, in this study, we aimed to investigate the potential protective effect of HNG in HF using a mouse model. HF was induced in mice through intraperitoneal injection of isoproterenol or transverse aortic constriction, followed by separate administration of HNG to assess its therapeutic impact. Our results revealed that HNG treatment significantly delayed the onset of cardiac dysfunction and structural remodeling in the HF mouse model. Furthermore, HNG administration was associated with reduced infiltration of inflammatory cells, improved myocardial fibrosis, and attenuation of cardiomyocyte apoptosis in the treated cardiac tissues. Additionally, we identified the involvement of the transforming growth factor-beta signaling pathway in the beneficial effects of HNG in isoproterenol-induced HF mice. Collectively, these findings underscore the therapeutic potential of HNG in preventing the progression of HF, as demonstrated in two distinct HF mouse models.