Ibrutinib Contributes to Atrial Arrhythmia through the Autophagic Degradation of Connexins by Inhibiting the PI3K-AKT-mTOR Signaling Pathway.

BACKGROUND: Ibrutinib could increase the risk of atrial fibrillation (AF) in chronic lymphocytic leukemia (CLL) patients. However, the precise mechanism underlying ibrutinib-induced AF remains incompletely elucidated. METHODS: We investigated the proportion of ibrutinib-treated CLL patients with new-onset AF. Optical mapping was conducted to reveal the proarrhythmic effect of ibrutinib on HL-1 cells. Fluorescence staining and western blot were used to compare connexins 43 and 40 expression in ibrutinib-treated and control groups. To identify autophagy phenotypes, we used western blot to detect autophagy-related proteins, transmission electron microscopy to picture autophagosomes, and transfected mCherry-GFP-LC3 virus to label autophagosomes and lysosomes. Hydroxychloroquine as an autophagy inhibitor was administered to rescue ibrutinib-induced Cx43 and Cx40 degradation. RESULTS: About 2.67% of patients developed atrial arrhythmias after ibrutinib administration. HL-1 cells treated with ibrutinib exhibited diminished conduction velocity and a higher incidence of reentry-like arrhythmias compared to controls. Cx43 and Cx40 expression reduced along with autophagy markers increased in HL-1 cells treated with ibrutinib. Inhibiting autophagy upregulated Cx43 and Cx40. CONCLUSIONS: The off-target effect of ibrutinib on the PI3K-AKT-mTOR signaling pathway caused connexin degradation and atrial arrhythmia via promoting autophagy. CLINICAL TRIAL REGISTRATION: ChiCTR2100046062, https://clin.larvol.com/trial-detail/ChiCTR2100046062.

The crucial relationship between miRNA-27 and CSE/H2S, and the mechanism of action of GLP-1 in myocardial hypertrophy.

Cardiac hypertrophy is the most prevalent compensatory heart disease that ultimately leads to spontaneous heart failure. Mounting evidence suggests that microRNAs (miRs) and endogenous hydrogen sulfide (H2S) play a crucial role in the regulation of cardiac hypertrophy. In this study, we aimed to investigate whether inhibition of miR-27a could protect against cardiac hypertrophy by modulating H2S signaling. We established a model of cardiac hypertrophy by obtaining hypertrophic tissue from mice subjected to transverse aortic constriction (TAC) and from cells treated with angiotensin-II. Molecular alterations in the myocardium were quantified using quantitative real time PCR (qRT-PCR), Western blotting, and ELISA. Morphological changes were characterized by hematoxylin and eosin (HE) staining and Masson's trichrome staining. Functional myocardial changes were assessed using echocardiography. Our results demonstrated that miR-27a levels were elevated, while H2S levels were reduced in TAC mice and myocardial hypertrophy. Further luciferase and target scan assays confirmed that cystathionine-γ-lyase (CSE) was a direct target of miR-27a and was negatively regulated by it. Notably, enhancement of H2S expression in the heart was observed in mice injected with recombinant adeno-associated virus vector 9 (rAAV9)-anti-miR-27a and in cells transfected with a miR-27a inhibitor during cardiac hypertrophy. However, this effect was abolished by co-transfection with CSE siRNA and the miR-27a inhibitor. Conversely, injecting rAAV9-miR-27a yielded opposite results. Interestingly, our findings demonstrated that glucagon-like peptide-1 (GLP-1) agonists could mitigate myocardial damage by down-regulating miR-27a and up-regulating CSE. In summary, our study suggests that inhibition of miR-27a holds therapeutic promise for the treatment of cardiac hypertrophy by increasing H2S levels. Furthermore, our findings unveil a novel mechanism of GLP-1 agonists involving the miR-27a/H2S pathway in the management of cardiac hypertrophy.

Deep Survival Analysis With Latent Clustering and Contrastive Learning.

Survival analysis is employed to analyze the time before the event of interest occurs, which is broadly applied in many fields. The existence of censored data with incomplete supervision information about survival outcomes is one key challenge in survival analysis tasks. Although some progress has been made on this issue recently, the present methods generally treat the instances as separate ones while ignoring their potential correlations, thus rendering unsatisfactory performance. In this study, we propose a novel Deep Survival Analysis model with latent Clustering and Contrastive learning (DSACC). Specifically, we jointly optimize representation learning, latent clustering and survival prediction in a unified framework. In this way, the clusters distribution structure in latent representation space is revealed, and meanwhile the structure of the clusters is well incorporated to improve the ability of survival prediction. Besides, by virtue of the learned clusters, we further propose a contrastive loss function, where the uncensored data in each cluster are set as anchors, and the censored data are treated as positive/negative sample pairs according to whether they belong to the same cluster or not. This design enables the censored data to make full use of the supervision information of the uncensored samples. Through extensive experiments on four popular clinical datasets, we demonstrate that our proposed DSACC achieves advanced performance in terms of both C-index (0.6722, 0.6793, 0.6350, and 0.7943) and Integrated Brier Score (IBS) (0.1616, 0.1826, 0.2028, and 0.1120).

Anticancer peptides from induced tumor-suppressing cells for inhibiting osteosarcoma cells.

Osteosarcoma (OS) is the most frequent primary bone cancer, which is mainly suffered by children and young adults. While the current surgical treatment combined with chemotherapy is effective for the early stage of OS, advanced OS preferentially metastasizes to the lung and is difficult to treat. Here, we examined the efficacy of ten anti-OS peptide candidates from a trypsin-digested conditioned medium that was derived from the secretome of induced tumor-suppressing cells (iTSCs). Using OS cell lines, the antitumor capabilities of the peptide candidates were evaluated by assaying the alterations in metabolic activities, proliferation, motility, and invasion of OS cells. Among ten candidates, peptide P05 (ADDGRPFPQVIK), a fragment of aldolase A (ALDOA), presented the most potent OS-suppressing capabilities. Its efficacy was additive with standard-of-care chemotherapeutic agents such as cisplatin and doxorubicin, and it downregulated oncoproteins such as epidermal growth factor receptor (EGFR), Snail, and Src in OS cells. Interestingly, P05 did not present inhibitory effects on non-OS skeletal cells such as mesenchymal stem cells and osteoblast cells. Collectively, this study demonstrated that iTSC-derived secretomes may provide a source for identifying anticancer peptides, and P05 may warrant further evaluations for the treatment of OS.

Arrhythmogenic Right Ventricular Cardiomyopathy With Extensive Abnormal Substrate: Is Isolation Possible?

BACKGROUND: In arrhythmogenic right ventricular cardiomyopathy (ARVC) patients with extensive right ventricular free wall (RVFW) abnormal substrate, large-area homogenization with combined epicardial and endocardial approach is time consuming and often inadequate for modification. OBJECTIVES: This study aimed to explore the feasibility and efficacy of RVFW abnormal substrate isolation in such patients to control ventricular tachycardia (VT). METHODS: Eight consecutive ARVC patients with VT who had extensive abnormal RVFW substrate were included. VT induction was performed before substrate mapping and modification. Detailed voltage mapping was done during sinus rhythm. A circumferential linear lesion was deployed along the border zone of low-voltage area on the RVFW to achieve electrical isolation. Other small areas with fractionated or late potentials were further homogenized. RESULTS: All 8 patients had RVFW endocardial low-voltage area. The entire RV low-voltage area was 113.8 ± 84.1 cm2 (49.6% ± 29.8%) and the dense scar was 59.6 ± 39.8 cm2 (25.0% ± 14.1%). Electrical isolation of abnormal substrate was achieved in 5 of 8 (62.5%) patients via endocardial approach alone and 3 of 8 (37.5%) patients via a combination of endocardial and epicardial approach. Electrical isolation was verified by slow automaticity (5 of 8, 62.5%) or RV noncapture (3 of 8, 37.5%) during high-output pacing inside the encircled area. VTs were induced in 6 patients before ablation, and all patients were rendered noninducible after ablation. During a median follow-up of 43 months (range: 24-53 months), 7 of 8 (87.5%) patients remained free of sustained VT. CONCLUSIONS: Electrical isolation of RVFW is feasible and can be the option in ARVC patients with extensive abnormal substrate.

"Dual Anta-Inhibitors" of the A2A Adenosine Receptor and Casein Kinase CK1delta: Synthesis, Biological Evaluation, and Molecular Modeling Studies.

Based on a screening of a chemical library of A2A adenosine receptor (AR) antagonists, a series of di- and tri-substituted adenine derivatives were synthesized and tested for their ability to inhibit the activity of the enzyme casein kinase 1 delta (CK1δ) and to bind adenosine receptors (ARs). Some derivatives, here called "dual anta-inhibitors", demonstrated good CK1δ inhibitory activity combined with a high binding affinity, especially for the A2AAR. The N6-methyl-(2-benzimidazolyl)-2-dimethyamino-9-cyclopentyladenine (17, IC50 = 0.59 µM and KiA2A = 0.076 µM) showed the best balance of A2AAR affinity and CK1δ inhibitory activity. Computational studies were performed to simulate, at the molecular level, the protein-ligand interactions involving the compounds of our series. Hence, the dual anta-inhibitor 17 could be considered the lead compound of new therapeutic agents endowed with synergistic effects for the treatment of chronic neurodegenerative and cancer diseases.

Macrophage Involvement in Aging-Associated Skeletal Muscle Regeneration.

The skeletal muscle is a dynamic organ composed of contractile muscle fibers, connective tissues, blood vessels and nerve endings. Its main function is to provide motility to the body, but it is also deeply involved in systemic metabolism and thermoregulation. The skeletal muscle frequently encounters microinjury or trauma, which is primarily repaired by the coordinated actions of muscle stem cells (satellite cells, SCs), fibro-adipogenic progenitors (FAPs), and multiple immune cells, particularly macrophages. During aging, however, the capacity of skeletal muscle to repair and regenerate declines, likely contributing to sarcopenia, an age-related condition defined as loss of muscle mass and function. Recent studies have shown that resident macrophages in skeletal muscle are highly heterogeneous, and their phenotypes shift during aging, which may exacerbate skeletal muscle deterioration and inefficient regeneration. In this review, we highlight recent insight into the heterogeneity and functional roles of macrophages in skeletal muscle regeneration, particularly as it declines with aging.

Dexmedetomidine improves DM-induced oxidative stress injury to protect liver function through Nrf2 pathway.

OBJECTIVE: Diabetes mellitus-induced oxidative stress (OS) causes liver injury. Intraoperative pumping of dexmedetomidine (DEX) effectively reduced the postoperative OS response in patients with type 2 diabetes mellitus (T2DM) and had a certain protective effect on liver function. However, the mechanisms of the protective effect on the liver remained unclear. In this study, we investigated the antagonistic effects and the possible mechanism of DEX on T2DM-induced liver injury in the mouse model and Palmitic acid (Pal)-induced injury in hepatocellular carcinoma cells (HepG2). METHODS: Seven wt/wt mice served as Control group, and 28 db/db mice were randomly divided into four groups using a random number table method: Model group (n=7), D25 group (n=7), D50 group (n=7) and D75 group (n=7). Different concentrations of DEX were injected intraperitoneally in the D25 group, D50 group and D75 group, while the Control group and the Model group were intraperitoneally injected with the same amount of normal saline for 3 weeks. In the cell intervention experiments, HepG2 cell line was used. The control group (Con group), the palmitic acid group (Pal group) and the DEX treatment group (Pal + Dex group) were set up. The test results were compared among mice groups and cell groups, respectively. RESULTS: DEX alleviated the increase of alanine aminotransferase, triglyceride, total cholesterol and aspartate aminotransferase contents induced by high fat or T2DM. DEX reversed the decrease of nuclear factor E2 related factor 2 (Nrf2) in the nuclear translocation and the lower transcriptional activity of Nrf2 to inhibit the expression of heme oxygenase-1, NADPH quinone oxidoreductase-1 and superoxide dismutase 2 and reduced the activity of superoxide dismutase to increase reactive oxygen species content induced by high fat or T2DM. CONCLUSION: By attenuating the high-fat or T2DM-induced Nrf2 pathway impairment, DEX can reduce OS injury and inhibit the disorder of lipid anabolism and protect liver function. This study provides a theoretical basis for the protection of liver function by DEX in clinical T2DM patients.

Study on pharmacokinetic interactions between SHR2554 and itraconazole in healthy subjects: A single-center, open-label phase I trial.

BACKGROUND: SHR2554, a novel oral Enhancer of Zeste Homolog 2 inhibitor, shows broad-spectrum anti-tumor efficacy in preclinical studies. As SHR2554 is mainly metabolized by CYP3A4, it is helpful to conduct research on the effects of itraconazole, a strong inhibitor of CYP3A4-metabolizing enzymes, on the pharmacokinetic characteristics and safety of SHR2554. METHODS: We conducted a single-center, open-label pharmacokinetic study of itraconazole on SHR2554 in 18 healthy Chinese subjects. Subjects were orally administrated SHR2554 50 mg on Day 1, itraconazole 200 mg Quaque Die (QD) from Days 4 to 7, SHR2554 50 mg co-administrated with itraconazole 200 mg on Day 8, and itraconazole 200 mg QD from Days 9 to 12. Then, 4 ml of venous blood was collected at predetermined time points. Plasma SHR2554 concentrations were analyzed using a validated high-performance liquid chromatography tandem mass spectrometry method. Pharmacokinetic parameters were calculated using Phoenix WinNonlin v8.1. RESULTS: The Cmax of SHR2554 alone and in combination was 10.197 ± 7.0262 ng·ml-1 versus 70.538 ± 25.0219 ng·ml-1 , AUC0-∞ was 50.99 ± 19.358 h·ng·ml-1 versus 641.53 ± 319.538 h·ng·ml-1 , and AUC0-t was 28.70 ± 18.913 h·ng·ml-1 versus 612.13 ± 315.720 h·ng·ml-1 . Co-administration of SHR2554 and itraconazole caused 7.73-, 12.47-, and 23.75-fold adjusted geometric mean ratios increases in SHR2554 Cmax , AUC0-∞ and AUC0-t respectively. The co-administration regimen was well tolerated and had a good safety profile. CONCLUSIONS: Compared with a single dose of SHR2554 50 mg, the exposure of SHR2554 in vivo was significantly affected by the combined administration of itraconazole.

Single-cell analysis of skeletal muscle macrophages reveals age-associated functional subpopulations.

Tissue-resident macrophages represent a group of highly responsive innate immune cells that acquire diverse functions by polarizing toward distinct subpopulations. The subpopulations of macrophages that reside in skeletal muscle (SKM) and their changes during aging are poorly characterized. By single-cell transcriptomic analysis with unsupervised clustering, we found 11 distinct macrophage clusters in male mouse SKM with enriched gene expression programs linked to reparative, proinflammatory, phagocytic, proliferative, and senescence-associated functions. Using a complementary classification, membrane markers LYVE1 and MHCII identified four macrophage subgroups: LYVE1-/MHCIIhi (M1-like, classically activated), LYVE1+/MHCIIlo (M2-like, alternatively activated), and two new subgroups, LYVE1+/MHCIIhi and LYVE1-/MHCIIlo. Notably, one new subgroup, LYVE1+/MHCIIhi, had traits of both M2 and M1 macrophages, while the other new subgroup, LYVE1-/MHCIIlo, displayed strong phagocytic capacity. Flow cytometric analysis validated the presence of the four macrophage subgroups in SKM and found that LYVE1- macrophages were more abundant than LYVE1+ macrophages in old SKM. A striking increase in proinflammatory markers (S100a8 and S100a9 mRNAs) and senescence-related markers (Gpnmb and Spp1 mRNAs) was evident in macrophage clusters from older mice. In sum, we have identified dynamically polarized SKM macrophages and propose that specific macrophage subpopulations contribute to the proinflammatory and senescent traits of old SKM.

Novel Split Intein-Mediated Enzymatic Channeling Accelerates the Multimeric Bioconversion Pathway of Ginsenoside.

Cascade reaction systems, such as protein fusion and synthetic protein scaffold systems, can both spatially control the metabolic flux and boost the productivity of multistep enzymatic reactions. Despite many efforts to generate fusion proteins, this task remains challenging due to the limited expression of complex enzymes. Therefore, we developed a novel fusion system that bypasses the limited expression of complex enzymes via a post-translational linkage. Here, we report a split intein-mediated cascade system wherein orthogonal split inteins serve as adapters for protein ligation. A genetically programmable, self-assembled, and traceless split intein was utilized to generate a biocatalytic cascade to produce the ginsenoside compound K (CK) with various pharmacological activities, including anticarcinogenic, anti-inflammatory, and antidiabetic effects. We used two types of split inteins, consensus atypical (Cat) and Rma DnaB, to form a covalent scaffold with the three enzymes involved in the CK conversion pathway. The multienzymatic complex with a size greater than 240 kDa was successfully assembled in a soluble form and exhibited specific activity toward ginsenoside conversion. Furthermore, our split intein cascade system significantly increased the CK conversion rate and reduced the production time by more than 2-fold. Our multienzymatic cascade system that uses split inteins can be utilized as a platform for regulating multimeric bioconversion pathways and boosting the production of various high-value substances.

A randomized, double-blind, parallel control study to evaluate the biosimilarity of QL1209 with Perjeta® in healthy male subjects.

Purpose: This is the first study to compare the pharmacokinetics, safety and, immunogenicity of QL1209, a biosimilar of Perjeta®. Methods: This study was a randomized, double-blind, parallel-controlled clinical trial evaluating the biosimilarity between QL1209 (specification: 420 mg:14 ml, single use via, manufacturer: Qilu Pharmaceutical Co., Ltd., batch number: 201808001KJL) and Perjeta® (specification: 420 mg: 14 ml, single use via, manufacturer: Roche Pharma AG, batch number: H0309H02). The trial period was 99 days (blood samples for PK were collected 99 days after infusion). Serum concentrations were determined using a validated assay. PK parameters were calculated using a non-compartmental model and analyzed statistically. Anti-drug antibody (ADA)-positive samples were further tested for the presence of neutralization antibody detection (NAb). Results: A total of 137 healthy subjects were administrated. The subjects were randomized 1:1 to receive QL1209 or Perjeta® 420 mg intravenously. The geometric mean ratio (GMRs) for QL1209 versus Perjeta® are 104.14%, 104.09%, and 110.59% for Cmax, AUC0-t, and AUC0-∞, respectively, and their 90% confidence interval (CIs) all fell within the predefined bioequivalence margin 80.00-125%. The incidence of drug-related adverse events was 95.6% and 95.5% in the QL1209 and Perjeta® groups, respectively, also comparable between the two groups. Conclusion: The results of this comparative clinical pharmacology study demonstrated the PK similarity of QL1209 (420 mg: 14 ml) and Perjeta® (420 mg: 14 ml) and there was no significant difference in safety and immunogenicity between QL1209 and Perjeta® manufactured by Roche Pharma AG.

Progesterone Reduces ATP-Induced Pyroptosis of SH-SY5Y Cells.

Aim: To investigate the mechanism of progesterone inhibiting the scorch death of SH-SY5Y cells induced by exogenous adenosine triphosphate (ATP). Methods: SH-SY5Y cells with good logarithmic growth were used in the experiment. The cells were randomly divided into 5 groups: normal control group, DMSO group, BBG group, ATP group, and ATP+progesterone group. The cell survival rate of each group was measured by CCK-8 method. The expressions of P2X7 receptor, caspase-1, caspase-11, and IL-1ß were detected by western blotting. Results: (1) After SH-SY5Y cells were treated with ATP at different concentrations (1, 3, 6, and 9 mmol/L) for 2 hours, the cell survival rate decreased in a concentration-dependent manner compared with the normal blank group. The results showed that the optimal lethal concentration of ATP was 6 mmol/L. SH-SY5Y cells were preincubated with progesterone at different concentrations (3, 10, 30, and 100 nmol/L) for 30 minutes and then incubated with 6 mmol/L ATP. The cell survival rate of this group was significantly improved (P < 0.01). The optimal concentration of progesterone to improve cell survival and inhibit cell death was 30 nmol/L. (2) Compared to the control group, there was no significant difference (P > 0.05) in P2X7 receptor, caspase-1, caspase-11, and IL-1ß with the DMSO group (0.001% DMSO, 24 h) and BBG group (bbg1 mmol/L, 24 h). (3) In the ATP group, the expression of P2X7 receptor and caspase-1 (the key protein of classical cell death pathway) increased significantly (P < 0.01), which was related to inflammatory factor IL-1ß with consistent performance (P < 0.01). There was no significant change in caspase-11 (the key protein of nonclassical focal death pathway) (P > 0.05). (4) The expression of P2X7 receptor, caspase-1, and inflammatory factor IL-1ß in the progesterone+ATP group was significantly downregulated (P < 0.01). There was no significant change in caspase-11 (P > 0.05). Conclusion: Certain dose of progesterone can inhibit the focal death of SH-SY5Y cells induced by extracellular high concentration ATP. It can reduce the expression of P2X7 receptor, inhibit the conduction pathway of cell death, reduce the release of inflammatory factor IL-1ß, and improve cell survival.

Maternal supplementation with Artemisia annua L. ameliorates intestinal inflammation via inhibiting the TLR4/NF-κB and MAPK pathways and improves the oxidative stability of offspring.

Artemisia annua L. (A. annua) contains artemisinin, which attracts attention on account of its anti-inflammatory and anti-oxidant effects. Increased intestinal inflammation, oxidative stress, and hypoimmunity commonly occur in neonatal and early-weaning piglets. Abundant evidence suggests that maternal nutritional interventions during pregnancy modify the offspring's long-term gut development. The present study was conducted to investigate the effects of maternal A. annua extract (AAE) supplementation on the offspring's intestinal inflammation and redox status. A total of 90 pregnant sows were assigned randomly and equally into the control (CON) group (fed with a basal diet) and the 0.1% (AAE) group (basal diet with 1.0 g kg-1 AAE) during late gestation and lactation. The results showed that 0.1% AAE supplementation significantly decreased the contents and relative mRNA expressions of interleukin (IL)-1ß, IL-6, and IL-12, and tumor necrosis factor-α in the small intestine of the newborn and weaned piglets (offspring) (P < 0.05). There were higher activities of total antioxidant capacity and total superoxide dismutase, whereas a lower concentration of malondialdehyde in the small instestine of offspring in the 0.1% AAE group than that in the CON group (P < 0.05). Furthermore, the 0.1% AAE group decreased the mRNA and protein expressions of Toll-like receptor 4 (TLR4) and inhibited the activation of TLR4-mediated nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways (P < 0.05). The mRNA expression of peroxisome proliferator activated receptor γ (PPARγ), porcine beta-defensin (PBD)-1, PBD-2, PBD-3, mucin (MUC)-1, MUC-2 and MUC-4 was significantly enhanced in the small intestine of both neonatal and weanling piglets (P < 0.05). Together, these results showed that maternal 0.1% AAE supplementation improved the redox status and attenuated the neonatal and early-weaning associated inflammatory response in the offspring's small intestine, possibly by suppressing the activation of the TLR4/NF-κB and MAPK inflammatory pathways, and stimulated expressions of beta-defensins, mucins, and PPARγ to promote inflammation resolution and innate immunity response.

Febuxostat Increases Ventricular Arrhythmogenesis Through Calcium Handling Dysregulation in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Febuxostat is a xanthine oxidase inhibitor used to reduce the formation of uric acid and prevent gout attacks. Previous studies have suggested that febuxostat was associated with a higher risk of cardiovascular events, including atrial fibrillation, compared with allopurinol, another anti-hyperuricemia drug. Whereas in our clinical practice, we identified 2 cases of febuxostat-associated ventricular tachycardia (VT) events. The proarrhythmogenic effects of febuxostat on human cardiomyocytes and underlined mechanisms remain poorly understood. In this study, we employed real-time cell analysis and calcium transient to investigate the effects of febuxostat on the cytotoxicity and electrophysiology properties of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Up to 10 µM febuxostat treatment did not show toxicity to cell viability. However, 48-h febuxostat exposure generated dose-dependent increased irregular calcium transients and decreased calcium transient amplitude. Furthermore, RNA-seq analysis indicated that the MAPK signaling pathway was enriched in the febuxostat-treated group, especially the protein kinases c-Jun N-terminal kinase (JNK). Western blotting of 3 main protein kinases demonstrated that JNK activation is related to febuxostat-induced arrhythmia rather than extracellular signal regulated kinases (ERK) or p38. The dysfunctional calcium dynamics of febuxostat-treated hiPSC-CMs could be ameliorated by SP600125, the inhibitor of JNK. In conclusion, our study demonstrated that febuxostat increases the predisposition to ventricular arrhythmia by dysregulating calcium dynamics.

Ah-type Baroreceptor Neurons Expressing Estrogen Dependent mGluR7 Mediate Descending Inhibition of Cardiac Nociception.

Silent myocardial infarction (MI) is critical for clinical practice with increasing risk for women and the cause remains a medical mystery. Upon the discovery of female-specific Ah-type baroreceptor neurons (BRNs), we hypothesize that glutamate mediates depressor response through afferent-specific expression of particular glutamate receptors (mGluRs) leading descending inhibition of cardiac nociception. In vivo, tail-flick reflex and electromyography were assessed to evaluate glutamate-mediated blood pressure regulation, peripheral and cardiac nociception. The results showed that glutamate decreased mean arterial pressure (MAP) and increased peripheral nociception. Interestingly, glutamate-mediated capsaicin-induced cardiac nociception was strongly reduced in female rats compared with males. Furthermore, Nodose (NG) microinjection of mGluR7 agonist significantly increased MAP in males and slightly decreased that in females. Even though mGluR8 direct activation intensified baroreceptor activation, the sensitivity was similar between sexes. In vitro, the expression profiles of mGluRs were investigated using Western blot and identified BRNs using single-cell qRT-PCR under ischemic conditions. Glutamate in serum, NG and nucleus tractus solitary (NTS) was raised significantly in the model rats of both sexes vs. sham-controls. Female-specific expression of mGluR7 in the baroreflex afferent pathway, especially higher expression in Ah-type BRNs, contributes significantly to cardiac analgesia, which may explain that the pathogenesis of silent MI occurs mainly in female patients. Therefore, higher expression of mGluR7 in female-specific subpopulation of Ah-type BRNs plays a critical role in cardiac analgesia and peripheral nociception.

Atrial Lesions in a Pedigree With PRKAG2 Cardiomyopathy: Involvement of Disrupted AMP-Activated Protein Kinase Signaling.

PRKAG2 cardiomyopathy is a rare progressive disease characterized by increased ventricular wall thickness and preexcitation. Dysfunction of the protein 5'-AMP-activated protein kinase (AMPK) plays a decisive role in the progression of ventricular lesions. Although patients with the PRKAG2-R302Q mutation have a high incidence of atrial fibrillation (AF), the molecular mechanism contributing to the disease remains unclear. We carried out whole-genome sequencing with linkage analysis in three affected members of a family. Atrial samples were obtained from the proband via surgical intervention. Control atrium biopsies were obtained from patients with persistent AF. Pathological changes were analyzed using the hematoxylin and eosin (H&E), Masson, and periodic acid-Schiff (PAS) staining. The AMPK signaling pathway was investigated by western blot. A murine atrial cardiomyocyte cell line (HL-1) and human induced pluripotent stem derived atrial cardiomyocytes (hiPSC-ACMs) were transfected with an adenovirus carrying the same mutation. We used enzyme linked immunosorbent assay (ELISA) to determine the AMPK activity in HL-1 cells and hiPSC-ACMs overexpressing PRKAG2-R302Q. Pathological results showed a large quantity of glycogen accumulation and vacuolization in cardiomyocytes from the proband atrial tissue. Western blot analysis revealed that the AMPK activity was significantly downregulated compared with that of the controls. Furthermore, remarkable glycogen deposition and impairment of AMPK activity were reproduced in HL-1 cells overexpressing PRKAG2-R302Q. Taken together, PRKAG2-R302Q mutation directly impair atrial cardiomyocytes. PRKAG2-R302Q mutation lead to glycogen deposition and promote the growth of atrial lesions by disrupting the AMPK pathway.

Early SRC activation skews cell fate from apoptosis to senescence.

Cells responding to DNA damage implement complex adaptive programs that often culminate in one of two distinct outcomes: apoptosis or senescence. To systematically identify factors driving each response, we analyzed human IMR-90 fibroblasts exposed to increasing doses of the genotoxin etoposide and identified SRC as a key kinase contributing early to this dichotomous decision. SRC was activated by low but not high levels of etoposide. With low DNA damage, SRC-mediated activation of p38 critically promoted expression of cell survival and senescence proteins, while SRC-mediated repression of p53 prevented a rise in proapoptotic proteins. With high DNA damage, failure to activate SRC led to elevation of p53, inhibition of p38, and apoptosis. In mice exposed to DNA damage, pharmacologic inhibition of SRC prevented the accumulation of senescent cells in tissues. We propose that inhibiting SRC could be exploited to favor apoptosis over senescence in tissues to improve health outcomes.

Estrogen-dependent depressor response of melatonin via baroreflex afferent function and intensification of PKC-mediated Nav1.9 activation.

Recent studies suggest that melatonin (Mel) plays an important role in the regulation of blood pressure (BP) via the aortic baroreflex pathway. In this study, we investigated the interaction between the baroreflex afferent pathway and Mel-mediated BP regulation in rats under physiological and hypertensive conditions. Mel (0.1, 0.3, and 1.0 mg/mL) was microinjected into the nodose ganglia (NG) of rats. We showed that Mel-induced reduction of mean arterial pressure in female rats was significantly greater than that in male and in ovariectomized rats under physiological condition. Consistently, the expression of Mel receptors (MTNRs) in the NG of female rats was significantly higher than that of males. In L-NAME-induced hypertensive and spontaneously hypertensive rat models, MTNRs were upregulated in males but downregulated in female models. Interestingly, Mel-induced BP reduction was found in male hypertensive models. In whole-cell recording from identified baroreceptor neurons (BRNs) in female rats, we found that Mel (0.1 µM) significantly increased the excitability of a female-specific subpopulation of Ah-type BRNs by increasing the Nav1.9 current density via a PKC-mediated pathway. Similar results were observed in baroreceptive neurons of the nucleus tractus solitarius, showing the facilitation of spontaneous and evoked excitatory post-synaptic currents in Ah-type neurons. Collectively, this study reveals the estrogen-dependent effect of Mel/MTNRs under physiological and hypertensive conditions is mainly mediated by Ah-type BRNs, which may provide new theoretical basis and strategies for the gender-specific anti-hypertensive treatment in clinical practice.

Leucine supplementation during late gestation globally alters placental metabolism and nutrient transport via modulation of the PI3K/AKT/mTOR signaling pathway in sows.

In a previously published study we reported that sow dietary leucine supplementation during late pregnancy significantly improved newborn piglet birth weight by stimulating protein synthesis in the longissimus dorsi muscle. However, there is still limited knowledge as to whether leucine can exert its effects on the placenta, one of the most important temporal organs during pregnancy, to promote maternal-fetal nutrient supply and thus contribute to fetal intrauterine development. Therefore, we tested this hypothesis in the present study. In total, 150 sows at day 90 of gestation were divided into three groups and fed with either a control diet (CON), CON + 0.4% Leu or CON + 0.8% Leu, respectively, until parturition. Placental metabolomics, full spectrum amino acids and nutrient transporters were systematically analyzed after sample collection. The results indicated that Leu supplementation led to an altered placental metabolism with an increased number of metabolites related to glycolysis and the oxidation of fatty acids, as well as elevated levels of amino acid accumulation in the placenta. In addition, nutrient transporters of amino acids, glucose and fatty acids in the placenta were globally up-regulated and several enzymes related to energy metabolism, including hexokinase, succinate dehydrogenase, lactated hydrogenase, glycogen phosphorylase and hydroxyacyl-CoA-dehydrogenase, were also significantly increased with no change observed in the antioxidative status of those groups with Leu supplementation. Furthermore, the phosphorylation of PI3K, Akt, and mTOR was enhanced in the placenta of sows undergoing Leu treatment. Collectively, we concluded that supplementing the diets of sows with Leu during late gestation globally altered placental metabolism and promoted maternal-fetus nutrient transport (amino acids, glucose, and fatty acids) via modulation of the PI3K/Akt/mTOR signaling pathway.