MRP1-Dependent Extracellular Release of Glutathione Induces Cardiomyocyte Ferroptosis After Ischemia-Reperfusion.

BACKGROUND: The membrane components of cardiomyocytes are rich in polyunsaturated fatty acids, which are easily oxidized. Thus, an efficient glutathione-based lipid redox system is essential for maintaining cellular functions. However, the relationship between disruption of the redox system during ischemia-reperfusion (IR), oxidized lipid production, and consequent cell death (ferroptosis) remains unclear. We investigated the mechanisms underlying the disruption of the glutathione-mediated reduction system related to ferroptosis during IR and developed intervention strategies to suppress ferroptosis. METHODS: In vivo fluctuations of both intra- and extracellular metabolite levels during IR were explored via microdialysis and tissue metabolome analysis. Oxidized phosphatidylcholines were assessed using liquid chromatography high-resolution mass spectrometry. The areas at risk following IR were assessed using triphenyl-tetrazolium chloride/Evans blue stain. RESULTS: Metabolomic analysis combined with microdialysis revealed a significant release of glutathione from the ischemic region into extracellular spaces during ischemia and after reperfusion. The release of glutathione into extracellular spaces and a concomitant decrease in intracellular glutathione concentrations were also observed during anoxia-reperfusion in an in vitro cardiomyocyte model. This extracellular glutathione release was prevented by chemical inhibition or genetic suppression of glutathione transporters, mainly MRP1 (multidrug resistance protein 1). Treatment with MRP1 inhibitor reduced the intracellular reactive oxygen species levels and lipid peroxidation, thereby inhibiting cell death. Subsequent in vivo evaluation of endogenously oxidized phospholipids following IR demonstrated the involvement of ferroptosis, as levels of multiple oxidized phosphatidylcholines were significantly elevated in the ischemic region 12 hours after reperfusion. Inhibition of the MRP1 transporter also alleviated intracellular glutathione depletion in vivo and significantly reduced the generation of oxidized phosphatidylcholines. Administration of MRP1 inhibitors significantly attenuated infarct size after IR injury. CONCLUSIONS: Glutathione was released continuously during IR, primarily in an MRP1-dependent manner, and induced ferroptosis. Suppression of glutathione release attenuated ferroptosis and reduced myocardial infarct size following IR.

Multinational Federated Learning Approach to Train ECG and Echocardiogram Models for Hypertrophic Cardiomyopathy Detection.

BACKGROUND: Novel targeted treatments increase the need for prompt hypertrophic cardiomyopathy (HCM) detection. However, its low prevalence (0.5%) and resemblance to common diseases present challenges that may benefit from automated machine learning-based approaches. We aimed to develop machine learning models to detect HCM and to differentiate it from other cardiac conditions using ECGs and echocardiograms, with robust generalizability across multiple cohorts. METHODS: Single-institution HCM ECG models were trained and validated on external data. Multi-institution models for ECG and echocardiogram were trained on data from 3 academic medical centers in the United States and Japan using a federated learning approach, which enables training on distributed data without data sharing. Models were validated on held-out test sets for each institution and from a fourth academic medical center and were further evaluated for discrimination of HCM from aortic stenosis, hypertension, and cardiac amyloidosis. Last, automated detection was compared with manual interpretation by 3 cardiologists on a data set with a realistic HCM prevalence. RESULTS: We identified 74 376 ECGs for 56 129 patients and 8392 echocardiograms for 6825 patients at the 4 academic medical centers. Although ECG models trained on data from each institution displayed excellent discrimination of HCM on internal test data (C statistics, 0.88-0.93), the generalizability was limited, most notably for a model trained in Japan and tested in the United States (C statistic, 0.79-0.82). When trained in a federated manner, discrimination of HCM was excellent across all institutions (C statistics, 0.90-0.96 and 0.90-0.96 for ECG and echocardiogram model, respectively), including for phenotypic subgroups. The models further discriminated HCM from hypertension, aortic stenosis, and cardiac amyloidosis (C statistics, 0.84, 0.83, and 0.88, respectively, for ECG and 0.93, 0.94, 0.85, respectively, for echocardiogram). Analysis of electrocardiography-echocardiography paired data from 11 823 patients from an external institution indicated a higher sensitivity of automated HCM detection at a given positive predictive value compared with cardiologists (0.98 versus 0.81 at a positive predictive value of 0.01 for ECG and 0.78 versus 0.59 at a positive predictive value of 0.24 for echocardiogram). CONCLUSIONS: Federated learning improved the generalizability of models that use ECGs and echocardiograms to detect and differentiate HCM from other causes of hypertrophy compared with training within a single institution.

Improved prediction of sudden cardiac death in patients with heart failure through digital processing of electrocardiography.

AIMS: Available predictive models for sudden cardiac death (SCD) in heart failure (HF) patients remain suboptimal. We assessed whether the electrocardiography (ECG)-based artificial intelligence (AI) could better predict SCD, and also whether the combination of the ECG-AI index and conventional predictors of SCD would improve the SCD stratification among HF patients. METHODS AND RESULTS: In a prospective observational study, 4 tertiary care hospitals in Tokyo enrolled 2559 patients hospitalized for HF who were successfully discharged after acute decompensation. The ECG data during the index hospitalization were extracted from the hospitals' electronic medical record systems. The association of the ECG-AI index and SCD was evaluated with adjustment for left ventricular ejection fraction (LVEF), New York Heart Association (NYHA) class, and competing risk of non-SCD. The ECG-AI index plus classical predictive guidelines (i.e. LVEF ≤35%, NYHA Class II and III) significantly improved the discriminative value of SCD [receiver operating characteristic area under the curve (ROC-AUC), 0.66 vs. 0.59; P = 0.017; Delong's test] with good calibration (P = 0.11; Hosmer-Lemeshow test) and improved net reclassification [36%; 95% confidence interval (CI), 9-64%; P = 0.009]. The Fine-Gray model considering the competing risk of non-SCD demonstrated that the ECG-AI index was independently associated with SCD (adjusted sub-distributional hazard ratio, 1.25; 95% CI, 1.04-1.49; P = 0.015). An increased proportional risk of SCD vs. non-SCD with an increasing ECG-AI index was also observed (low, 16.7%; intermediate, 18.5%; high, 28.7%; P for trend = 0.023). Similar findings were observed in patients aged ≤75 years with a non-ischaemic aetiology and an LVEF of >35%. CONCLUSION: To improve risk stratification of SCD, ECG-based AI may provide additional values in the management of patients with HF.

Prevalence and associated factors of psychological distress among young adult cancer patients in Japan.

OBJECTIVES: Adolescent and young adult (AYA) cancer patients may be at high risk of experiencing psychological distress because their diagnosis came during a key time of consolidation of identity and social growth. This study aimed (1) to examine the prevalence of psychological distress among AYA cancer patients within a year of diagnosis to long-term survivors and (2) to describe socio-demographic and cancer-related characteristics associated with psychological distress. METHODS: In a cross-sectional web-based survey, patients who scored 5 or more on the Kessler Psychological Distress Scale were assessed for significant psychological distress. Logistic regression examined whether demographics, clinical variables, and social support were associated with psychological distress. RESULTS: A total of 206 young adult cancer patients participated. The median age at the survey was 34.5 years (range: 22-39 years), and 87.4% were female. The prevalence of psychological distress was 55.3%. Psychological distress among patients diagnosed within a year and long-term survivors (≥10 years since diagnosis) was significantly higher than patients 1-4 years since diagnosis. Pain, decrease in income after a cancer diagnosis, experience of negative change in work/school after a cancer diagnosis and poor social support were significantly associated with psychological distress. SIGNIFICANCE OF RESULTS: Over half of young adult patients had significant psychological distress in Japan. Our findings potentially contribute to the intervention components for distress management among AYA cancer survivors.

Association of mitochondrial DNA haplogroup and hearing impairment with aging in Japanese general population of the Iwaki Health Promotion Project.

Age-related hearing loss (ARHL) is a complex multifactorial disorder. Studies in animals, including mitochondria-mutator mice, and in human suggest that oxidative stress and mitochondrial disturbance play an important role in the pathoetiology of ARHL. Mitochondrial DNA (mtDNA) haplogroups are populations with genetically similar traits, and they have been reported to affect the mitochondrial function of oxidative phosphorylation. To gain further insights into the relationships between mitochondrial haplotypes and the susceptibility to cochlear aging, in this study, we aimed to elucidate how the differences in mtDNA haplogroups may affect ARHL development in Japanese general population. We focused on early onset ARHL, as the same mtDNA haplogroup can show either a negative or positive effect on systemic co-morbidities of ARHL that appear later in life. A total of 1167 participants of the Iwaki Health Promotion Project were surveyed in 2014, and 12 major haplotype groups (D4a, D4b, D5, G1, G2, M7a, M7b, A, B4, B5, N9, and F) were selected for the analysis. A total of 698 subjects aged 30 to 65 years were included in the statistical analysis, and the hearing loss group consisted of 112 males (40.3%) and 111 females (26.4%). Multiple logistic regression analysis showed that the male subjects belonging to haplogroup A had a significantly increased risk of hearing loss, whereas the female subjects belonging to haplogroup N9 had a significantly decreased risk of hearing loss. These results suggested that the mtDNA haplogroup may be an indicator for future risk of morbidity associated with ARHL.

Validation of reliability and predictivity of membrane septum length measurements for pacemaker need after transcatheter aortic valve replacement.

OBJECTIVES: To assess the inter methodological agreement of membrane septum (MS) length measurement and additive value for risk stratification of new pacemaker implantation (PMI) over the established predictors after transcatheter aortic valve replacement (TAVR). BACKGROUND: Recent studies have suggested MS length and implantation depth (ID) as predictors for PMI after TAVR. However, the measurement of MS length is neither uniform nor validated in different cohort. METHODS: We retrospectively analyzed patients who underwent TAVR at five centers. The MS length was measured by two previously proposed methods (coronal and annular view method). Predictive ability of risk factors, including MS length and ID, for new PMI within 30 days after TAVR were evaluated. RESULTS: Among 754 patients of study population, 31 patients (4.1%) required new PMI within 30 days of TAVR. There was a weak correlation (ρ = 0.47) and a poor agreement between the two methods. The ID and the difference between MS length and ID (ΔMSID), were independent predictors for new PMI, whereas MS length alone was not. Further, for predicting new PMI after TAVR, discrimination performance was not significantly improved when MS length was added to the model with ID alone (integrated discrimination improvement = 0, p= 0.99; continuous net-reclassification improvement = 0.10, p= 0.62). CONCLUSIONS: External validity and predictive accuracy of MS length for PMI after TAVR were not sufficient to provide better risk stratification over the established predictors in our cohort. Moreover, the ID and ΔMSID, but not MS length alone, are predictive of future PMI after TAVR.

Contributing role of mitochondrial energy metabolism on platelet adhesion, activation and thrombus formation under blood flow conditions.

Platelets have an active energy metabolism mediated by mitochondria. However, the role of mitochondria in platelet adhesion, activation, and thrombus formation under blood flow conditions remains to be elucidated. Blood specimens were obtained from healthy adult volunteers. The consumption of glucose molecules by platelets was measured after 24 hours. Platelet adhesion, activation, and thrombus formation on collagen fibrils and immobilized von Willebrand factor (VWF) at a wall shear rate of 1,500 s-1 were detected by fluorescence microscopy with an ultrafast laser confocal unit in the presence or absence of mitochondrial functional inhibitors of carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), antimycin A, and oligomycin. Consumption of glucose molecules within the first 24 h of 4.21 × 10-15 ± 4.46 x 10-15 (n = 6) increased to 13.82 × 10-15 ± 3.46 x 10-15 (n = 4) in the presence of FCCP, 12.11 × 10-15 ± 2.33 x 10-15 (n = 4) in the presence of antimycin A, and 11.87 × 10-15 ± 3.56 x 10-15 (n = 4) in the presence of oligomycin (p < .05). These mitochondrial functional blockers did not influence both surface area coverage by platelets and the 3-dimensional size of platelet thrombi formed on the collagen fibrils. However, a rapid increase in the intracellular calcium ion concentration ([Ca2+]i) upon adhering on immobilized VWF decreased significantly from 405.5 ± 86.2 nM in control to 198.0 ± 79.2 nM in the presence of FCCP (p < .005). A similar decrease in the rapid increase in ([Ca2+]i) was observed in the presence of antimycin A and oligomycin. Mitochondrial function is necessary for platelet activation represented by a rapid increase in [Ca2+]i after platelet adhesion on VWF. However, the influence could not be detected as changes in platelet adhesion or 3-dimensional growth of platelet thrombi on collagen fibrils.

MITOL/MARCH5 determines the susceptibility of cardiomyocytes to doxorubicin-induced ferroptosis by regulating GSH homeostasis.

MITOL/MARCH5 is an E3 ubiquitin ligase that plays a crucial role in the control of mitochondrial quality and function. However, the significance of MITOL in cardiomyocytes under physiological and pathological conditions remains unclear. First, to determine the significance of MITOL in unstressed hearts, we assessed the cellular changes with the reduction of MITOL expression by siRNA in neonatal rat primary ventricular cardiomyocytes (NRVMs). MITOL knockdown in NRVMs induced cell death via ferroptosis, a newly defined non-apoptotic programmed cell death, even under no stress conditions. This phenomenon was observed only in NRVMs, not in other cell types. MITOL knockdown markedly reduced mitochondria-localized GPX4, a key enzyme associated with ferroptosis, promoting accumulation of lipid peroxides in mitochondria. In contrast, the activation of GPX4 in MITOL knockdown cells suppressed lipid peroxidation and cell death. MITOL knockdown reduced the glutathione/oxidized glutathione (GSH/GSSG) ratio that regulated GPX4 expression. Indeed, the administration of GSH or N-acetylcysteine improved the expression of GPX4 and viability in MITOL-knockdown NRVMs. MITOL-knockdown increased the expression of the glutathione-degrading enzyme, ChaC glutathione-specific γ-glutamylcyclotransferase 1 (Chac1). The knockdown of Chac1 restored the GSH/GSSG ratio, GPX4 expression, and viability in MITOL-knockdown NRVMs. Further, in cultured cardiomyocytes stressed with DOX, both MITOL and GPX4 were reduced, whereas forced-expression of MITOL suppressed DOX-induced ferroptosis by maintaining GPX4 content. Additionally, MITOL knockdown worsened vulnerability to DOX, which was almost completely rescued by treatment with ferrostatin-1, a ferroptosis inhibitor. In vivo, cardiac-specific depletion of MITOL did not produce obvious abnormality, but enhanced susceptibility to DOX toxicity. Finally, administration of ferrostatin-1 suppressed exacerbation of DOX-induced myocardial damage in MITOL-knockout hearts. The present study demonstrates that MITOL determines the cell fate of cardiomyocytes via the ferroptosis process and plays a key role in regulating vulnerability to DOX treatment. (288/300).

Imeglimin prevents heart failure with preserved ejection fraction by recovering the impaired unfolded protein response in mice subjected to cardiometabolic stress.

The pathogenesis of heart failure with preserved ejection fraction (HFpEF) in obese diabetic patients has been implicated in metainflammation. Increased expression of inducible nitric oxide synthase (iNOS) and dysfunction of the unfolded protein response (UPR), especially inositol-requiring enzyme 1α-X-box binding protein 1 (IRE1α-Xbp1s) signaling in the heart, have been associated with HFpEF. We investigated the effect of imeglimin, a potential new treatment for type 2 diabetes, on the pathogenesis of HFpEF. We induced obesity, impaired glucose tolerance, and cardiac hypertrophy with fibrosis, fat accumulation, and diastolic dysfunction in wild-type mice with a high-fat diet (HFD) and the nitric oxide synthase (NOS) inhibitor l-NAME for 16 weeks. Treatment with imeglimin starting at 10 weeks not only improved their abnormal systemic glucose metabolism and visceral obesity but also their cardiac abnormalities. We found that imeglimin suppressed the upregulation of iNOS, and restored the expression of Xbp1s and the expression of the E3 ubiquitin ligase STIP1 homology and U-box-containing protein 1 (STUB1), which is responsible for the degradation of Forkhead box protein O1 (FoxO1), a direct transcriptional target of Xbp1s. It also suppressed the excessive transcriptional activity of FoxO1, which is located downstream of Xbp1s and is involved in the form development of HFpEF and cardiac adipogenesis. Imeglimin also restored the expression of Glutathione peroxidase 4 (GPX4), which protects cells against excess lipid peroxidation and governs a novel form of programmed cell death, called ferroptosis.

Computer Simulation of Platelet Adhesion around Stent Struts in the Presence and Absence of Tissue Defects around Them.

AIM: To predict platelet accumulation around stent struts in the presence or absence of tissue defects around them. METHODS: Computer simulations were performed using virtual platelets implementing the function of the three membrane proteins: glycoprotein (GP) Ibα, GPIIb/IIIa, and GPVI. These platelets were perfused around the stent struts implanted into the vessel wall in the presence or absence of tissue defects around them using within the simulation platform. The number of platelets that adhered around stent struts was calculated by solving the blood flow using Navier-Stokes equation along with the adhesion of membrane protein modeled within the platform. RESULTS: Platelet accumulation around stent struts occurred mostly at the downstream region of the stent strut array. The majority of platelets adhered at the downstream of the first bend regardless of the tissue defect status. Platelet adhesion around stent struts occurred more rapidly in the presence of tissue defects. CONCLUSION: Computer simulation using virtual platelets suggested a higher rate of platelet adhesion in the presence of tissue defects around stent struts.

Differences in subtrochanteric and diaphyseal atypical femoral fractures in a super-aging prefectural area: YamaCAFe Study.

INTRODUCTION: Atypical femoral fractures (AFFs) have been correlated with long-term use of bisphosphonates (BPs), glucocorticoids (GCs), and femoral geometry. We investigated the incidence and characteristics of subtrochanteric (ST) and diaphyseal (DP) AFFs in all institutes in a super-aging prefectural area. MATERIALS AND METHODS: We performed a blinded analysis of radiographic data in 87 patients with 98 AFFs in all institutes in Yamagata prefectural area from 2009 to 2014. Among the 98 AFFs, 57 AFFs comprising 11 ST fractures in 9 patients and 46 DP fractures in 41 patients with adequate medical records and X-rays were surveyed for time to bone healing and geometry. RESULTS: Of the 87 patients, 67 received BPs/denosumab (77%) and 10 received GCs (11%). Surgery was performed in 94 AFFs. Among 4 AFFs with conservative therapy, 3 required additional surgery. In univariate regression analyses for ST group versus DP group, male-to-female ratio was 2/7 versus 1/40, mean age at fracture was 58.2 (37-75) versus 78 (60-89) years, rheumatic diseases affected 55.5% (5/9) versus 4.9% (2/41), femoral lateral bowing angle was 1.7 (0-6) versus 11.8 (0.8-24)°, GC usage was 67% (6/9) versus 4.9% (2/41), and bone healing time was 12.1 (6-20) versus 8.1 (3-38) months (p < 0.05). In multivariate analyses, higher male-to-female ratio, younger age, greater proportion affected by rheumatic diseases, and higher GC usage remained significant (p < 0.05). CONCLUSIONS: The incidence of AFFs in our prefectural area was 1.43 cases/100,000 persons/year. This study suggests that the onset of ST AFFs have greater correlation with the worse bone quality, vice versa, the onset of DP AFFs correlated with the bone geometry. The developmental mechanisms of AFFs may differ significantly between ST and DP fractures.

Time-Series Transcriptome Analysis Reveals the miR-27a-5p-Ppm1l Axis as a New Pathway Regulating Macrophage Alternative Polarization After Myocardial Infarction.

BACKGROUND: Timely differentiation of monocytes into M2-like macrophages is important in the cardiac healing process after myocardial infarction (MI), but molecular mechanisms governing M2-like macrophage differentiation at the transcriptional level after MI have not been fully understood.Methods and Results:A time-series microarray analysis of mRNAs and microRNAs in macrophages isolated from the infarcted myocardium was performed to identify the microRNAs involved in regulating the process of differentiation to M2-like macrophages. Correlation analysis revealed 7 microRNAs showing negative correlations with the progression of polarity changes towards M2-like subsets. Next, correlation coefficients for the changes in expression of mRNAs and miRNAs over time were calculated for all combinations. As a result, miR-27a-5p was extracted as a possible regulator of the largest number of genes in the pathway for the M2-like polarization. By selecting mouse mRNAs and human mRNAs possessing target sequences of miR-27a-5p and showing expression patterns inversely correlated with that of miR-27a-5p, 8 potential targets of miR-27a-5p were identified, includingPpm1l. Using the mouse bone marrow-derived macrophages undergoing differentiation into M2-like subsets by interleukin 4 stimulation, we confirmed that miR-27a-5p suppressed M2-related genes by negatively regulatingPpm1lexpression. CONCLUSIONS: Ppm1land miR-27a-5p may be the key molecules regulating M2-like polarization, with miR-27a-5p inhibiting the M2-like polarization through downregulation ofPpm1lexpression.

Palmitate induces cardiomyocyte death via inositol requiring enzyme-1 (IRE1)-mediated signaling independent of X-box binding protein 1 (XBP1).

Overloading of the saturated fatty acid (SFA) palmitate induces cardiomyocyte death. The purpose of this study is to elucidate signaling pathways contributing to palmitate-induced cardiomyocyte death. Palmitate-induced cardiomyocyte death was induced in Toll-like receptor 2/4 double-knockdown cardiomyocytes to a similar extent as wild-type cardiomyocytes, while cardiomyocyte death was canceled out by triacsin C, a long-chain acyl-CoA synthetase inhibitor. These results indicated that palmitate induced cytotoxicity after entry and conversion into palmitoyl-CoA. Palmitoyl-CoA is not only degraded by mitochondrial oxidation but also taken up as a component of membrane phospholipids. Palmitate overloading causes cardiomyocyte membrane fatty acid (FA) saturation, which is associated with the activation of endoplasmic reticulum (ER) unfolded protein response (UPR) signaling. We focused on the ER UPR signaling as a possible mechanism of cell death. Palmitate loading activates the UPR signal via membrane FA saturation, but not via unfolded protein overload in the ER since the chemical chaperone 4-phenylbutyrate failed to suppress palmitate-induced ER UPR. The mammalian UPR relies on three ER stress sensors named inositol requiring enzyme-1 (IRE1), PKR-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6). Palmitate loading activated only IRE1 and PERK. Knockdown of PERK did not affect palmitate-induced cardiomyocyte death, while knockdown of IRE1 suppressed palmitate-induced cardiomyocyte death. However, knockdown of X-box binding protein 1 (XBP1), the downstream effector of IRE1, did not affect palmitate-induced cardiomyocyte death. These results were validated by pharmacological inhibitor experiments. In conclusion, we identified that palmitate-induced cardiomyocyte death was triggered by IRE1-mediated signaling independent of XBP1.

Adrenal cortex hypoxia modulates aldosterone production in heart failure.

Plasma aldosterone concentration increases in proportion to the severity of heart failure, even during treatment with renin-angiotensin system inhibitors. This study investigated alternative regulatory mechanisms of aldosterone production that are significant in heart failure. Dahl salt-sensitive rats on a high-salt diet, a rat model of heart failure with cardio-renal syndrome, had high plasma aldosterone levels and elevated ß3-adrenergic receptor expression in hypoxic zona glomerulosa cells. In H295R cells (a human adrenocortical cell line), hypoxia-induced ß3-adrenergic receptor expression. Hypoxia-mediated ß3-adrenergic receptor expression augmented aldosterone production by facilitating hydrolysis of lipid droplets though ERK-mediated phosphorylation of hormone-sensitive lipase, also known as cholesteryl ester hydrolase. Hypoxia also accelerated the synthesis of cholesterol esters by acyl-CoA:cholesterol acyltransferase, thereby increasing the cholesterol ester content in lipid droplets. Thus, hypoxia enhanced aldosterone production by zona glomerulosa cells via promotion of the accumulation and hydrolysis of cholesterol ester in lipid droplets. In conclusion, hypoxic zona glomerulosa cells with heart failure show enhanced aldosterone production via increased catecholamine responsiveness and activation of cholesterol trafficking, irrespective of the renin-angiotensin system.

Pressure overload inhibits glucocorticoid receptor transcriptional activity in cardiomyocytes and promotes pathological cardiac hypertrophy.

Glucocorticoid receptor (GR) is abundantly expressed in cardiomyocytes. However, the role of GR in regulating cardiac hypertrophy and heart failure in response to pressure overload remains unclear. Cardiomyocyte-specific GR knockout (GRcKO) mice, mineralocorticoid receptor (MR) knockout (MRcKO), and GR and MR double KO (GRMRdcKO) mice were generated using the Cre-lox system. In response to pressure overload, GRcKO mice displayed worse cardiac remodeling compared to control (GRf/f) mice, including a greater increase in heart weight to body weight ratio with a greater increase in cardiomyocytes size, a greater decline in left ventricular contractility, and higher reactivation of fetal genes. MRcKO mice showed a comparable degree of cardiac remodeling compared to control (MRf/f) mice. The worse cardiac remodeling in pressure overloaded GRcKO mice is not due to compensatory activation of cardiomyocyte MR, since pressure overloaded GRMRdcKO mice displayed cardiac remodeling to the same extent as GRcKO mice. Pressure overload suppressed GR-target gene expression in the heart. Although plasma corticosterone levels and subcellular localization of GR (nuclear/cytoplasmic GR) were not changed, a chromatin immunoprecipitation assay revealed that GR recruitment onto the promoter of GR-target genes was significantly suppressed in response to pressure overload. Rescue of the expression of GR-target genes to the same extent as sham-operated hearts attenuated adverse cardiac remodeling in pressure-overloaded hearts. Thus, GR works as a repressor of adverse cardiac remodeling in response to pressure overload, but GR-mediated transcription is suppressed under pressure overload. Therapies that maintain GR-mediated transcription in cardiomyocytes under pressure overload can be a promising therapeutic strategy for heart failure.

Sirt1 counteracts decrease in membrane phospholipid unsaturation and diastolic dysfunction during saturated fatty acid overload.

BACKGROUND: The fatty acid (FA) composition of membrane phospholipid reflects at least in part dietary fat composition. Saturated FA (SFA) suppress Sirt1 activity, while monounsaturated FA (MUFA) counteract this effect. OBJECTIVE: We explored a role of Sirt1 in homeostatic control of the fatty acid composition of membrane phospholipid in the presence of SFA overload. METHODS AND RESULTS: Sirt1 deficiency in cardiomyocytes decreased the expression levels of liver X receptor (LXR)-target genes, particularly stearoyl-CoA desaturase-1 (Scd1), a rate-limiting enzyme in the cellular synthesis of MUFA from SFA, increased membrane SFA/MUFA ratio, and worsened left ventricular (LV) diastolic function in mice fed an SFA-rich high fat diet. In cultured cardiomyocytes, Sirt1 knockdown (KD) exacerbated the palmitate overload-induced increase in membrane SFA/MUFA ratio, which was associated with decrease in the expression of LXR-target genes, including Scd1. Forced overexpression of Scd1 in palmitate-overloaded Sirt1KD cardiomyocytes lowered the SFA/MUFA ratio. Nicotinamide mononucleotide (NMN) increased Sirt1 activity and Scd1 expression, thereby lowering membrane SFA/MUFA ratio in palmitate-overloaded cardiomyocytes. These effects of NMN were not observed for Scd1KD cardiomyocytes. LXRα/ßKD exacerbated palmitate overload-induced increase in membrane SFA/MUFA ratio, while LXR agonist T0901317 alleviated it. NMN failed to rescue Scd1 protein expression and membrane SFA/MUFA ratio in palmitate-overloaded LXRα/ßKD cardiomyocytes. The administration of NMN or T0901317 showed a dramatic reversal in membrane SFA/MUFA ratio and LV diastolic function in SFA-rich HFD-fed mice. CONCLUSION: Cardiac Sirt1 counteracted SFA overload-induced decrease in membrane phospholipid unsaturation and diastolic dysfunction via regulating LXR-mediated transcription of the Scd1 gene.

IL (Interleukin)-10-STAT3-Galectin-3 Axis Is Essential for Osteopontin-Producing Reparative Macrophage Polarization After Myocardial Infarction.

BACKGROUND: Both osteopontin (OPN) and galectin-3 have been implicated in phagocytic clearance of dead cells and reparative fibrosis during wound healing. CD206+ macrophages are involved in tissue repair through phagocytosis and fibrosis after myocardial infarction (MI). However, the relationship among OPN, galectin-3, and macrophage polarization in the context of MI remains unclear. METHODS: The time course of Spp1 (encoding OPN) expression in the heart after MI showed a strong activation of Spp1 on day 3 after MI. To identify where in the body and in which cells the transcriptional activity of Spp1 increased after MI, we analyzed EGFP (enhanced green fluorescent protein)- Spp1 knockin reporter mice on day 3 after MI. RESULTS: The transcriptional activity of Spp1 increased only in CD206+ macrophages in the infarct myocardium, and most of CD206+ macrophages have strong transcriptional activation of Spp1 after MI. The temporal expression pattern of Lgal3 (encoding galectin-3) in cardiac macrophages after MI was similar to that of Spp1, and OPN is almost exclusively produced by galectin-3hiCD206+ macrophages. Although both interleukin (IL)-4 and IL-10 were reported to promote CD206+ macrophage-mediated cardiac repair after MI, IL-10- but not IL-4-stimulated CD11b+Ly6G- cells could differentiate into OPN-producing galectin-3hiCD206+ macrophages and showed enhanced phagocytic ability. Inhibition of STAT3 tyrosine phosphorylation suppressed IL-10-induced expression of intracellular galectin-3 and transcriptional activation of Spp1. Knockdown of galectin-3 suppressed their ability to differentiate into OPN-producing cells, but not STAT3 activation. The tyrosine phosphorylation of STAT3 and the appearance rate of galectin-3hiCD206+ cells on cardiac CD11b+Ly6G- cells in Spp1 knockout mice were the same as those in wild-type mice. Spp1 knockout mice showed vulnerability to developing post-MI left ventricular chamber dilatation and the terminal deoxynucleo-tidyltransferase 2'-Deoxyuridine-5'-triphosphate nick-end labeling (TUNEL)-positive cells in the infarcted myocardium after MI remained higher in number in Spp1 knockout mice than in wild-type mice. CONCLUSIONS: OPN is almost exclusively produced by galectin-3hiCD206+ macrophages, which specifically appear in the infarct myocardium after MI. The IL-10-STAT3-galectin-3 axis is essential for OPN-producing reparative macrophage polarization after myocardial infarction, and these macrophages contribute to tissue repair by promoting fibrosis and clearance of apoptotic cells. These results suggest that galectin-3 may contribute to reparative fibrosis in the infarct myocardium by controlling OPN levels.

Detecting intertrochanteric hip fractures with orthopedist-level accuracy using a deep convolutional neural network.

OBJECTIVE: To compare performances in diagnosing intertrochanteric hip fractures from proximal femoral radiographs between a convolutional neural network and orthopedic surgeons. MATERIALS AND METHODS: In total, 1773 patients were enrolled in this study. Hip plain radiographs from these patients were cropped to display only proximal fractured and non-fractured femurs. Images showing pseudarthrosis after femoral neck fracture and those showing artificial objects were excluded. This yielded a total of 3346 hip images (1773 fractured and 1573 non-fractured hip images) that were used to compare performances between the convolutional neural network and five orthopedic surgeons. RESULTS: The convolutional neural network and orthopedic surgeons had accuracies of 95.5% (95% CI = 93.1-97.6) and 92.2% (95% CI = 89.2-94.9), sensitivities of 93.9% (95% CI = 90.1-97.1) and 88.3% (95% CI = 83.3-92.8), and specificities of 97.4% (95% CI = 94.5-99.4) and 96.8% (95% CI = 95.1-98.4), respectively. CONCLUSIONS: The performance of the convolutional neural network exceeded that of orthopedic surgeons in detecting intertrochanteric hip fractures from proximal femoral radiographs under limited conditions. The convolutional neural network has a significant potential to be a useful tool for screening for fractures on plain radiographs, especially in the emergency room, where orthopedic surgeons are not readily available.