SDHAF1 confers metabolic resilience to aging hematopoietic stem cells by promoting mitochondrial ATP production.

Aging generally predisposes stem cells to functional decline, impairing tissue homeostasis. Here, we report that hematopoietic stem cells (HSCs) acquire metabolic resilience that promotes cell survival. High-resolution real-time ATP analysis with glucose tracing and metabolic flux analysis revealed that old HSCs reprogram their metabolism to activate the pentose phosphate pathway (PPP), becoming more resistant to oxidative stress and less dependent on glycolytic ATP production at steady state. As a result, old HSCs can survive without glycolysis, adapting to the physiological cytokine environment in bone marrow. Mechanistically, old HSCs enhance mitochondrial complex II metabolism during stress to promote ATP production. Furthermore, increased succinate dehydrogenase assembly factor 1 (SDHAF1) in old HSCs, induced by physiological low-concentration thrombopoietin (TPO) exposure, enables rapid mitochondrial ATP production upon metabolic stress, thereby improving survival. This study provides insight into the acquisition of resilience through metabolic reprogramming in old HSCs and its molecular basis to ameliorate age-related hematopoietic abnormalities.

Context-dependent modification of PFKFB3 in hematopoietic stem cells promotes anaerobic glycolysis and ensures stress hematopoiesis.

Metabolic pathways are plastic and rapidly change in response to stress or perturbation. Current metabolic profiling techniques require lysis of many cells, complicating the tracking of metabolic changes over time after stress in rare cells such as hematopoietic stem cells (HSCs). Here, we aimed to identify the key metabolic enzymes that define differences in glycolytic metabolism between steady-state and stress conditions in murine HSCs and elucidate their regulatory mechanisms. Through quantitative 13C metabolic flux analysis of glucose metabolism using high-sensitivity glucose tracing and mathematical modeling, we found that HSCs activate the glycolytic rate-limiting enzyme phosphofructokinase (PFK) during proliferation and oxidative phosphorylation (OXPHOS) inhibition. Real-time measurement of ATP levels in single HSCs demonstrated that proliferative stress or OXPHOS inhibition led to accelerated glycolysis via increased activity of PFKFB3, the enzyme regulating an allosteric PFK activator, within seconds to meet ATP requirements. Furthermore, varying stresses differentially activated PFKFB3 via PRMT1-dependent methylation during proliferative stress and via AMPK-dependent phosphorylation during OXPHOS inhibition. Overexpression of Pfkfb3 induced HSC proliferation and promoted differentiated cell production, whereas inhibition or loss of Pfkfb3 suppressed them. This study reveals the flexible and multilayered regulation of HSC glycolytic metabolism to sustain hematopoiesis under stress and provides techniques to better understand the physiological metabolism of rare hematopoietic cells.

MBTD1 preserves adult hematopoietic stem cell pool size and function.

Mbtd1 (mbt domain containing 1) encodes a nuclear protein containing a zinc finger domain and four malignant brain tumor (MBT) repeats. We previously generated Mbtd1-deficient mice and found that MBTD1 is highly expressed in fetal hematopoietic stem cells (HSCs) and sustains the number and function of fetal HSCs. However, since Mbtd1-deficient mice die soon after birth possibly due to skeletal abnormalities, its role in adult hematopoiesis remains unclear. To address this issue, we generated Mbtd1 conditional knockout mice and analyzed adult hematopoietic tissues deficient in Mbtd1. We observed that the numbers of HSCs and progenitors increased and Mbtd1-deficient HSCs exhibited hyperactive cell cycle, resulting in a defective response to exogenous stresses. Mechanistically, we found that MBTD1 directly binds to the promoter region of FoxO3a, encoding a forkhead protein essential for HSC quiescence, and interacts with components of TIP60 chromatin remodeling complex and other proteins involved in HSC and other stem cell functions. Restoration of FOXO3a activity in Mbtd1-deficient HSCs in vivo rescued cell cycle and pool size abnormalities. These findings indicate that MBTD1 is a critical regulator for HSC pool size and function, mainly through the maintenance of cell cycle quiescence by FOXO3a.

Distinct roles of the preparatory and payoff phases of glycolysis in hematopoietic stem cells.

In physiological conditions, most adult hematopoietic stem cells (HSCs) maintain a quiescent state. Glycolysis is a metabolic process that can be divided into preparatory and payoff phases. Although the payoff phase maintains HSC function and properties, the role of the preparatory phase remains unknown. In this study, we aimed to investigate whether the preparatory or payoff phases of glycolysis were required for maintenance of quiescent and proliferative HSCs. We used glucose-6-phosphate isomerase (Gpi1) as a representative gene for the preparatory phase and glyceraldehyde-3-phosphate dehydrogenase (Gapdh) as a representative gene for the payoff phase of glycolysis. First, we identified that stem cell function and survival were impaired in Gapdh-edited proliferative HSCs. Contrastingly, cell survival was maintained in quiescent Gapdh- and Gpi1-edited HSCs. Gapdh- and Gpi1-defective quiescent HSCs maintained adenosine-triphosphate (ATP) levels by increasing mitochondrial oxidative phosphorylation (OXPHOS), whereas ATP levels were decreased in Gapdh-edited proliferative HSCs. Interestingly, Gpi1-edited proliferative HSCs maintained ATP levels independent of increased OXPHOS. Oxythiamine, a transketolase inhibitor, impaired proliferation of Gpi1-edited HSCs, suggesting that the nonoxidative pentose phosphate pathway (PPP) is an alternative means to maintain glycolytic flux in Gpi1-defective HSCs. Our findings suggest that OXPHOS compensated for glycolytic deficiencies in quiescent HSCs, and that in proliferative HSCs, nonoxidative PPP compensated for defects in the preparatory phase of glycolysis but not for defects in the payoff phase. These findings provide new insights into regulation of HSC metabolism, which could have implications for development of novel therapies for hematologic disorders.

Mitf is required for T cell maturation by regulating dendritic cell homing to the thymus.

Thymic dendritic cells (DCs) promote immune tolerance by regulating negative selection of autoreactive T cells in the thymus. How DC homing to the thymus is transcriptionally regulated is still unclear. Microphthalmia-associated transcription factor (Mitf) is broadly expressed and plays essential roles in the hematopoietic system. Here, we used Mitf-mutated mice (Mitfvit/vit) and found enlargement of the thymus and expansion of CD4/CD8 double-positive T cells. Mitf was highly expressed in a subset of thymic DCs among the hematopoietic system. Genetic mutation or pharmacological inhibition of Mitf in DCs decreased the expression levels of Itga4, which are critical molecules for the homing of DCs to the thymus. Further, inhibition of Mitf decreased thymic DC number. These results suggest a pivotal role of Mitf in the maintenance of T cell differentiation by regulating the homing of DC subsets within the thymus.

A culture platform to study quiescent hematopoietic stem cells following genome editing.

Other than genetically engineered mice, few reliable platforms are available for the study of hematopoietic stem cell (HSC) quiescence. Here we present a platform to analyze HSC cell cycle quiescence by combining culture conditions that maintain quiescence with a CRISPR-Cas9 genome editing system optimized for HSCs. We demonstrate that preculture of HSCs enhances editing efficiency by facilitating nuclear transport of ribonucleoprotein complexes. For post-editing culture, mouse and human HSCs edited based on non-homologous end joining and cultured under low-cytokine, low-oxygen, and high-albumin conditions retain their phenotypes and quiescence better than those cultured under the proliferative conditions. Using this approach, HSCs regain quiescence even after editing by homology-directed repair. Our results show that low-cytokine culture conditions for gene-edited HSCs are a useful approach for investigating HSC quiescence ex vivo.

OGT Regulates Hematopoietic Stem Cell Maintenance via PINK1-Dependent Mitophagy.

O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) is a unique enzyme introducing O-GlcNAc moiety on target proteins, and it critically regulates various cellular processes in diverse cell types. However, its roles in hematopoietic stem and progenitor cells (HSPCs) remain elusive. Here, using Ogt conditional knockout mice, we show that OGT is essential for HSPCs. Ogt is highly expressed in HSPCs, and its disruption induces rapid loss of HSPCs with increased reactive oxygen species and apoptosis. In particular, Ogt-deficient hematopoietic stem cells (HSCs) lose quiescence, cannot be maintained in vivo, and become vulnerable to regenerative and competitive stress. Interestingly, Ogt-deficient HSCs accumulate defective mitochondria due to impaired mitophagy with decreased key mitophagy regulator, Pink1, through dysregulation of H3K4me3. Furthermore, overexpression of PINK1 restores mitophagy and the number of Ogt-deficient HSCs. Collectively, our results reveal that OGT critically regulates maintenance and stress response of HSCs by ensuring mitochondrial quality through PINK1-dependent mitophagy.

A case of Type-C Wolff-Parkinson-White syndrome with severe left ventricular dysfunction: Efficacy of catheter ablation.

The present case report describes a 59-year-old female with manifest Wolff-Parkinson-White syndrome and severe left ventricular (LV) dysfunction, however, there was no indication of heart palpitations. The polarity of delta is consistent with the features of the right anteroseptal accessory pathways (APs). The echocardiography showed a remarkable dyssynchrony of the LV wall motion. To circumvent the cardiac dysfunctions, radiofrequency catheter ablation (RFCA) was successfully performed to disconnect the AP. Thereafter, the dyssynchrony disappeared, and the clinical reports observed 6 months following RFCA showed that the LV ejection fraction had been improved from 13% up to 48%, in addition to the improvement in other parameters. The RFCA prevented her from receiving a cardiac resynchronization therapy defibrillator as well as a heart transplantation. .

Mast Cells Play an Important Role in the Pathogenesis of Hyperglycemia-Induced Atrial Fibrillation.

BACKGROUND AND OBJECTIVES: Recently, it was reported that mast cells (MCs) could underlie the mechanisms of several cardiovascular diseases. However, the role of MCs in diabetes-induced atrial fibrillation (AF) has not been notably investigated. We tested the hypothesis that MC deficiency attenuates hyperglycemia-induced AF in mice. METHODS AND RESULTS: Mast cell-deficient W/W(v)  mice, and congenic +/+ littermates (WT) were divided into either the vehicle (VEH)-injection group or the streptozotocin (STZ)-injection group (MCKO-VEH, MCKO-STZ, WT-VEH, and WT-STZ groups). On day 28 of our studies, we observed that (1) STZ-induced hyperglycemia increased MC infiltration in the left atrium (LA) in WT mice (P < 0.01), (2) atrium isolated from the WT-STZ group showed inhomogeneous interstitial fibrosis, abundant infiltration of macrophages, and enhanced apoptosis compared to the WT-VEH group (P < 0.01, P < 0.01, P < 0.05, respectively). However, the changes observed in the WT-STZ group were significantly attenuated in the MCKO-STZ mice. In addition, we observed that (3) messenger RNA levels of tumor necrosis factor-α, monocyte chemoattractant protein-1, interleukin-1ß, transforming growth factor-ß, and collagen-1 in the LA were increased in the WT-STZ group, but not in the MCKO-STZ group, (4) STZ-induced hyperglycemia increased AF induction and prolonged interatrial conduction time in the WT mice, which were not observed in the MCKO mice, and that (5) hyperglycemia-enhanced atrial production of reactive oxygen species (ROS) was equally observed in the WT and MCKO mice. CONCLUSIONS: Our results suggest that MCs contribute to the pathogenesis of hyperglycemia-induced AF via enhancement of inflammation and fibrosis.

A case of anomalous origin of the left coronary artery from the pulmonary artery presenting with sudden cardiac arrest due to coronary artery steal generated by excessive exercise.

This case report describes a 43-year-old man who temporarily survived cardiac arrest that was prospectively related to ventricular fibrillation due to the anomalous origin of the left coronary artery from the pulmonary artery (ALCAPA). Prior to admission to our hospital, he was asymptomatic for ALCAPA syndrome. Emergent coronary angiography revealed that the dilated right coronary artery was connected with extensive collateral vessels to the left coronary artery. The origin of the latter was in the pulmonary artery. Moreover, coronary steal phenomenon was identified by examining the pulmonary arterial blood oxygen saturation. The patient later died of acute decompensated acidosis. .

[Relationships of the stages of behavior change in dietary habits of the mothers of school-age children with the breakfast intake of the children and the health-associated behavior of the family].

OBJECTIVES: We aimed to clarify the relationships of the different stages of behavior change in dietary habits followed by the mothers of school-age children with the actual breakfast intake of these children and the health-associated behavior of the family. METHODS: We carried out a questionnaire-based survey of 1949 children at 18 elementary schools and of 881 families with children attending seven elementary schools in Kagoshima prefecture. We were supplied with information about children's breakfast intake and content on the day they took the survey and information about mothers' breakfast intake and the stage of behavior change in dietary habits to which they belonged, for which five stages were defined using the stage-of-change model. RESULTS: The collection rates were 83.3% and 83.1% among children and mothers respectively. Of the children, 83.1% ate breakfast every day, while 15.1% were not in the habit of having breakfast. Furthermore, 98.6% children had eaten breakfast on the day of the survey, but 15.1% had eaten only staple foods such as rice or bread; only 34.0% children combined staple foods, a main dish, and vegetables/fruits in their breakfast. Regarding dietary stage, 28.1% of the mothers belonged to the "maintenance" stage; 24.0%, the "action" stage; 6.9%, the "preparation" stage; 9.8%, the "contemplation" stage; and 5.7%, the "precontemplation" stage. Mothers belonging to the first two stages constituted the "action group," because they were already taking care of their dietary habits, and mothers belonging to the latter three stages constituted the "no-action group", because they were not taking care of their dietary habits. The mothers who could provide no answers to the question constituted the "no-answer group" (25.5%). A comparison of the three groups revealed that mothers belonging to the no-answer group had more children who went without breakfast than the action group (P = 0.000). The children of mothers belonging to the no-action group (P = 0.003) and the no-answer group (P = 0.036) were not in general eating vegetables/fruits in their breakfast, in contrast with the action group. Furthermore, in the case of families with mothers belonging to the no-action and no-answer groups, the families did not often talk about diet, and the incidence of smokers among the fathers was high. CONCLUSION: In this study, the breakfast habits of children and the health behavior of families differed by stage of dietary behavior change to which the mother belonged.