5,10-Methylenetetrahydrofolate reductase becomes phosphorylated during meiotic maturation in mouse oocytes.

The enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR) links the folate cycle that produces one-carbon units with the methionine cycle that converts these into S-adenosylmethionine (SAM), the universal methyl donor for almost all methyltransferases. Previously, MTHFR has been shown to be regulated by phosphorylation, which suppresses its activity. SAM levels have been shown to increase substantially soon after initiation of meiotic maturation of the mouse germinal vesicle (GV) stage oocyte and then decrease back to their original low level in mature second meiotic metaphase (MII) eggs. As MTHFR controls the entry of one-carbon units into the methionine cycle, it is a candidate regulator of the SAM levels in oocytes and eggs. Mthfr transcripts are expressed in mouse oocytes and preimplantation embryos and MTHFR protein is present at each stage. In mature MII eggs, the apparent molecular weight of MTHFR was increased compared with GV oocytes, which we hypothesized was due to increased phosphorylation. The increase in apparent molecular weight was reversed by treatment with lambda protein phosphatase (LPP), indicating that MTHFR is phosphorylated in MII eggs. In contrast, LPP had no effect on MTHFR from GV oocytes, 2-cell embryos, or blastocysts. MTHFR was progressively phosphorylated after initiation of meiotic maturation, reaching maximal levels in MII eggs before decreasing again after egg activation. As phosphorylation suppresses MTHFR activity, it is predicted that MTHFR becomes inactive during meiotic maturation and is minimally active in MII eggs, which is consistent with the reported changes in SAM levels during mouse oocyte maturation.

Betaine is accumulated via transient choline dehydrogenase activation during mouse oocyte meiotic maturation.

Betaine (N,N,N-trimethylglycine) plays key roles in mouse eggs and preimplantation embryos first in a novel mechanism of cell volume regulation and second as a major methyl donor in blastocysts, but its origin is unknown. Here, we determined that endogenous betaine was present at low levels in germinal vesicle (GV) stage mouse oocytes before ovulation and reached high levels in the mature, ovulated egg. However, no betaine transport into oocytes was detected during meiotic maturation. Because betaine can be synthesized in mammalian cells via choline dehydrogenase (CHDH; EC 1.1.99.1), we assessed whether this enzyme was expressed and active. Chdh transcripts and CHDH protein were expressed in oocytes. No CHDH enzyme activity was detected in GV oocyte lysate, but CHDH became highly active during oocyte meiotic maturation. It was again inactive after fertilization. We then determined whether oocytes synthesized betaine and whether CHDH was required. Isolated maturing oocytes autonomously synthesized betaine in vitro in the presence of choline, whereas this failed to occur in Chdh-/- oocytes, directly demonstrating a requirement for CHDH for betaine accumulation in oocytes. Overall, betaine accumulation is a previously unsuspected physiological process during mouse oocyte meiotic maturation whose underlying mechanism is the transient activation of CHDH.

PREVEntion and regReSsive Effect of weight-loss and risk factor modification on Atrial Fibrillation: the REVERSE-AF study.

Aims: Atrial fibrillation (AF) is a progressive disease. Obesity is associated with progression of AF. This study evaluates the impact of weight and risk factor management (RFM) on progression of the AF. Methods and results: As described in the Long-Term Effect of Goal-Directed Weight Management in an Atrial Fibrillation Cohort: A Long-Term Follow-Up (LEGACY) Study, of 1415 consecutive AF patients, 825 had body mass index ≥ 27 kg/m2 and were offered weight and RFM. After exclusion, 355 were included for analysis. Weight loss was categorized as: Group 1 (<3%), Group 2 (3-9%), and Group 3 (≥10%). Change in AF type was determined by clinical review and 7-day Holter yearly. Atrial fibrillation type was categorized as per the Heart Rhythm Society consensus. There were no differences in baseline characteristic or follow-up duration between groups (P = NS). In Group 1, 41% progressed from paroxysmal to persistent and 26% from persistent to paroxysmal or no AF. In Group 2, 32% progressed from paroxysmal to persistent and 49% reversed from persistent to paroxysmal or no AF. In Group 3, 3% progressed to persistent and 88% reversed from persistent to paroxysmal or no AF (P < 0.001). Increased weight loss was significantly associated with greater AF freedom: 45 (39%) in Group 1, 69 (67%) in Group 2, and 116 (86%) in Group 3 (P ≤ 0.001). Conclusion: Obesity is associated with progression of the AF disease. This study demonstrates the dynamic relationship between weight/risk factors and AF. Weight-loss management and RFM reverses the type and natural progression of AF.

Acute cell volume regulation by Janus kinase 2-mediated sodium/hydrogen exchange activation develops at the late one-cell stage in mouse preimplantation embryos.

Early preimplantation embryos are extremely sensitive to dysregulation of cell volume, which can lead to developmental arrest. It was previously shown that mouse embryos at the two-cell stage respond to a cell volume decrease by quickly activating Na+/H+ exchange via a signaling mechanism that involves the tyrosine kinase Janus kinase 2 (JAK2). However, it was not known whether this mechanism is active at the one-cell stage, when embryos are most sensitive to perturbed cell volume. Na+/H+ exchanger activity elicited by an induced cell volume decrease was significantly lower at the mid one-cell stage than at the late one-cell stage or during the two-cell stage. This activity could be completely blocked by the broad specificity tyrosine kinase inhibitor genistein at either stage, but only at the two-cell stage was there a substantial component of activity that was sensitive to low concentrations of the JAK2-selective inhibitors TG101348 or ruxolitinib. Western blots to detect active JAK2 phosphorylated on tyrosine Y1007/8 revealed that JAK2 became substantially phosphorylated in response to a cell volume decrease at the mid two-cell, but not mid one-cell stage. Such cell volume decrease-induced JAK2 phosphorylation appeared by the late one-cell stage. At least in part this appears to be due to an increase in total JAK2 protein at the late one-cell stage. Furthermore, TG101348 impaired maintenance of cell volume at the two-cell, but not mid one-cell, stages. Thus, cell volume homeostasis requiring Na+/H+ exchange signaled by JAK2 first becomes prominent during mouse embryonic development at the late one-cell stage.

Growing Mouse Oocytes Transiently Activate Folate Transport via Folate Receptors As They Approach Full Size.

The folate cycle is central to cellular one-carbon metabolism, where folates are carriers of one-carbon units that are critical for synthesis of purines, thymidylate, and S-adenosylmethionine, the universal methyl donor that forms the cellular methyl pool. Although folates are well-known to be important for early embryo and fetal development, their role in oogenesis has not been clearly established. Here, folate transport proteins were detected in developing neonatal ovaries and growing oocytes by immunohistochemistry, Western blot, and immunofluorescence. The folate receptors FOLR1 and FOLR2 as well as reduced folate carrier 1 (RFC1, SLC19A1 protein) each appeared to be present in follicular cells including granulosa cells. In growing oocytes, however, only FOLR2 immunoreactivity appeared abundant. Localization of apparent FOLR2 immunofluorescence near the plasma membrane increased with oocyte growth and peaked in oocytes as they neared full size. We assessed folate transport using the model folate leucovorin (folinic acid). Unexpectedly, there was a transient burst of folate transport activity for a brief period during oocyte growth as they neared full size, while folate transport was otherwise undetectable for the rest of oogenesis and in fully grown germinal vesicle stage oocytes. This folate transport was inhibited by dynasore, an inhibitor of endocytosis, but insensitive to the anion transport inhibitor stilbene 4-acetamido-40-isothiocyanato-stilbene-2,20-disulfonic acid, consistent with folate receptor-mediated transport but not with RFC1-mediated transport. Thus, near the end of their growth, growing oocytes may take up folates that could support the final stage of oogenesis or be stored to provide the endogenous folates needed in early embryogenesis.

Cost-Effectiveness and Clinical Effectiveness of the Risk Factor Management Clinic in Atrial Fibrillation: The CENT Study.

BACKGROUND: Atrial fibrillation (AF) imposes a substantial cost burden on the healthcare system. Weight and risk factor management (RFM) reduces AF burden and improves the outcomes of AF ablation. OBJECTIVES: This study sought to evaluate the cost and clinical effectiveness of integrating RFM into the overall management of AF. METHODS: Of 1,415 consecutive patients with symptomatic AF, 825 patients had body mass index ≥27 kg/m2. After screening for exclusion criteria, the final cohort comprised 355 patients: 208 patients who opted for RFM and 147 control subjects and were followed by 3 to 6 monthly clinic review, 7-day Holter monitoring, and AF Symptom Score. A decision analytical model calculated the incremental cost-effectiveness ratios of cost per unit of global well-being gained and unit of AF burden reduced. RESULTS: There were no differences in baseline characteristics or follow-up duration (p = NS). Arrhythmia-free survival was better in the RFM compared with control subjects (Kaplan-Meier: 79% vs. 44%; p < 0.001). At follow-up, RFM group had less unplanned specialist visits (0.19 ± 0.40 vs. 1.94 ± 2.00; p < 0.001), hospitalizations (0.74 ± 1.3 vs. 1.05 ± 1.60; p = 0.03), cardioversions (0.89 ± 1.50 vs. 1.51 ± 2.30; p = 0.002), emergency presentations (0.18 ± 0.50 vs. 0.76 ± 1.20; p < 0.001), and ablation procedures (0.60 ± 0.69 vs. 0.72 ± 0.86; p = 0.03). Antihypertensive (0.53 ± 0.70 vs. 0.78 ± 0.60; p = 0.04) and antiarrhythmic (0.26 ± 0.50 vs. 0.91 ± 0.60; p = 0.003) use declined in RFM. The RFM group had an increase of 0.1930 quality-adjusted life years and a cost saving of $12,094 (incremental cost-effectiveness ratios of $62,653 saved per quality-adjusted life years gained). CONCLUSIONS: A structured physician-directed RFM program is clinically effective and cost saving.

Long-Term Effect of Goal-Directed Weight Management in an Atrial Fibrillation Cohort: A Long-Term Follow-Up Study (LEGACY).

BACKGROUND: Obesity and atrial fibrillation (AF) frequently coexist. Weight loss reduces the burden of AF, but whether this is sustained, has a dose effect, or is influenced by weight fluctuation is unknown. OBJECTIVES: This study sought to evaluate the long-term impact of weight loss and weight fluctuation on rhythm control in obese individuals with AF. METHODS: Of 1,415 consecutive patients with AF, 825 had a body mass index ≥ 27 kg/m(2) and were offered weight management. After screening for exclusion criteria, 355 were included in this analysis. Weight loss was categorized as group 1 (≥ 10%), group 2 (3% to 9%), and group 3 (<3%). Weight trend and/or fluctuation was determined by yearly follow-up. We determined the impact on the AF severity scale and 7-day ambulatory monitoring. RESULTS: There were no differences in baseline characteristics or follow-up among the groups. AF burden and symptom severity decreased more in group 1 compared with groups 2 and 3 (p < 0.001 for all). Arrhythmia-free survival with and without rhythm control strategies was greatest in group 1 compared with groups 2 and 3 (p < 0.001 for both). In multivariate analyses, weight loss and weight fluctuation were independent predictors of outcomes (p < 0.001 for both). Weight loss ≥ 10% resulted in a 6-fold (95% confidence interval: 3.4 to 10.3; p < 0.001) greater probability of arrhythmia-free survival compared with the other 2 groups. Weight fluctuation >5% partially offset this benefit, with a 2-fold (95% confidence interval: 1.0 to 4.3; p = 0.02) increased risk of arrhythmia recurrence. CONCLUSIONS: Long-term sustained weight loss is associated with significant reduction of AF burden and maintenance of sinus rhythm. (Long-Term Effect of Goal directed weight management on Atrial Fibrillation Cohort: A 5 Year follow-up study [LEGACY Study]; ACTRN12614001123639).

Impact of CARDIOrespiratory FITness on Arrhythmia Recurrence in Obese Individuals With Atrial Fibrillation: The CARDIO-FIT Study.

BACKGROUND: Obesity begets atrial fibrillation (AF). Although cardiorespiratory fitness is protective against incident AF in obese individuals, its effect on AF recurrence or the benefit of cardiorespiratory fitness gain is unknown. OBJECTIVES: This study sought to evaluate the role of cardiorespiratory fitness and the incremental benefit of cardiorespiratory fitness improvement on rhythm control in obese individuals with AF. METHODS: Of 1,415 consecutive patients with AF, 825 had a body mass index ≥27 kg/m(2) and were offered risk factor management and participation in a tailored exercise program. After exclusions, 308 patients were included in the analysis. Patients underwent exercise stress testing to determine peak metabolic equivalents (METs). To determine a dose response, cardiorespiratory fitness was categorized as: low (<85%), adequate (86% to 100%), and high (>100%). Impact of cardiorespiratory fitness gain was ascertained by the objective gain in fitness at final follow-up (≥2 METs vs. <2 METs). AF rhythm control was determined using 7-day Holter monitoring and AF severity scale questionnaire. RESULTS: There were no differences in baseline characteristics or follow-up duration between the groups defined by cardiorespiratory fitness. Arrhythmia-free survival with and without rhythm control strategies was greatest in patients with high cardiorespiratory fitness compared to adequate or low cardiorespiratory fitness (p < 0.001 for both). AF burden and symptom severity decreased significantly in the group with cardiorespiratory fitness gain ≥2 METs as compared to <2 METs group (p < 0.001 for all). Arrhythmia-free survival with and without rhythm control strategies was greatest in those with METs gain ≥2 compared to those with METs gain <2 in cardiorespiratory fitness (p < 0.001 for both). CONCLUSIONS: Cardiorespiratory fitness predicts arrhythmia recurrence in obese individuals with symptomatic AF. Improvement in cardiorespiratory fitness augments the beneficial effects of weight loss. (Evaluating the Impact of a Weight Loss on the Burden of Atrial Fibrillation [AF] in Obese Patients; ACTRN12614001123639).