Off-target depletion of plasma tryptophan by allosteric inhibitors of BCKDK.

The activation of branched chain amino acid (BCAA) catabolism has garnered interest as a potential therapeutic approach to improve insulin sensitivity, enhance recovery from heart failure, and blunt tumor growth. Evidence for this interest relies in part on BT2, a small molecule that promotes BCAA oxidation and is protective in mouse models of these pathologies. BT2 and other analogs allosterically inhibit branched chain ketoacid dehydrogenase kinase (BCKDK) to promote BCAA oxidation, which is presumed to underlie the salutary effects of BT2. Potential "off-target" effects of BT2 have not been considered, however. We therefore tested for metabolic off-target effects of BT2 in Bckdk-/- animals. As expected, BT2 failed to activate BCAA oxidation in these animals. Surprisingly, however, BT2 strongly reduced plasma tryptophan levels and promoted catabolism of tryptophan to kynurenine in both control and Bckdk-/- mice. Mechanistic studies revealed that none of the principal tryptophan catabolic or kynurenine-producing/consuming enzymes (TDO, IDO1, IDO2, or KATs) were required for BT2-mediated lowering of plasma tryptophan. Instead, using equilibrium dialysis assays and mice lacking albumin, we show that BT2 avidly binds plasma albumin and displaces tryptophan, releasing it for catabolism. These data confirm that BT2 activates BCAA oxidation via inhibition of BCKDK but also reveal a robust off-target effect on tryptophan metabolism via displacement from serum albumin. The data highlight a potential confounding effect for pharmaceutical compounds that compete for binding with albumin-bound tryptophan.

Clinical predictors of right ventricular dysfunction and association with adverse outcomes in peripartum cardiomyopathy.

AIMS: We sought to identify factors associated with right ventricular (RV) dysfunction and elevated pulmonary artery systolic pressure (PASP) and association with adverse outcomes in peripartum cardiomyopathy (PPCM). METHODS AND RESULTS: We conducted a multi-centre cohort study to identify subjects with PPCM with the following criteria: left ventricular ejection fraction (LVEF) < 40%, development of heart failure within the last month of pregnancy or 5 months of delivery, and no other identifiable cause of heart failure with reduced ejection fraction. Outcomes included a composite of (i) major adverse events (need for extracorporeal membrane oxygenation, ventricular assist device, orthotopic heart transplantation, or death) or (ii) recurrent heart failure hospitalization. RV function was obtained from echocardiogram reports. In total, 229 women (1993-2017) met criteria for PPCM. Mean age was 32.4 ± 6.8 years, 28% were of African descent, 50 (22%) had RV dysfunction, and 38 (17%) had PASP ≥ 30 mmHg. After a median follow-up of 3.4 years (interquartile range 1.0-8.8), 58 (25%) experienced the composite outcome of adverse events. African descent, family history of cardiomyopathy, LVEF, and PASP were significant predictors of RV dysfunction. Using Cox proportional hazards models, we found that women with RV dysfunction were three times more likely to experience the adverse composite outcome: hazard ratio 3.21 (95% confidence interval: 1.11-9.28), P = 0.03, in a multivariable model adjusting for age, race, body mass index, preeclampsia, hypertension, diabetes, kidney disease, and LVEF. Women with PASP ≥ 30 mmHg had a lower probability of survival free from adverse events (log-rank P = 0.04). CONCLUSIONS: African descent and family history of cardiomyopathy were significant predictors of RV dysfunction. RV dysfunction and elevated PASP were significantly associated with a composite of major adverse cardiac events. This at-risk group may prompt closer monitoring or early referral for advanced therapies.

The mitochondrial Ca2+ channel MCU is critical for tumor growth by supporting cell cycle progression and proliferation.

Introduction: The mitochondrial uniporter (MCU) Ca2+ ion channel represents the primary means for Ca2+ uptake by mitochondria. Mitochondrial matrix Ca2+ plays critical roles in mitochondrial bioenergetics by impinging upon respiration, energy production and flux of biochemical intermediates through the TCA cycle. Inhibition of MCU in oncogenic cell lines results in an energetic crisis and reduced cell proliferation unless media is supplemented with nucleosides, pyruvate or α-KG. Nevertheless, the roles of MCU-mediated Ca2+ influx in cancer cells remain unclear, in part because of a lack of genetic models. Methods: MCU was genetically deleted in transformed murine fibroblasts for study in vitro and in vivo. Tumor formation and growth were studied in murine xenograft models. Proliferation, cell invasion, spheroid formation and cell cycle progression were measured in vitro. The effects of MCU deletion on survival and cell-death were determined by probing for live/death markers. Mitochondrial bioenergetics were studied by measuring mitochondrial matrix Ca2+ concentration, membrane potential, global dehydrogenase activity, respiration, ROS production and inactivating-phosphorylation of pyruvate dehydrogenase. The effects of MCU rescue on metabolism were examined by tracing of glucose and glutamine utilization for fueling of mitochondrial respiration. Results: Transformation of primary fibroblasts in vitro was associated with increased MCU expression, enhanced MCU-mediated Ca2+ uptake, altered mitochondrial matrix Ca2+ concentration responses to agonist stimulation, suppression of inactivating-phosphorylation of pyruvate dehydrogenase and a modest increase of mitochondrial respiration. Genetic MCU deletion inhibited growth of HEK293T cells and transformed fibroblasts in mouse xenograft models, associated with reduced proliferation and delayed cell-cycle progression. MCU deletion inhibited cancer stem cell-like spheroid formation and cell invasion in vitro, both predictors of metastatic potential. Surprisingly, mitochondrial matrix [Ca2+], membrane potential, global dehydrogenase activity, respiration and ROS production were unaffected. In contrast, MCU deletion elevated glycolysis and glutaminolysis, strongly sensitized cell proliferation to glucose and glutamine limitation, and altered agonist-induced cytoplasmic Ca2+ signals. Conclusion: Our results reveal a dependence of tumorigenesis on MCU, mediated by a reliance on MCU for cell metabolism and Ca2+ dynamics necessary for cell-cycle progression and cell proliferation.

The mitochondrial Ca 2+ channel MCU is critical for tumor growth by supporting cell cycle progression and proliferation.

The mitochondrial uniporter (MCU) Ca 2+ ion channel represents the primary means for Ca 2+ uptake into mitochondria. Here we employed in vitro and in vivo models with MCU genetically eliminated to understand how MCU contributes to tumor formation and progression. Transformation of primary fibroblasts in vitro was associated with increased MCU expression, enhanced mitochondrial Ca 2+ uptake, suppression of inactivating-phosphorylation of pyruvate dehydrogenase, a modest increase of basal mitochondrial respiration and a significant increase of acute Ca 2+ -dependent stimulation of mitochondrial respiration. Inhibition of mitochondrial Ca 2+ uptake by genetic deletion of MCU markedly inhibited growth of HEK293T cells and of transformed fibroblasts in mouse xenograft models. Reduced tumor growth was primarily a result of substantially reduced proliferation and fewer mitotic cells in vivo , and slower cell proliferation in vitro associated with delayed progression through S-phase of the cell cycle. MCU deletion inhibited cancer stem cell-like spheroid formation and cell invasion in vitro , both predictors of metastatic potential. Surprisingly, mitochondrial matrix Ca 2+ concentration, membrane potential, global dehydrogenase activity, respiration and ROS production were unchanged by genetic deletion of MCU in transformed cells. In contrast, MCU deletion elevated glycolysis and glutaminolysis, strongly sensitized cell proliferation to glucose and glutamine limitation, and altered agonist-induced cytoplasmic Ca 2+ signals. Our results reveal a dependence of tumorigenesis on MCU, mediated by a reliance on mitochondrial Ca 2+ uptake for cell metabolism and Ca 2+ dynamics necessary for cell-cycle progression and cell proliferation.

Riding up the escaLEC-TOR for valvular health.

Endothelial-lined valves assure unidirectional flow in the lymphatic system. In this issue, Saygili Demir et al. (2023. J. Cell Biol.https://doi.org/10.1083/jcb.202207049) demonstrate how continuous repair of these valves occur, beginning with mTOR-activated cell replication in valve sinuses, and followed by cell migration to cover the valve surface.

Metabolite signaling in the heart.

The heart is the most metabolically active organ in the body, sustaining a continuous and high flux of nutrient catabolism via oxidative phosphorylation. The nature and relative contribution of these fuels have been studied extensively for decades. By contrast, less attention has been placed on how intermediate metabolites generated from this catabolism affect intracellular signaling. Numerous metabolites, including intermediates of glycolysis and the tricarboxylic acid (TCA) cycle, nucleotides, amino acids, fatty acids and ketones, are increasingly appreciated to affect signaling in the heart, via various mechanisms ranging from protein-metabolite interactions to modifying epigenetic marks. We review here the current state of knowledge of intermediate metabolite signaling in the heart.

Glutamine uptake and catabolism is required for myofibroblast formation and persistence.

BACKGROUND: Fibrosis and extracellular matrix remodeling are mediated by resident cardiac fibroblasts (CFs). In response to injury, fibroblasts activate, differentiating into specialized synthetic and contractile myofibroblasts producing copious extracellular matrix proteins (e.g., collagens). Myofibroblast persistence in chronic diseases, such as HF, leads to progressive cardiac dysfunction and maladaptive remodeling. We recently reported that an increase in αKG (alpha-ketoglutarate) bioavailability, which contributes to enhanced αKG-dependent lysine demethylase activity and chromatin remodeling, is required for myofibroblast formation. Therefore, we aimed to determine the substrates and metabolic pathways contributing to αKG biosynthesis and their requirement for myofibroblast formation. METHODS: Stable isotope metabolomics identified glutaminolysis as a key metabolic pathway required for αKG biosynthesis and myofibroblast formation, therefore we tested the effects of pharmacologic inhibition (CB-839) or genetic deletion of glutaminase (Gls1-/-) on myofibroblast formation in both murine and human cardiac fibroblasts. We employed immunofluorescence staining, functional gel contraction, western blotting, and bioenergetic assays to determine the myofibroblast phenotype. RESULTS: Carbon tracing indicated enhanced glutaminolysis mediating increased αKG abundance. Pharmacological and genetic inhibition of glutaminolysis prevented myofibroblast formation indicated by a reduction in αSMA+ cells, collagen gel contraction, collagen abundance, and the bioenergetic response. Inhibition of glutaminolysis also prevented TGFß-mediated histone demethylation and supplementation with cell-permeable αKG rescued the myofibroblast phenotype. Importantly, inhibition of glutaminolysis was sufficient to prevent myofibroblast formation in CFs isolated from the human failing heart. CONCLUSIONS: These results define glutaminolysis as necessary for myofibroblast formation and persistence, providing substantial rationale to evaluate several new therapeutic targets to treat cardiac fibrosis.

Animal Models of Cardiovascular Complications of Pregnancy.

Cardiovascular complications of pregnancy have risen substantially over the past decades, and now account for the majority of pregnancy-induced maternal deaths, as well as having substantial long-term consequences on maternal cardiovascular health. The causes and pathophysiology of these complications remain poorly understood, and therapeutic options are limited. Preclinical models represent a crucial tool for understanding human disease. We review here advances made in preclinical models of cardiovascular complications of pregnancy, including preeclampsia and peripartum cardiomyopathy, with a focus on pathological mechanisms elicited by the models and on relevance to human disease.

Comprehensive nutrient consumption estimation and metabolic profiling during ketogenic diet and relationship with myocardial glucose uptake on FDG-PET.

AIMS: The ketogenic diet (KD) is standard-of-care to achieve myocardial glucose suppression (MGS) for assessing inflammation using fluorine-18 fluorodeoxyglucose-positron emission tomography (FDG-PET). As KD protocols remain highly variable between centres (including estimation of nutrient intake by dietary logs for adequacy of dietary preparation), we aimed to assess the predictive utility of nutrient intake in achieving MGS. METHODS AND RESULTS: Nineteen healthy participants underwent short-term KD, with FDG-PET performed after 1 and 3 days of KD (goal carbohydrate intake <20 g/day). Nutrient consumption was estimated from dietary logs using nutrition research software. The area under receiver operating characteristics (AUROC) of macronutrients (carbohydrate, fat, and protein intake) for predicting MGS was analysed. The association between 133 nutrients and 4 biomarkers [beta-hydroxybutyrate (BHB), non-esterified fatty acids, insulin, and glucagon] with myocardial glucose uptake was assessed using mixed effects regression with false discovery rate (FDR) correction. Median (25th-75th percentile) age was 29 (25-34) years, 47% were women, and 42% were non-white. Median (25th-75th percentile) carbohydrate intake (g) was 18.7 (13.1-30.7), 16.9 (10.4-28.7), and 21.1 (16.6-29.0) on Days 1-3. No macronutrient intake (carbohydrate, fat, or protein) predicted MGS (c-statistic 0.45, 0.53, 0.47, respectively). Of 133 nutrients and 4 biomarkers, only BHB was associated with myocardial glucose uptake after FDR correction (corrected P-value 0.003). CONCLUSIONS: During highly supervised, short-term KD, approximately half of patients meet strict carbohydrate goals. Yet, in healthy volunteers, dietary review does not provide reassurance for adequacy of myocardial preparation since no clear thresholds for carbohydrate or fat intake reliably predict MGS.

Folliculin promotes substrate-selective mTORC1 activity by activating RagC to recruit TFE3.

Mechanistic target of rapamycin complex I (mTORC1) is central to cellular metabolic regulation. mTORC1 phosphorylates a myriad of substrates, but how different substrate specificity is conferred on mTORC1 by different conditions remains poorly defined. Here, we show how loss of the mTORC1 regulator folliculin (FLCN) renders mTORC1 specifically incompetent to phosphorylate TFE3, a master regulator of lysosome biogenesis, without affecting phosphorylation of other canonical mTORC1 substrates, such as S6 kinase. FLCN is a GTPase-activating protein (GAP) for RagC, a component of the mTORC1 amino acid (AA) sensing pathway, and we show that active RagC is necessary and sufficient to recruit TFE3 onto the lysosomal surface, allowing subsequent phosphorylation of TFE3 by mTORC1. Active mutants of RagC, but not of RagA, rescue both phosphorylation and lysosomal recruitment of TFE3 in the absence of FLCN. These data thus advance the paradigm that mTORC1 substrate specificity is in part conferred by direct recruitment of substrates to the subcellular compartments where mTORC1 resides and identify potential targets for specific modulation of specific branches of the mTOR pathway.

Endothelial Lipid Metabolism.

Endothelial cells (ECs) line all vessels of all vertebrates and are fundamental to organismal metabolism. ECs rely on their metabolism both to transport nutrients in and out of underlying parenchyma, and to support their own cellular activities, including angiogenesis. ECs primarily consume glucose, and much is known of how ECs transport and consume glucose and other carbohydrates. In contrast, how lipids are transported, and the role of lipids in normal EC function, has garnered less attention. We review here recent developments on the role of lipids in endothelial metabolism, with a focus on lipid uptake and transport in quiescent endothelium, and the use of lipid pathways during angiogenesis.

Integrated landscape of cardiac metabolism in end-stage human nonischemic dilated cardiomyopathy.

Heart failure (HF) is a leading cause of mortality. Failing hearts undergo profound metabolic changes, but a comprehensive evaluation in humans is lacking. We integrate plasma and cardiac tissue metabolomics of 678 metabolites, genome-wide RNA-sequencing, and proteomic studies to examine metabolic status in 87 explanted human hearts from 39 patients with end-stage HF compared with 48 nonfailing donors. We confirm bioenergetic defects in human HF and reveal selective depletion of adenylate purines required for maintaining ATP levels. We observe substantial reductions in fatty acids and acylcarnitines in failing tissue, despite plasma elevations, suggesting defective import of fatty acids into cardiomyocytes. Glucose levels, in contrast, are elevated. Pyruvate dehydrogenase, which gates carbohydrate oxidation, is de-repressed, allowing increased lactate and pyruvate burning. Tricarboxylic acid cycle intermediates are significantly reduced. Finally, bioactive lipids are profoundly reprogrammed, with marked reductions in ceramides and elevations in lysoglycerophospholipids. These data unveil profound metabolic abnormalities in human failing hearts.

Truncated titin proteins in dilated cardiomyopathy.

Truncating variants in TTN (TTNtvs) are the most common known cause of nonischemic dilated cardiomyopathy (DCM), but how TTNtvs cause disease has remained controversial. Efforts to detect truncated titin proteins in affected human DCM hearts have failed, suggesting that disease is caused by haploinsufficiency, but reduced amounts of titin protein have not yet been demonstrated. Here, we leveraged a collection of 184 explanted posttransplant DCM hearts to show, using specialized electrophoretic gels, Western blotting, allelic phasing, and unbiased proteomics, that truncated titin proteins can quantitatively be detected in human DCM hearts. The sizes of truncated proteins corresponded to that predicted by their respective TTNtvs; the truncated proteins were encoded by the TTNtv-bearing allele; and no degradation fragments from protein encoded by either allele were detectable. In parallel, full-length titin was less abundant in TTNtv+ than in TTNtv− DCM hearts. Disease severity or need for transplantation did not correlate with TTNtv location. Transcriptomic profiling revealed few differences in splicing or allelic imbalance of the TTN transcript between TTNtv+ and TTNtv− DCM hearts. Studies with isolated human adult cardiomyocytes revealed no defects in contractility in cells from TTNtv+ compared to TTNtv− DCM hearts. Together, these data demonstrate the presence of truncated titin protein in human TTNtv+ DCM, show reduced amounts of full-length titin protein in TTNtv+ DCM hearts, and support combined dominant-negative and haploinsufficiency contributions to disease.

Peripartum cardiomyopathy: from genetics to management.

Peripartum cardiomyopathy (PPCM) is a disease that occurs globally in all ethnic groups and should be suspected in any peripartum women presenting with symptoms and signs of heart failure, towards the end of pregnancy or in the months following delivery, with confirmed left ventricular dysfunction. After good history taking, all women should be thoroughly assessed, and alternative causes should be excluded. Urgent cardiac investigations with electrocardiogram and natriuretic peptide measurement (if available) should be performed. Echocardiography follows as the next step in investigation. Patients with abnormal cardiac investigations should be urgently referred to a cardiology team for expert management. Referral for genetic work-up should be considered if there is a family history of cardiomyopathy or sudden death. PPCM is a disease with substantial maternal and neonatal morbidity and mortality. Maternal mortality rates range widely, from 0% to 30%, depending on the ethnic background and geographic region. Just under half of women experience myocardial recovery. Remarkable advances in the comprehension of the pathogenesis and in patient management and therapy have been achieved, largely due to team efforts and close collaboration between basic scientists, cardiologists, intensive care specialists, and obstetricians. This review summarizes current knowledge of PPCM genetics, pathophysiology, diagnostic approach, management, and outcome.

Neighborhood education status drives racial disparities in clinical outcomes in PPCM.

BACKGROUND: Peripartum cardiomyopathy (PPCM) disproportionately affects women of African ancestry. Additionally, clinical outcomes are worse in this subpopulation compared to White women with PPCM.  The extent to which socioeconomic parameters contribute to these racial disparities is not known. METHODS: We aimed to quantify the association between area-based proxies of socioeconomic status (SES) and clinical outcomes in PPCM, and to determine the potential contribution of these factors to racial disparities in outcomes. A retrospective cohort study was performed at the University of Pennsylvania Health System, a tertiary referral center serving a population with a high proportion of Black individuals. The cohort included 220 women with PPCM, 55% of whom were Black or African American. Available data included clinical and demographic characteristics as well as residential address georeferenced to US Census-derived block group measures of SES. Rates of sustained cardiac dysfunction (defined as persistent LVEF <50%, LVAD placement, transplant, or death) were compared by race and block group-level measures of SES, and a composite neighborhood concentrated disadvantage index (NDI). The contributions of area-based socioeconomic parameters to the association between race and sustained cardiac dysfunction were quantified. RESULTS: Black race and higher NDI were both independently associated with sustained cardiac dysfunction (relative risk [RR] 1.63, confidence interval [CI] 1.13-2.36; and RR 1.29, CI 1.08-1.53, respectively). Following multivariable adjustment, effect size for NDI remained statistically significant, but effect size for Black race did not. The impact of low neighborhood education on racial disparities in outcomes was stronger than that of low neighborhood income (explaining 45% and 0% of the association with black race, respectively). After multivariate adjustment, only low area-based education persisted as significantly correlating with sustained cardiac dysfunction (RR 1.49; CI 1.02-2.17). CONCLUSIONS: Both Black race and NDI independently associate with adverse outcomes in women with PPCM in a single center study. Of the specific components of NDI, neighborhood low education was most strongly associated with clinical outcome and partially explained differences in race. These results suggest interventions targeting social determinants of health in disadvantaged communities may help to mitigate outcome disparities.

Genetic and Phenotypic Landscape of Peripartum Cardiomyopathy.

BACKGROUND: Peripartum cardiomyopathy (PPCM) occurs in ≈1:2000 deliveries in the United States and worldwide. The genetic underpinnings of PPCM remain poorly defined. Approximately 10% of women with PPCM harbor truncating variants in TTN (TTNtvs). Whether mutations in other genes can predispose to PPCM is not known. It is also not known if the presence of TTNtvs predicts clinical presentation or outcomes. Nor is it known if the prevalence of TTNtvs differs in women with PPCM and preeclampsia, the strongest risk factor for PPCM. METHODS: Women with PPCM were retrospectively identified from several US and international academic centers, and clinical information and DNA samples were acquired. Next-generation sequencing was performed on 67 genes, including TTN, and evaluated for burden of truncating and missense variants. The impact of TTNtvs on the severity of clinical presentation, and on clinical outcomes, was evaluated. RESULTS: Four hundred sixty-nine women met inclusion criteria. Of the women with PPCM, 10.4% bore TTNtvs (odds ratio=9.4 compared with 1.2% in the reference population; Bonferroni-corrected P [P*]=1.2×10-46). We additionally identified overrepresentation of truncating variants in FLNC (odds ratio=24.8, P*=7.0×10-8), DSP (odds ratio=14.9, P*=1.0×10-8), and BAG3 (odds ratio=53.1, P*=0.02), genes not previously associated with PPCM. This profile is highly similar to that found in nonischemic dilated cardiomyopathy. Women with TTNtvs had lower left ventricular ejection fraction on presentation than did women without TTNtvs (23.5% versus 29%, P=2.5×10-4), but did not differ significantly in timing of presentation after delivery, in prevalence of preeclampsia, or in rates of clinical recovery. CONCLUSIONS: This study provides the first extensive genetic and phenotypic landscape of PPCM and demonstrates that predisposition to heart failure is an important risk factor for PPCM. The work reveals a degree of genetic similarity between PPCM and dilated cardiomyopathy, suggesting that gene-specific therapeutic approaches being developed for dilated cardiomyopathy may also apply to PPCM, and that approaches to genetic testing in PPCM should mirror those taken in dilated cardiomyopathy. Last, the clarification of genotype/phenotype associations has important implications for genetic counseling.

Soluble Flt1 levels are associated with cardiac dysfunction in Black women with and without severe preeclampsia.

Background: We evaluate soluble fms-like tyrosine kinase-1 (sFlt-1) levels and cardiac function during pregnancy and postpartum among Black women with and without preeclampsia. Study design: Prospective longitudinal cohort study from 2015 to 2017 of Black women with preterm severe preeclampsia and normotensive pregnant controls.We obtained echocardiograms and sFlt-1 levels during pregnancy and postpartum. Results: 93 Black women were included (43 cases, 50 controls). Higher sFlt1 levels were correlated with worse longitudinal strain, diastolic dysfunction, decreased ventricular-arterial coupling, and increased chamber and arterial elastance at the time of preeclampsia diagnosis and postpartum. Conclusions: Higher sFlt1 levels are associated with cardiovascular dysfunction during pregnancy and postpartum.