In the embryonic heart, the activation of the mitochondrial electron transport chain (ETC) coincides with the closure of the cyclophilin D (CypD) regulated mitochondrial permeability transition pore ...(mPTP). However, it remains to be established whether the absence of CypD has a regulatory effect on mitochondria during cardiac development. Using a variety of assays to analyze cardiac tissue from wildtype and CypD knockout mice from embryonic day (E)9.5 to adult, we found that mitochondrial structure, function, and metabolism show distinct transitions. Deletion of CypD altered the timing of these transitions as the mPTP was closed at all ages, leading to coupled ETC activity in the early embryo, decreased citrate synthase activity, and an altered metabolome particularly after birth. Our results suggest that manipulating CypD activity may control myocyte proliferation and differentiation and could be a tool to increase ATP production and cardiac function in immature hearts.
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•Bioenergetic capacity increases during cardiac development•These changes correlate to the physiologic state and energetic demands•CypD deletion alters this metabolic trajectory, notably in the embryo and newborn•Manipulation of CypD activity might increase cardiac function in immature hearts
Biological sciences; Physiology; Cell biology; Metabolomics
Dysregulated metabolism of bioactive sphingolipids, including ceramides and sphingosine-1-phosphate, has been implicated in cardiovascular disease, although the specific species, disease contexts, ...and cellular roles are not completely understood. Sphingolipids are produced by the serine palmitoyltransferase enzyme, canonically composed of 2 subunits, SPTLC1 (serine palmitoyltransferase long chain base subunit 1) and SPTLC2 (serine palmitoyltransferase long chain base subunit 2). Noncanonical sphingolipids are produced by a more recently described subunit, SPTLC3 (serine palmitoyltransferase long chain base subunit 3).
The noncanonical (d16) and canonical (d18) sphingolipidome profiles in cardiac tissues of patients with end-stage ischemic cardiomyopathy and in mice with ischemic cardiomyopathy were analyzed by targeted lipidomics. Regulation of SPTLC3 by HIF1α under ischemic conditions was determined with chromatin immunoprecipitation. Transcriptomics, lipidomics, metabolomics, echocardiography, mitochondrial electron transport chain, mitochondrial membrane fluidity, and mitochondrial membrane potential were assessed in the cSPTLC3
transgenic mice we generated. Furthermore, morphological and functional studies were performed on cSPTLC3
mice subjected to permanent nonreperfused myocardial infarction.
Herein, we report that SPTLC3 is induced in both human and mouse models of ischemic cardiomyopathy and leads to production of atypical sphingolipids bearing 16-carbon sphingoid bases, resulting in broad changes in cell sphingolipid composition. This induction is in part attributable to transcriptional regulation by HIF1α under ischemic conditions. Furthermore, cardiomyocyte-specific depletion of SPTLC3 in mice attenuates oxidative stress, fibrosis, and hypertrophy in chronic ischemia, and mice demonstrate improved cardiac function and increased survival along with increased ketone and glucose substrate metabolism utilization. Depletion of SPTLC3 mechanistically alters the membrane environment and subunit composition of mitochondrial complex I of the electron transport chain, decreasing its activity.
Our findings suggest a novel essential role for SPTLC3 in electron transport chain function and a contribution to ischemic injury by regulating complex I activity.
In embryonic myocytes, closure of the mitochondrial permeability transition pore (PTP) drives mitochondrial maturation and cardiac myocyte differentiation. Since neonatal cardiac myocytes remain ...relatively immature, we hypothesized that inducing PTP closure at this age, by inhibiting the PTP regulator, cyclophilin D (CyPD), genetically or with Cyclosporin A (CsA) and NIM811, would increase cardiac function by increasing mitochondrial maturation and myocyte differentiation.
Cultured neonatal myocytes or neonatal mice were treated for 5 d with vehicle, CsA or NIM811. Mitochondrial function and structure were measured in vitro. Myocyte differentiation was assessed by immunolabeling for contractile proteins. Cardiac function was determined using echocardiography.
The probability of PTP opening was high in WT neonatal myocytes. Treatment with CsA or NIM811 in vitro increased mitochondrial structural complexity and membrane potential, decreased reactive oxygen species levels, and increased myocyte differentiation. WT mice treated with either CsA or NIM811 in vivo for the first 5 d of life had higher ejection fractions. Deleting CyPD had similar effects as CsA and NIM811 on all parameters.
It may be feasible to inhibit the PTP using available drugs to increase mitochondrial maturation, myocyte differentiation, and cardiac function in neonates.
Preterm birth increases the risk for pulmonary hypertension and heart failure in adulthood. Oxygen therapy can damage the immature cardiopulmonary system and may be partially responsible for the ...cardiovascular disease in adults born preterm. We previously showed that exposing newborn mice to hyperoxia causes pulmonary hypertension by 1 year of age that is preceded by a poorly understood loss of pulmonary vein cardiomyocyte proliferation. We now show that hyperoxia also reduces cardiomyocyte proliferation and survival in the left atrium and causes diastolic heart failure by disrupting its filling of the left ventricle. Transcriptomic profiling showed that neonatal hyperoxia permanently suppressed fatty acid synthase (Fasn), stearoyl-CoA desaturase 1 (Scd1), and other fatty acid synthesis genes in the atria of mice, the HL-1 line of mouse atrial cardiomyocytes, and left atrial tissue explanted from human infants. Suppressing Fasn or Scd1 reduced HL-1 cell proliferation and increased cell death, while overexpressing these genes maintained their expansion in hyperoxia, suggesting that oxygen directly inhibits atrial cardiomyocyte proliferation and survival by repressing Fasn and Scd1. Pharmacologic interventions that restore Fasn, Scd1, and other fatty acid synthesis genes in atrial cardiomyocytes may, thus, provide a way of ameliorating the adverse effects of supplemental oxygen on preterm infants.
Objective
Persons with congenital heart disease (CHD) are at increased risk of neurodevelopmental disabilities, including impairments to executive function. Sulcal pattern features correlate with ...executive function in adolescents with single‐ventricle heart disease and tetralogy of Fallot. However, the interaction of sulcal pattern features with genetic and participant factors in predicting executive dysfunction is unknown.
Methods
We studied sulcal pattern features, participant factors, and genetic risk for executive function impairment in a cohort with multiple CHD types using stepwise linear regression and machine learning.
Results
Genetic factors, including predicted damaging de novo or rare inherited variants in neurodevelopmental disabilities risk genes, apolipoprotein E genotype, and principal components of sulcal pattern features were associated with executive function measures after adjusting for age at testing, sex, mother's education, and biventricular versus single‐ventricle CHD in a linear regression model. Using regression trees and bootstrap validation, younger participant age and larger alterations in sulcal pattern features were consistently identified as important predictors of decreased cognitive flexibility with left hemisphere graph topology often selected as the most important predictor. Inclusion of both sulcal pattern and genetic factors improved model fit compared to either alone.
Interpretation
We conclude that sulcal measures remain important predictors of cognitive flexibility, and the model predicting executive outcomes is improved by inclusion of potential genetic sources of neurodevelopmental risk. If confirmed, measures of sulcal patterning may serve as early imaging biomarkers to identify those at heightened risk for future neurodevelopmental disabilities.
Cardiac energy demands during early embryonic periods are sufficiently met through glycolysis, but as development proceeds, the oxidative phosphorylation in mitochondria becomes increasingly vital. ...Adrenergic hormones are known to stimulate metabolism in adult mammals and are essential for embryonic development, but relatively little is known about their effects on metabolism in the embryonic heart. Here, we show that embryos lacking adrenergic stimulation have ∼10-fold less cardiac ATP compared with littermate controls. Despite this deficit in steady-state ATP, neither the rates of ATP formation nor degradation was affected in adrenergic hormone-deficient hearts, suggesting that ATP synthesis and hydrolysis mechanisms were fully operational. We thus hypothesized that adrenergic hormones stimulate metabolism of glucose to provide chemical substrates for oxidation in mitochondria. To test this hypothesis, we employed a metabolomics-based approach using LC/MS. Our results showed glucose 1-phosphate and glucose 6-phosphate concentrations were not significantly altered, but several downstream metabolites in both glycolytic and pentose–phosphate pathways were significantly lower compared with controls. Furthermore, we identified glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphate dehydrogenase as key enzymes in those respective metabolic pathways whose activity was significantly (p < 0.05) and substantially (80 and 40%, respectively) lower in adrenergic hormone-deficient hearts. Addition of pyruvate and to a lesser extent ribose led to significant recovery of steady-state ATP concentrations. These results demonstrate that without adrenergic stimulation, glucose metabolism in the embryonic heart is severely impaired in multiple pathways, ultimately leading to insufficient metabolic substrate availability for successful transition to aerobic respiration needed for survival.
In myocytes, calcium plays an important role in intracellular signaling and contraction. However, the ability of calcium to
modulate the differentiation of striated muscle cells is poorly understood. ...To examine this issue we studied C2C12 cells,
which is a myoblast cell line that differentiates in vitro . First, we observed that the L-type calcium channel blockers nifedipine and verapamil effectively inhibited electrically
induced calcium transients. Next, C2C12 cells were exposed to these agents during conditions that induce myocyte differentiation.
In the presence of nifedipine and verapamil, myoblasts failed to form myotubes. Dantrolene and thapsigargin, which decrease
intracellular calcium by different mechanisms, also inhibited differentiation. In addition, nifedipine and verapamil inhibited
the expression of myosin heavy chain and myogenin, two markers of skeletal myoblast differentiation. In contrast, levels of
the transcriptional factor Myf5, which is expressed in undifferentiated myoblasts, did not decline. Calcium channel blockade
also prevented the expression of a reporter driven by the skeletal muscle α-actin promoter. These data demonstrate that lowering
intracellular calcium levels inhibits the differentiation of skeletal myoblasts into mature myotubes.
Congenital heart disease (CHD) is the most common major congenital anomaly and causes significant morbidity and mortality. Epidemiologic evidence supports a role of genetics in the development of ...CHD. Genetic diagnoses can inform prognosis and clinical management. However, genetic testing is not standardized among individuals with CHD. We sought to develop a list of validated CHD genes using established methods and to evaluate the process of returning genetic results to research participants in a large genomic study.
Two-hundred ninety-five candidate CHD genes were evaluated using a ClinGen framework. Sequence and copy number variants involving genes in the CHD gene list were analyzed in Pediatric Cardiac Genomics Consortium participants. Pathogenic/likely pathogenic results were confirmed on a new sample in a clinical laboratory improvement amendments-certified laboratory and disclosed to eligible participants. Adult probands and parents of probands who received results were asked to complete a post-disclosure survey.
A total of 99 genes had a strong or definitive clinical validity classification. Diagnostic yields for copy number variants and exome sequencing were 1.8% and 3.8%, respectively. Thirty-one probands completed clinical laboratory improvement amendments-confirmation and received results. Participants who completed postdisclosure surveys reported high personal utility and no decision regret after receiving genetic results.
The application of ClinGen criteria to CHD candidate genes yielded a list that can be used to interpret clinical genetic testing for CHD. Applying this gene list to one of the largest research cohorts of CHD participants provides a lower bound for the yield of genetic testing in CHD.
Known genetic causes of congenital heart disease (CHD) explain <40% of CHD cases, and interpreting the clinical significance of variants with uncertain functional impact remains challenging. We aim ...to improve diagnostic classification of variants in patients with CHD by assessing the impact of noncanonical splice region variants on RNA splicing.
We tested de novo variants from trio studies of 2649 CHD probands and their parents, as well as rare (allele frequency, <2×10
) variants from 4472 CHD probands in the Pediatric Cardiac Genetics Consortium through a combined computational and in vitro approach.
We identified 53 de novo and 74 rare variants in CHD cases that alter splicing and thus are loss of function. Of these, 77 variants are in known dominant, recessive, and candidate CHD genes, including
and
. In 1 case, we confirmed the variant's predicted impact on RNA splicing in RNA transcripts from the proband's cardiac tissue. Two probands were found to have 2 loss-of-function variants for recessive CHD genes
and
. In addition, SpliceAI-a predictive algorithm for altered RNA splicing-has a positive predictive value of ≈93% in our cohort.
Through assessment of RNA splicing, we identified a new loss-of-function variant within a CHD gene in 78 probands, of whom 69 (1.5%; n=4472) did not have a previously established genetic explanation for CHD. Identification of splice-altering variants improves diagnostic classification and genetic diagnoses for CHD.
URL: https://clinicaltrials.gov; Unique identifier: NCT01196182.
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