The transcription factor EBF1 non-cell-autonomously regulates cardiac growth and differentiation
Reciprocal interactions between non-myocytes and cardiomyocytes regulate cardiac growth and differentiation. Here, we report that the transcription factor Ebf1 is highly expressed in non-myocytes and potently regulates heart development. Ebf1-deficient hearts display myocardial hypercellularity and reduced cardiomyocyte size, ventricular conduction system hypoplasia, and conduction system disease. Growth abnormalities in Ebf1 knockout hearts are observed as early as embryonic day 13.5. Transcriptional profiling of Ebf1-deficient embryonic cardiac non-myocytes demonstrates dysregulation of Polycomb repressive complex 2 targets, and ATAC-Seq reveals altered chromatin accessibility near many of these same genes. Gene set enrichment analysis of differentially expressed genes in cardiomyocytes isolated from E13.5 hearts of wild-type and mutant mice reveals significant enrichment of MYC targets and, consistent with this finding, we observe increased abundance of MYC in mutant hearts. EBF1-deficient non-myocytes, but not wild-type non-myocytes, are sufficient to induce excessive accumulation of MYC in co-cultured wild-type cardiomyocytes. Finally, we demonstrate that BMP signaling induces Ebf1 expression in embryonic heart cultures and controls a gene program enriched in EBF1 targets. These data reveal a previously unreported non-cell-autonomous pathway controlling cardiac growth and differentiation.
TGFβ-2 Haploinsufficiency Causes Early Death in Mice with Marfan Syndrome
To assess the contribution of individual TGF-β isoforms to aortopathy in Marfan syndrome (MFS), we quantified the survival and phenotypes of mice with a combined fibrillin1 (the gene defective in MFS) hypomorphic mutation and a TGF-β1, 2, or 3 heterozygous null mutation. The loss of TGF-β2, and only TGF-β2, resulted in 80% of the double mutant animals dying earlier, by post-natal day 20, than MFS only mice. Death was not from thoracic aortic rupture, as observed in MFS mice, but was associated with hyperplastic aortic valve leaflets, aortic regurgitation, enlarged aortic root, increased heart weight, and impaired lung alveolar septation. Thus, there appears to be a relationship between loss of fibrillin1 and TGF-β2 in the post-natal development of the heart, aorta and lungs.
Purkinje Cardiomyocytes of the Adult Ventricular Conduction System Are Highly Diploid but Not Uniquely Regenerative
Adult hearts are characterized by inefficient regeneration after injury, thus, the features that support or prevent cardiomyocyte (CM) proliferation are important to clarify. Diploid CMs are a candidate cell type that may have unique proliferative and regenerative competence, but no molecular markers are yet known that selectively identify all or subpopulations of diploid CMs. Here, using the conduction system expression marker Cntn2-GFP and the conduction system lineage marker Etv1CreERT2, we demonstrate that Purkinje CMs that comprise the adult ventricular conduction system are disproportionately diploid (33%, vs. 4% of bulk ventricular CMs). These, however, represent only a small proportion (3%) of the total diploid CM population. Using EdU incorporation during the first postnatal week, we demonstrate that bulk diploid CMs found in the later heart enter and complete the cell cycle during the neonatal period. In contrast, a significant fraction of conduction CMs persist as diploid cells from fetal life and avoid neonatal cell cycle activity. Despite their high degree of diploidy, the Purkinje lineage had no enhanced competence to support regeneration after adult heart infarction.
Chronic Kidney Disease Induces Proarrhythmic Remodeling
BACKGROUND:Patients with chronic kidney disease (CKD) are at increased risk of developing cardiac arrhythmogenesis and sudden cardiac death; however, the basis for this association is incompletely known. METHODS:Here, using murine models of CKD, we examined interactions between kidney disease progression and structural, electrophysiological, and molecular cardiac remodeling. RESULTS:C57BL/6 mice with adenine supplemented in their diet developed progressive CKD. Electrocardiographically, CKD mice developed significant QT prolongation and episodes of bradycardia. Optical mapping of isolated-perfused hearts using voltage-sensitive dyes revealed significant prolongation of action potential duration with no change in epicardial conduction velocity. Patch-clamp studies of isolated ventricular cardiomyocytes revealed changes in sodium and potassium currents consistent with action potential duration prolongation. Global transcriptional profiling identified dysregulated expression of cellular stress response proteins RBM3 (RNA-binding motif protein 3) and CIRP (cold-inducible RNA-binding protein) that may underlay the ion channel remodeling. Unexpectedly, we found that female sex is a protective factor in the progression of CKD and its cardiac sequelae. CONCLUSIONS:Our data provide novel insights into the association between CKD and pathologic proarrhythmic cardiac remodeling. Cardiac cellular stress response pathways represent potential targets for pharmacologic intervention for CKD-induced heart rhythm disorders.
Sex differences in the prognostic value of troponin and D-dimer in COVID-19 illness
BACKGROUND:Male sex, elevated troponin levels, and elevated D-dimer levels are associated with more complicated COVID-19 illness and greater mortality; however, while there are known sex differences in the prognostic value of troponin and D-dimer in other disease states, it is unknown whether they exist in the setting of COVID-19. OBJECTIVE:We assessed whether sex modified the relationship between troponin, D-dimer, and severe COVID-19 illness (defined as mechanical ventilation, ICU admission or transfer, discharge to hospice, or death). METHODS:We conducted a retrospective cohort study of patients hospitalized with COVID-19 at a large, academic health system. We used multivariable regression to assess associations between sex, troponin, D-dimer, and severe COVID-19 illness, adjusting for demographic, clinical, and laboratory covariates. To test whether sex modified the relationship between severe COVID-19 illness and troponin or D-dimer, models with interaction terms were utilized. RESULTS:Among 4,574 patients hospitalized with COVID-19, male sex was associated with higher levels of troponin and greater odds of severe COVID-19 illness, but lower levels of initial D-dimer when compared with female sex. While sex did not modify the relationship between troponin level and severe COVID-19 illness, peak D-dimer level was more strongly associated with severe COVID-19 illness in male patients compared to female patients (males: OR=2.91, 95%CI=2.63-2.34, p<0.001; females: OR=2.31, 95%CI=2.04-2.63, p<0.001; p-interaction=0.005). CONCLUSION/CONCLUSIONS:Sex did not modify the association between troponin level and severe COVID-19 illness, but did modify the association between peak D-dimer and severe COVID-19 illness, suggesting greater prognostic value for D-dimer in males with COVID-19.
Contrasting Ionic Mechanisms of Impaired Conduction in FHF1- and FHF2-Deficient Hearts [Letter]
Partial and complete loss of myosin binding protein H-like cause cardiac conduction defects
A premature truncation of MYBPHL in humans and a loss of Mybphl in mice is associated with dilated cardiomyopathy, atrial and ventricular arrhythmias, and atrial enlargement. MYBPHL encodes myosin binding protein H-like (MyBP-HL). Prior work in mice indirectly identified Mybphl expression in the atria and in small puncta throughout the ventricle. Because of its genetic association with human and mouse cardiac conduction system disease, we evaluated the anatomical localization of MyBP-HL and the consequences of loss of MyBP-HL on conduction system function. Immunofluorescence microscopy of normal adult mouse ventricles identified MyBP-HL-positive ventricular cardiomyocytes that co-localized with the ventricular conduction system marker contactin-2 near the atrioventricular node and in a subset of Purkinje fibers. Mybphl heterozygous ventricles had a marked reduction of MyBP-HL-positive cells compared to controls. Lightsheet microscopy of normal perinatal day 5 mouse hearts showed enrichment of MyBP-HL-positive cells within and immediately adjacent to the contactin-2-positive ventricular conduction system, but this association was not apparent in Mybphl heterozygous hearts. Surface telemetry of Mybphl-null mice revealed atrioventricular block and atrial bigeminy, while intracardiac pacing revealed a shorter atrial relative refractory period and atrial tachycardia. Calcium transient analysis of isolated Mybphl-null atrial cardiomyocytes demonstrated an increased heterogeneity of calcium release and faster rates of calcium release compared to wild type controls. Super-resolution microscopy of Mybphl heterozygous and homozygous null atrial cardiomyocytes showed ryanodine receptor disorganization compared to wild type controls. Abnormal calcium release, shorter atrial refractory period, and atrial dilation seen in Mybphl null, but not wild type control hearts, agree with the observed atrial arrhythmias, bigeminy, and atrial tachycardia, whereas the proximity of MyBP-HL-positive cells with the ventricular conduction system provides insight into how a predominantly atrial expressed gene contributes to ventricular arrhythmias and ventricular dysfunction.
The Purkinje-myocardial junction is the anatomical origin of ventricular arrhythmia in CPVT
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an arrhythmia syndrome due to gene mutations that render RYR2 calcium release channels hyperactive, causing spontaneous calcium release and delayed afterdepolarizations (DADs). What remains unknown is the cellular source of ventricular arrhythmia triggered by DADs - Purkinje cells in the conduction system or ventricular cardiomyocytes in the working myocardium. To answer this question, we used a genetic approach in mice to knock out cardiac calsequestrin either in Purkinje cells or in ventricular cardiomyocytes. Total loss of calsequestrin in the heart causes a severe CPVT phenotype in mice and humans. We found that loss of calsequestrin only in ventricular myocytes produced a full-blown CPVT phenotype, whereas mice with loss of calsequestrin only in Purkinje cells were comparable to wild-type mice. Subendocardial chemical ablation or restoration of calsequestrin expression in subendocardial cardiomyocytes neighboring Purkinje cells was sufficient to protect against catecholamine-induced arrhythmias. In silico modeling demonstrated that DADs in ventricular myocardium can trigger full action potentials in the Purkinje fiber, but not vice versa. Hence, ectopic beats in CPVT are likely generated at the Purkinje-myocardial junction via a heretofore unrecognized tissue mechanism, whereby DADs in the ventricular myocardium trigger full action potentials in adjacent Purkinje cells.
A pilot open-label study of aldose reductase inhibition with AT-001 (caficrestat) in patients hospitalized for COVID-19 infection: Results from a registry-based matched-control analysis
BACKGROUND AND AIMS/OBJECTIVE:Cardiometabolic disease may confer increased risk of adverse outcomes in COVID-19 patients by activation of the aldose reductase pathway. We hypothesized that aldose reductase inhibition with AT-001 might reduce viral inflammation and risk of adverse outcomes in diabetic patients with COVID-19. METHODS:We conducted an open-label prospective phase 2 clinical trial to assess safety, tolerability and efficacy of AT-001 in patients hospitalized with COVID-19 infection, history of diabetes mellitus and chronic heart disease. Eligible participants were prospectively enrolled and treated with AT-001 1500Â mg BID for up to 14 days. Safety, tolerability, survival and length of hospital stay (LOS) were collected from the electronic medical record and compared with data from two matched control groups (MC1 and MC2) selected from a deidentified registry of COVID-19 patients at the same institution. RESULTS:AT-001 was safe and well tolerated in the 10 participants who received the study drug. In-hospital mortality observed in the AT-001 group was 20% vs. 31% in MC1 and 27% in MC2. Mean LOS observed in the AT-001 group was 5 days vs. 10 days in MC1 and 25 days in MC2. CONCLUSIONS:In hospitalized patients with COVID-19 and co-morbid diabetes mellitus and heart disease, treatment with AT-001 was safe and well tolerated. Exposure to AT-001 was associated with a trend of reduced mortality and shortened LOS. While the observed trend did not reach statistical significance, the present study provides the rationale for investigating potential benefit of AT-001 in COVID 19 affected patients in future studies.
Early onset epilepsy and sudden unexpected death in epilepsy with cardiac arrhythmia in mice carrying the early infantile epileptic encephalopathy 47 gain-of-function FHF1(FGF12) missense mutation
OBJECTIVE:Fibroblast growth factor homologous factors (FHFs) are brain and cardiac sodium channel-binding proteins that modulate channel density and inactivation gating. A recurrent de novo gain-of-function missense mutation in the FHF1(FGF12) gene (p.Arg52His) is associated with early infantile epileptic encephalopathy 47 (EIEE47; Online Mendelian Inheritance in Man database 617166). To determine whether the FHF1 missense mutation is sufficient to cause EIEE and to establish an animal model for EIEE47, we sought to engineer this mutation into mice. METHODS:protein. RESULTS:mice prior to seizure. SIGNIFICANCE/CONCLUSIONS:1.6 functional axis underlying altered brain sodium channel gating in epileptic encephalopathy.