Single cell sequencing data identify distinct B cell and fibroblast populations in stricturing Crohn's disease.

Single cell RNA sequencing of human full thickness Crohn's disease (CD) small bowel resection specimens was used to identify potential therapeutic targets for stricturing (S) CD. Using an unbiased approach, 16 cell lineages were assigned within 14,539 sequenced cells from patient-matched SCD and non-stricturing (NSCD) preparations. SCD and NSCD contained identical cell types. Amongst immune cells, B cells and plasma cells were selectively increased in SCD samples. B cell subsets suggested formation of tertiary lymphoid tissue in SCD and compared with NSCD there was an increase in IgG, and a decrease in IgA plasma cells, consistent with their potential role in CD fibrosis. Two Lumican-positive fibroblast subtypes were identified and subclassified based on expression of selectively enriched genes as fibroblast clusters (C) 12 and C9. Cells within these clusters expressed the profibrotic genes Decorin (C12) and JUN (C9). C9 cells expressed ACTA2; ECM genes COL4A1, COL4A2, COL15A1, COL6A3, COL18A1 and ADAMDEC1; LAMB1 and GREM1. GO and KEGG Biological terms showed extracellular matrix and stricture organization associated with C12 and C9, and regulation of WNT pathway genes with C9. Trajectory and differential gene analysis of C12 and C9 identified four sub-clusters. Intra sub-cluster gene analysis detected 13 co-regulated gene modules that aligned along predicted pseudotime trajectories. CXCL14 and ADAMDEC1 were key markers in module 1. Our findings support further investigation of fibroblast heterogeneity and interactions with local and circulating immune cells at earlier time points in fibrosis progression. Breaking these interactions by targeting one or other population may improve therapeutic management for SCD.

Epigenetic and metabolic reprogramming of fibroblasts in Crohn's disease strictures reveals histone deacetylases as therapeutic targets.

BACKGROUND & AIMS: No effective therapeutic intervention exists for intestinal fibrosis in Crohn's disease [CD]. We characterised fibroblast subtypes, epigenetic and metabolic changes, and signalling pathways in CD fibrosis to inform future therapeutic strategies. METHODS: We undertook immunohistochemistry, metabolic, signalling pathway and Epigenetic [Transposase-Accessible Chromatin using sequencing] analyses associated with collagen production in CCD-18Co intestinal fibroblasts and primary fibroblasts isolated from stricturing [SCD] and non-stricturing [NSCD] CD small intestine. SCD/ NSCD fibroblasts were cultured with TGFß and valproic acid [VPA]. RESULTS: Stricturing CD was characterised by distinct histone deacetylase [HDAC] expression profiles, particularly HDAC1, HDAC2, and HDAC7. As a proxy for HDAC activity, reduced numbers of H3K27ac+ cells were found in SCD compared to NSCD sections. Primary fibroblasts had increased extracellular lactate [increased glycolytic activity] and intracellular hydroxyproline [increased collagen production] in SCD compared to NSCD cultures. The metabolic effect of TGFß-stimulation was reversed by the HDAC inhibitor VPA. SCD fibroblasts appear "metabolically primed" and responded more strongly to both TGFß and VPA. Treatment with VPA revealed TGFß-dependent and independent Collagen-I production in CCD-18Co cells and primary fibroblasts. VPA altered the epigenetic landscape with reduced chromatin accessibility at the COL1A1 and COL1A2 promoters. CONCLUSIONS: Increased HDAC expression profiles, H3K27ac hypoacetylation, a significant glycolytic phenotype, and metabolic priming, characterise SCD-derived as compared to NSCD fibroblasts. Our results reveal a novel epigenetic component to Collagen-I regulation and TGFß-mediated CD fibrosis. HDAC inhibitor therapy may 'reset' the epigenetic changes associated with fibrosis.

Fibulin-3 is necessary to prevent cardiac rupture following myocardial infarction.

Despite the high prevalence of heart failure in the western world, there are few effective treatments. Fibulin-3 is a protein involved in extracellular matrix (ECM) structural integrity, however its role in the heart is unknown. We have demonstrated, using single cell RNA-seq, that fibulin-3 was highly expressed in quiescent murine cardiac fibroblasts, with expression highest prior to injury and late post-infarct (from ~ day-28 to week-8). In humans, fibulin-3 was upregulated in left ventricular tissue and plasma of heart failure patients. Fibulin-3 knockout (Efemp1-/-) and wildtype mice were subjected to experimental myocardial infarction. Fibulin-3 deletion resulted in significantly higher rate of cardiac rupture days 3-6 post-infarct, indicating a weak and poorly formed scar, with severe ventricular remodelling in surviving mice at day-28 post-infarct. Fibulin-3 knockout mice demonstrated less collagen deposition at day-3 post-infarct, with abnormal collagen fibre-alignment. RNA-seq on day-3 infarct tissue revealed upregulation of ECM degradation and inflammatory genes, but downregulation of ECM assembly/structure/organisation genes in fibulin-3 knockout mice. GSEA pathway analysis showed enrichment of inflammatory pathways and a depletion of ECM organisation pathways. Fibulin-3 originates from cardiac fibroblasts, is upregulated in human heart failure, and is necessary for correct ECM organisation/structural integrity of fibrotic tissue to prevent cardiac rupture post-infarct.

Characterising the RNA-binding protein atlas of the mammalian brain uncovers RBM5 misregulation in mouse models of Huntington's disease.

RNA-binding proteins (RBPs) are key players regulating RNA processing and are associated with disorders ranging from cancer to neurodegeneration. Here, we present a proteomics workflow for large-scale identification of RBPs and their RNA-binding regions in the mammalian brain identifying 526 RBPs. Analysing brain tissue from males of the Huntington's disease (HD) R6/2 mouse model uncovered differential RNA-binding of the alternative splicing regulator RBM5. Combining several omics workflows, we show that RBM5 binds differentially to transcripts enriched in pathways of neurodegeneration in R6/2 brain tissue. We further find these transcripts to undergo changes in splicing and demonstrate that RBM5 directly regulates these changes in human neurons derived from embryonic stem cells. Finally, we reveal that RBM5 interacts differently with several known huntingtin interactors and components of huntingtin aggregates. Collectively, we demonstrate the applicability of our method for capturing RNA interactor dynamics in the contexts of tissue and disease.

Quantitative trait and transcriptome analysis of genetic complexity underpinning cardiac interatrial septation in mice using an advanced intercross line.

Unlike single-gene mutations leading to Mendelian conditions, common human diseases are likely to be emergent phenomena arising from multilayer, multiscale, and highly interconnected interactions. Atrial and ventricular septal defects are the most common forms of cardiac congenital anomalies in humans. Atrial septal defects (ASD) show an open communication between the left and right atria postnatally, potentially resulting in serious hemodynamic consequences if untreated. A milder form of atrial septal defect, patent foramen ovale (PFO), exists in about one-quarter of the human population, strongly associated with ischaemic stroke and migraine. The anatomic liabilities and genetic and molecular basis of atrial septal defects remain unclear. Here, we advance our previous analysis of atrial septal variation through quantitative trait locus (QTL) mapping of an advanced intercross line (AIL) established between the inbred QSi5 and 129T2/SvEms mouse strains, that show extremes of septal phenotypes. Analysis resolved 37 unique septal QTL with high overlap between QTL for distinct septal traits and PFO as a binary trait. Whole genome sequencing of parental strains and filtering identified predicted functional variants, including in known human congenital heart disease genes. Transcriptome analysis of developing septa revealed downregulation of networks involving ribosome, nucleosome, mitochondrial, and extracellular matrix biosynthesis in the 129T2/SvEms strain, potentially reflecting an essential role for growth and cellular maturation in septal development. Analysis of variant architecture across different gene features, including enhancers and promoters, provided evidence for the involvement of non-coding as well as protein-coding variants. Our study provides the first high-resolution picture of genetic complexity and network liability underlying common congenital heart disease, with relevance to human ASD and PFO.

The rapid evolution of flagellar ion selectivity in experimental populations of E. coli.

Determining which cellular processes facilitate adaptation requires a tractable experimental model where an environmental cue can generate variants that rescue function. The bacterial flagellar motor (BFM) is an excellent candidate-an ancient and highly conserved molecular complex for bacterial propulsion toward favorable environments. Motor rotation is often powered by H+ or Na+ ion transit through the torque-generating stator subunit of the motor complex, and ion selectivity has adapted over evolutionary time scales. Here, we used CRISPR engineering to replace the native Escherichia coli H+-powered stator with Na+-powered stator genes and report the spontaneous reversion of our edit in a low-sodium environment. We followed the evolution of the stators during their reversion to H+-powered motility and used both whole-genome and RNA sequencing to identify genes involved in the cell's adaptation. Our transplant of an unfit protein and the cells' rapid response to this edit demonstrate the adaptability of the stator subunit and highlight the hierarchical modularity of the flagellar motor.

Myhre syndrome is caused by dominant-negative dysregulation of SMAD4 and other co-factors.

Myhre syndrome is a connective tissue disorder characterized by congenital cardiovascular, craniofacial, respiratory, skeletal, and cutaneous anomalies as well as intellectual disability and progressive fibrosis. It is caused by germline variants in the transcriptional co-regulator SMAD4 that localize at two positions within the SMAD4 protein, I500 and R496, with I500 V/T/M variants more commonly identified in individuals with Myhre syndrome. Here we assess the functional impact of SMAD4-I500V variant, identified in two previously unpublished individuals with Myhre syndrome, and provide novel insights into the molecular mechanism of SMAD4-I500V dysfunction. We show that SMAD4-I500V can dimerize, but its transcriptional activity is severely compromised. Our data show that SMAD4-I500V acts dominant-negatively on SMAD4 and on receptor-regulated SMADs, affecting transcription of target genes. Furthermore, SMAD4-I500V impacts the transcription and function of crucial developmental transcription regulator, NKX2-5. Overall, our data reveal a dominant-negative model of disease for SMAD4-I500V where the function of SMAD4 encoded on the remaining allele, and of co-factors, are perturbed by the continued heterodimerization of the variant, leading to dysregulation of TGF and BMP signaling. Our findings not only provide novel insights into the mechanism of Myhre syndrome pathogenesis but also extend the current knowledge of how pathogenic variants in SMAD proteins cause disease.

Standardised method for cardiomyocyte isolation and purification from individual murine neonatal, infant, and adult hearts.

Primary cardiomyocytes are invaluable for understanding postnatal heart development. However, a universal method to obtain freshly purified cardiomyocytes without using different age-dependent isolation procedures and cell culture, is lacking. Here, we report the development of a standardised method that allows rapid isolation and purification of high-quality cardiomyocytes from individual neonatal through to adult C57BL/6J murine hearts. Langendorff retrograde perfusion, which is currently limited to adult hearts, was adapted for use in neonatal and infant hearts by developing an easier in situ aortic cannulation technique. Tissue digestion conditions were optimised to achieve efficient digestion of hearts of all ages in a comparable timeframe (<14 min). This resulted in a high yield (1.56-2.2 × 106 cells/heart) and viability (~70-100%) of cardiomyocytes post-isolation. An immunomagnetic cell separation step was then applied to yield highly purified cardiomyocytes (~95%) as confirmed by immunocytochemistry, flow cytometry, and qRT-PCR. For cell type-specific studies, cardiomyocyte DNA, RNA, and protein could be extracted in sufficient yields to conduct molecular experiments. We generated transcriptomic datasets for neonatal cardiomyocytes from individual hearts, for the first time, which revealed nine sex-specific genes (FDR < 0.05) encoded on the sex chromosomes. Finally, we also developed an in situ fixation protocol that preserved the native cytoarchitecture of cardiomyocytes (~94% rod-shaped post-isolation), and used it to evaluate cell morphology during cardiomyocyte maturation, as well as capture spindle-shaped neonatal cells undergoing cytokinesis. Together, these procedures allow molecular and morphological profiling of high-quality cardiomyocytes from individual hearts of any postnatal age.

Exploring the Genetic Architecture of Spontaneous Coronary Artery Dissection Using Whole-Genome Sequencing.

BACKGROUND: Spontaneous coronary artery dissection (SCAD) is a cause of acute coronary syndrome that predominantly affects women. Its pathophysiology remains unclear but connective tissue disorders (CTD) and other vasculopathies have been observed in many SCAD patients. A genetic component for SCAD is increasingly appreciated, although few genes have been robustly implicated. We sought to clarify the genetic cause of SCAD using targeted and genome-wide methods in a cohort of sporadic cases to identify both common and rare disease-associated variants. METHODS: A cohort of 91 unrelated sporadic SCAD cases was investigated for rare, deleterious variants in genes associated with either SCAD or CTD, while new candidate genes were sought using rare variant collapsing analysis and identification of novel loss-of-function variants in genes intolerant to such variation. Finally, 2 SCAD polygenic risk scores were applied to assess the contribution of common variants. RESULTS: We identified 10 cases with at least one rare, likely disease-causing variant in CTD-associated genes, although only one had a CTD phenotype. No genes were significantly associated with SCAD from genome-wide collapsing analysis, however, enrichment for TGF (transforming growth factor)-ß signaling pathway genes was found with analysis of 24 genes harboring novel loss-of-function variants. Both polygenic risk scores demonstrated that sporadic SCAD cases have a significantly elevated genetic SCAD risk compared with controls. CONCLUSIONS: SCAD shares some genetic overlap with CTD, even in the absence of any major CTD phenotype. Consistent with a complex genetic architecture, SCAD patients also have a higher burden of common variants than controls.

Hif-1a suppresses ROS-induced proliferation of cardiac fibroblasts following myocardial infarction.

We report that cardiac fibroblasts (CFs) and mesenchymal progenitors are more hypoxic than other cardiac interstitial populations, express more hypoxia-inducible factor 1α (HIF-1α), and exhibit increased glycolytic metabolism. CF-specific deletion of Hif-1a resulted in decreased HIF-1 target gene expression and increased mesenchymal progenitors in uninjured hearts and increased CF activation without proliferation following sham injury, as demonstrated using single-cell RNA sequencing (scRNA-seq). After myocardial infarction (MI), however, there was ∼50% increased CF proliferation and excessive scarring and contractile dysfunction, a scenario replicated in 3D engineered cardiac microtissues. CF proliferation was associated with higher reactive oxygen species (ROS) as occurred also in wild-type mice treated with the mitochondrial ROS generator MitoParaquat (MitoPQ). The mitochondrial-targeted antioxidant MitoTEMPO rescued Hif-1a mutant phenotypes. Thus, HIF-1α in CFs provides a critical braking mechanism against excessive post-ischemic CF activation and proliferation through regulation of mitochondrial ROS. CFs are potential cellular targets for designer antioxidant therapies in cardiovascular disease.

Krüppel-like factor 1 is a core cardiomyogenic trigger in zebrafish.

Cardiac regeneration requires dedifferentiation and proliferation of mature cardiomyocytes, but the mechanisms underlying this plasticity remain unclear. Here, we identify a potent cardiomyogenic role for Krüppel-like factor 1 (Klf1/Eklf), which is induced in adult zebrafish myocardium upon injury. Myocardial inhibition of Klf1 function does not affect heart development, but it severely impairs regeneration. Transient Klf1 activation is sufficient to expand mature myocardium in uninjured hearts. Klf1 directs epigenetic reprogramming of the cardiac transcription factor network, permitting coordinated cardiomyocyte dedifferentiation and proliferation. Myocardial expansion is supported by Klf1-induced rewiring of mitochondrial metabolism from oxidative respiration to anabolic pathways. Our findings establish Klf1 as a core transcriptional regulator of cardiomyocyte renewal in adult zebrafish hearts.

Heterozygous loss of WBP11 function causes multiple congenital defects in humans and mice.

The genetic causes of multiple congenital anomalies are incompletely understood. Here, we report novel heterozygous predicted loss-of-function (LoF) and predicted damaging missense variants in the WW domain binding protein 11 (WBP11) gene in seven unrelated families with a variety of overlapping congenital malformations, including cardiac, vertebral, tracheo-esophageal, renal and limb defects. WBP11 encodes a component of the spliceosome with the ability to activate pre-messenger RNA splicing. We generated a Wbp11 null allele in mouse using CRISPR-Cas9 targeting. Wbp11 homozygous null embryos die prior to E8.5, indicating that Wbp11 is essential for development. Fewer Wbp11 heterozygous null mice are found than expected due to embryonic and postnatal death. Importantly, Wbp11 heterozygous null mice are small and exhibit defects in axial skeleton, kidneys and esophagus, similar to the affected individuals, supporting the role of WBP11 haploinsufficiency in the development of congenital malformations in humans. LoF WBP11 variants should be considered as a possible cause of VACTERL association as well as isolated Klippel-Feil syndrome, renal agenesis or esophageal atresia.

Sierra: discovery of differential transcript usage from polyA-captured single-cell RNA-seq data.

High-throughput single-cell RNA-seq (scRNA-seq) is a powerful tool for studying gene expression in single cells. Most current scRNA-seq bioinformatics tools focus on analysing overall expression levels, largely ignoring alternative mRNA isoform expression. We present a computational pipeline, Sierra, that readily detects differential transcript usage from data generated by commonly used polyA-captured scRNA-seq technology. We validate Sierra by comparing cardiac scRNA-seq cell types to bulk RNA-seq of matched populations, finding significant overlap in differential transcripts. Sierra detects differential transcript usage across human peripheral blood mononuclear cells and the Tabula Muris, and 3 'UTR shortening in cardiac fibroblasts. Sierra is available at https://github.com/VCCRI/Sierra .

Ularcirc: visualization and enhanced analysis of circular RNAs via back and canonical forward splicing.

Circular RNAs (circRNA) are a unique class of transcripts that can only be identified from sequence alignments spanning discordant junctions, commonly referred to as backsplice junctions (BSJ). Canonical splicing is also linked with circRNA biogenesis either from the parental transcript or internal to the circRNA, and is not fully utilized in circRNA software. Here we present Ularcirc, a software tool that integrates the visualization of both BSJ and forward splicing junctions and provides downstream analysis of selected circRNA candidates. Ularcirc utilizes the output of CIRI, circExplorer, or raw chimeric output of the STAR aligner and assembles BSJ count table to allow multi-sample analysis. We used Ularcirc to identify and characterize circRNA from public and in-house generated data sets and demonstrate how it can be used to (i) discover novel splicing patterns of parental transcripts, (ii) detect internal splicing patterns of circRNA, and (iii) reveal the complexity of BSJ formation. Furthermore, we identify circRNA that have potential open reading frames longer than their linear sequence. Finally, we detected and validated the presence of a novel class of circRNA generated from ApoA4 transcripts whose BSJ derive from multiple non-canonical splicing sites within coding exons. Ularcirc is accessed via https://github.com/VCCRI/Ularcirc.

Identification of clinically actionable variants from genome sequencing of families with congenital heart disease.

PURPOSE: Congenital heart disease (CHD) affects up to 1% of live births. However, a genetic diagnosis is not made in most cases. The purpose of this study was to assess the outcomes of genome sequencing (GS) of a heterogeneous cohort of CHD patients. METHODS: Ninety-seven families with probands born with CHD requiring surgical correction were recruited for genome sequencing. At minimum, a proband-parents trio was sequenced per family. GS data were analyzed via a two-tiered method: application of a high-confidence gene screen (hcCHD), and comprehensive analysis. Identified variants were assessed for pathogenicity using the American College of Medical Genetics and Genomics-Association for Molecular Pathology (ACMG-AMP) guidelines. RESULTS: Clinically relevant genetic variants in known and emerging CHD genes were identified. The hcCHD screen identified a clinically actionable variant in 22% of families. Subsequent comprehensive analysis identified a clinically actionable variant in an additional 9% of families in genes with recent disease associations. Overall, this two-tiered approach provided a clinically relevant variant for 31% of families. CONCLUSIONS: Interrogating GS data using our two-tiered method allowed identification of variants with high clinical utility in a third of our heterogeneous cohort. However, association of emerging genes with CHD etiology, and development of novel technologies for variant assessment and interpretation, will increase diagnostic yield during future reassessment of our GS data.

Orphan receptor GPR37L1 contributes to the sexual dimorphism of central cardiovascular control.

BACKGROUND: Over 100 mammalian G protein-coupled receptors are yet to be matched with endogenous ligands; these so-called orphans are prospective drug targets for the treatment of disease. GPR37L1 is one such orphan, abundant in the brain and detectable as mRNA in the heart and kidney. GPR37L1 ablation was reported to cause hypertension and left ventricular hypertrophy, and thus, we sought to further define the role of GPR37L1 in blood pressure homeostasis. METHODS: We investigated the cardiovascular effects of GPR37L1 using wild-type (GPR37L1wt/wt) and null (GPR37L1KO/KO) mice established on a C57BL/6J background, both under baseline conditions and during AngII infusion. We profiled GPR37L1 tissue expression, examining the endogenous receptor by immunoblotting and a ß-galactosidase reporter mouse by immunohistochemistry. RESULTS: GPR37L1 protein was abundant in the brain but not detectable in the heart and kidney. We measured blood pressure in GPR37L1wt/wt and GPR37L1KO/KO mice and found that deletion of GPR37L1 causes a female-specific increase in systolic, diastolic, and mean arterial pressures. When challenged with short-term AngII infusion, only male GPR37L1KO/KO mice developed exacerbated left ventricular hypertrophy and evidence of heart failure, while the female GPR37L1KO/KO mice were protected from cardiac fibrosis. CONCLUSIONS: Despite its absence in the heart and kidney, GPR37L1 regulates baseline blood pressure in female mice and is crucial for cardiovascular compensatory responses in males. The expression of GPR37L1 in the brain, yet absence from peripheral cardiovascular tissues, suggests this orphan receptor is a hitherto unknown contributor to central cardiovascular control.

A Screening Approach to Identify Clinically Actionable Variants Causing Congenital Heart Disease in Exome Data.

BACKGROUND: Congenital heart disease (CHD)-structural abnormalities of the heart that arise during embryonic development-is the most common inborn malformation, affecting ≤1% of the population. However, currently, only a minority of cases can be explained by genetic abnormalities. The goal of this study was to identify disease-causal genetic variants in 30 families affected by CHD. METHODS: Whole-exome sequencing was performed with the DNA of multiple family members. We utilized a 2-tiered whole-exome variant screening and interpretation procedure. First, we manually curated a high-confidence list of 90 genes known to cause CHD in humans, identified predicted damaging variants in genes on this list, and rated their pathogenicity using American College of Medical Genetics and Genomics-Association for Molecular Pathology guidelines. RESULTS: In 3 families (10%), we found pathogenic variants in known CHD genes TBX5, TFAP2B, and PTPN11, explaining the cardiac lesions. Second, exomes were comprehensively analyzed to identify additional predicted damaging variants that segregate with disease in CHD candidate genes. In 10 additional families (33%), likely disease-causal variants were uncovered in PBX1, CNOT1, ZFP36L2, TEK, USP34, UPF2, KDM5A, KMT2C, TIE1, TEAD2, and FLT4. CONCLUSIONS: The pathogenesis of CHD could be explained using our high-confidence CHD gene list for variant filtering in a subset of cases. Furthermore, our unbiased screening procedure of family exomes implicates additional genes and variants in the pathogenesis of CHD, which suggest themselves for functional validation. This 2-tiered approach provides a means of (1) identifying clinically actionable variants and (2) identifying additional disease-causal genes, both of which are essential for improving the molecular diagnosis of CHD.

NAD Deficiency, Congenital Malformations, and Niacin Supplementation.

BACKGROUND: Congenital malformations can be manifested as combinations of phenotypes that co-occur more often than expected by chance. In many such cases, it has proved difficult to identify a genetic cause. We sought the genetic cause of cardiac, vertebral, and renal defects, among others, in unrelated patients. METHODS: We used genomic sequencing to identify potentially pathogenic gene variants in families in which a person had multiple congenital malformations. We tested the function of the variant by using assays of in vitro enzyme activity and by quantifying metabolites in patient plasma. We engineered mouse models with similar variants using the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system. RESULTS: Variants were identified in two genes that encode enzymes of the kynurenine pathway, 3-hydroxyanthranilic acid 3,4-dioxygenase (HAAO) and kynureninase (KYNU). Three patients carried homozygous variants predicting loss-of-function changes in the HAAO or KYNU proteins (HAAO p.D162*, HAAO p.W186*, or KYNU p.V57Efs*21). Another patient carried heterozygous KYNU variants (p.Y156* and p.F349Kfs*4). The mutant enzymes had greatly reduced activity in vitro. Nicotinamide adenine dinucleotide (NAD) is synthesized de novo from tryptophan through the kynurenine pathway. The patients had reduced levels of circulating NAD. Defects similar to those in the patients developed in the embryos of Haao-null or Kynu-null mice owing to NAD deficiency. In null mice, the prevention of NAD deficiency during gestation averted defects. CONCLUSIONS: Disruption of NAD synthesis caused a deficiency of NAD and congenital malformations in humans and mice. Niacin supplementation during gestation prevented the malformations in mice. (Funded by the National Health and Medical Research Council of Australia and others.).

Longitudinal structural, functional, and cellular myocardial alterations with chronic centrifugal continuous-flow left ventricular assist device support.

BACKGROUND: Left ventricular assist device (LVAD) support triggers adaptations within failing hearts. The HeartWare (HeartWare International, Inc., Framingham, MA) LVAD exhibits different flow profiles and afterload dependence compared with previous-generation devices, which may alter remodelling patterns. We sought to characterize myocardial adaptation to third-generation centrifugal-flow LVADs at a functional, hemodynamic, and structural level in addition to profiling transcriptomal changes using next-generation sequencing platforms. METHODS: We studied 37 patients supported with the HeartWare device with paired measurements of invasive hemodynamics, serial longitudinal left ventricular (LV) and right ventricular (RV) 3-dimensional echocardiography, and N-terminal of the prohormone brain natriuretic peptide (NT-proBNP) measurements. Paired samples for comparison of histologic myocardial cellular size and transcriptomal profiling were performed on specimens taken at pump implant and transplantation. RESULTS: The mean support duration was 280 ± 163 days. Mechanical unloading after HeartWare support resulted in reduced filling pressures (mean pulmonary capillary wedge pressure 27.1 ± 6.6 to 14.8 ± 5.1 mm Hg, p < 0.0001). Mean LV cardiomyocyte cell size decreased from 2,789.7 ± 671.8 to 2,290.8 ± 494.2 µm2 (p = 0.02). LV and RV ejection fractions improved significantly (24% ± 8% to 35% ± 9% [p < 0.001] and 35% ± 11% to 40% ± 8% [p < 0.02], respectively). NT-proBNP levels fell 4.8-fold by Day 90 after support, consistent with a decrease in LV wall stress. Despite these concordant beneficial findings, the microRNA transcriptome did not change significantly across the group. CONCLUSIONS: Reverse remodelling is evident at multiple levels with chronic HeartWare support in the absence of changes in the microRNA transcriptome. Successful myocardial unloading is associated with a decrease in wall stress, regression of cardiomyocyte hypertrophy, and an improvement in LV and RV ejection fractions.