Burns C Blaxall’s research while affiliated with Cincinnati Children's Hospital Medical Center and other places

Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) regulates cardiac contraction through modulation of actomyosin interactions mediated by the protein's amino terminal (N′)-region (C0-C2 domains, 358 amino acids). On the other hand, dephosphorylation of cMyBP-C during myocardial injury results in cleavage of the 271 amino acid C0-C1f region and subsequent contractile dysfunction. Yet, our current understanding of amino terminus region of cMyBP-C in the context of regulating thin and thick filament interactions is limited. A novel cardiac-specific transgenic mouse model expressing cMyBP-C, but lacking its C0-C1f region (cMyBP-C∆C0-C1f), displayed dilated cardiomyopathy, underscoring the importance of the N′-region in cMyBP-C. Further exploring the molecular basis for this cardiomyopathy, in vitro studies revealed increased interfilament lattice spacing and rate of tension redevelopment, as well as faster actin-filament sliding velocity within the C-zone of the transgenic sarcomere. Moreover, phosphorylation of the unablated phosphoregulatory sites was increased, likely contributing to normal sarcomere morphology and myoarchitecture. These results led us to hypothesize that restoration of the N′-region of cMyBP-C would return actomyosin interaction to its steady state. Accordingly, we administered recombinant C0-C2 (rC0-C2) to permeabilized cardiomyocytes from transgenic, cMyBP-C null, and human heart failure biopsies, and we found that normal regulation of actomyosin interaction and contractility was restored. Overall, these data provide a unique picture of selective perturbations of the cardiac sarcomere that either lead to injury or adaptation to injury in the myocardium.

Methods for single-cell RNA sequencing (scRNA-seq) have greatly advanced in recent years. While droplet- and well-based methods have increased the capture frequency of cells for scRNA-seq, these technologies readily produce technical artifacts, such as doublet cell captures. Doublets occurring between distinct cell types can appear as hybrid scRNA-seq profiles, but do not have distinct transcriptomes from individual cell states. We introduce DoubletDecon, an approach that detects doublets with a combination of deconvolution analyses and the identification of unique cell-state gene expression. We demonstrate the ability of DoubletDecon to identify synthetic, mixed-species, genetic, and cell-hashing cell doublets from scRNA-seq datasets of varying cellular complexity with a high sensitivity relative to alternative approaches. Importantly, this algorithm prevents the prediction of valid mixed-lineage and transitional cell states as doublets by considering their unique gene expression. DoubletDecon has an easy-to-use graphical user interface and is compatible with diverse species and unsupervised population detection algorithms. : Multiplets are a source of confounding gene expression in single-cell RNA sequencing (scRNA-seq) that result from the simultaneous capture of multiple cells in a droplet. DePasquale et al. introduce DoubletDecon to identify putative doublets and to consider unique gene expression inherent to transitional states and progenitors to “rescue” singlet captures from inaccurate classification. Keywords: single-cell RNA-seq, multiplet, doublet, deconvolution, RNA-seq, bioinformatics, artifact detection

To understand the molecular pathogenesis of human disease, precision analyses to define alterations within and between disease-associated cell populations are desperately needed. Single-cell genomics represents an ideal platform to enable the identification and comparison of normal and diseased transcriptional cell populations. We created cellHarmony, an integrated solution for the unsupervised analysis, classification, and comparison of cell types from diverse single-cell RNA-Seq datasets. cellHarmony efficiently and accurately matches single-cell transcriptomes using a community-clustering and alignment strategy to compute differences in cell-type specific gene expression over potentially dozens of cell populations. Such transcriptional differences are used to automatically identify distinct and shared gene programs among cell-types and identify impacted pathways and transcriptional regulatory networks to understand the impact of perturbations at a systems level. cellHarmony is implemented as a python package and as an integrated workflow within the software AltAnalyze. We demonstrate that cellHarmony has improved or equivalent performance to alternative label projection methods, is able to identify the likely cellular origins of malignant states, stratify patients into clinical disease subtypes from identified gene programs, resolve discrete disease networks impacting specific cell-types, and illuminate therapeutic mechanisms. Thus, this approach holds tremendous promise in revealing the molecular and cellular origins of complex disease.

Fibrosis is a common feature of chronic kidney disease; however, no clinical therapies effectively target the progression of fibrosis. Inhibition of fibronectin polymerization with the small peptide pUR4 attenuates fibrosis in the liver and heart. Here, we show that pUR4 decreases renal fibrosis and tissue remodeling using a clinically relevant model of kidney injury, unilateral ischemia-reperfusion. This work highlights the benefits of inhibiting matrix polymerization, alone or in conjunction with cell-based therapies, as a novel approach to diminish the maladaptive responses to ischemic kidney injury that lead to chronic renal failure.

Cardiomyocyte-specific increases in phosphorylated Hsp20 (S16D-Hsp20) to levels similar to those observed in human failing hearts are associated with early fibrotic remodeling and depressed left ventricular function, symptoms which progress to heart failure and early death. The underlying mechanisms appear to involve translocation of phosphorylated Hsp20 to the nucleus and upregulation of interleukin (IL)-6, which subsequently activates cardiac fibroblasts in a paracrine fashion through transcription factor STAT3 signaling. Accordingly, treatment of S16D-Hsp20 mice with a rat anti-mouse IL-6 receptor monoclonal antibody (MR16-1) attenuated interstitial fibrosis and preserved cardiac function. These findings suggest that phosphorylated Hsp20 may be a potential therapeutic target in heart failure.

Background Primary sclerosing cholangitis, autoimmune hepatitis and autoimmune sclerosing cholangitis are forms of chronic, progressive autoimmune liver disease (AILD) that can affect the pediatric population. Objective To determine whether quantitative MRI- and laboratory-based biomarkers are associated with conventional imaging findings of portal hypertension (radiologic portal hypertension) in children and young adults with AILD. Materials and methods Forty-four patients with AILD enrolled in an institutional registry underwent a research abdominal MRI examination at 1.5 tesla (T). Five quantitative MRI techniques were performed: liver MR elastography, spleen MR elastography, liver iron-corrected T1 mapping, liver T2 mapping, and liver diffusion-weighted imaging (DWI, quantified as apparent diffusion coefficients). Two anatomical sequences were used to document splenomegaly, varices and ascites. We calculated aspartate aminotransferase (AST)-to-platelet ratio index (APRI) and fibrosis-4 (FIB-4) scores — laboratory-based biomarkers of liver fibrosis. We used receiver operating characteristic (ROC) curve analyses to establish the diagnostic performance of quantitative MRI and laboratory biomarkers for indicating the presence of radiologic portal hypertension. Results Twenty-three (52%) patients were male; mean age was 15.2±4.0 years. Thirteen (30%) patients had radiologic portal hypertension. Liver and spleen stiffness demonstrated the greatest diagnostic performance for indicating the presence of portal hypertension (area-under-the-ROC-curve [AUROC]=0.98 and 0.96, respectively). The APRI and FIB-4 scores also demonstrated good diagnostic performance (AUROC=0.87 and 0.88, respectively). Conclusion MRI-derived measures of liver and spleen stiffness as well as laboratory-based APRI and FIB-4 scores are highly associated with imaging findings of portal hypertension in children and young adults with AILD and thus might be useful for predicting portal hypertension impending onset and directing personalized patient management.

RNA binding proteins represent an emerging class of proteins with a role in cardiac dysfunction. We show that activation of the RNA binding protein human antigen R (HuR) is increased in the failing human heart. To determine the functional role of HuR in pathological cardiac hypertrophy, we created an inducible cardiomyocyte-specific HuR-deletion mouse and showed that HuR deletion reduces left ventricular hypertrophy, dilation, and fibrosis while preserving cardiac function in a transverse aortic constriction (TAC) model of pressure overload-induced hypertrophy. Assessment of HuR-dependent changes in global gene expression suggests that the mechanistic basis for this protection occurs through a reduction in fibrotic signaling, specifically through a reduction in TGF-β (Tgfb) expression. Finally, pharmacological inhibition of HuR at a clinically relevant time point following the initial development of pathological hypertrophy after TAC also yielded a significant reduction in pathological progression, as marked by a reduction in hypertrophy, dilation, and fibrosis and preserved function. In summary, this study demonstrates a functional role for HuR in the progression of pressure overload-induced cardiac hypertrophy and establishes HuR inhibition as a viable therapeutic approach for pathological cardiac hypertrophy and heart failure.

Rationale: Hypertrophic cardiomyopathy occurs with a frequency of about 1 in 500 people. Approximately 30% of those affected carry mutations within the gene encoding cMyBP-C (cardiac myosin binding protein C). Cardiac stress, as well as cMyBP-C mutations, can trigger production of a 40kDa truncated fragment derived from the amino terminus of cMyBP-C (Mybpc340kDa). Expression of the 40kDa fragment in mouse cardiomyocytes leads to hypertrophy, fibrosis, and heart failure. Here we use genetic approaches to establish a causal role for excessive myofibroblast activation in a slow, progressive genetic cardiomyopathy-one that is driven by a cardiomyocyte-intrinsic genetic perturbation that models an important human disease. Objective: TGFβ (transforming growth factor-β) signaling is implicated in a variety of fibrotic processes, and the goal of this study was to define the role of myofibroblast TGFβ signaling during chronic Mybpc340kDa expression. Methods and results: To specifically block TGFβ signaling only in the activated myofibroblasts in Mybpc340kDa transgenic mice and quadruple compound mutant mice were generated, in which the TGFβ receptor II (TβRII) alleles ( Tgfbr2) were ablated using the periostin ( Postn) allele, myofibroblast-specific, tamoxifen-inducible Cre ( Postnmcm) gene-targeted line. Tgfbr2 was ablated either early or late during pathological fibrosis. Early myofibroblast-specific Tgfbr2 ablation during the fibrotic response reduced cardiac fibrosis, alleviated cardiac hypertrophy, preserved cardiac function, and increased lifespan of the Mybpc340kDa transgenic mice. Tgfbr2 ablation late in the pathological process reduced cardiac fibrosis, preserved cardiac function, and prolonged Mybpc340kDa mouse survival but failed to reverse cardiac hypertrophy. Conclusions: Fibrosis and cardiac dysfunction induced by cardiomyocyte-specific expression of Mybpc340kDa were significantly decreased by Tgfbr2 ablation in the myofibroblast. Surprisingly, preexisting fibrosis was partially reversed if the gene was ablated subsequent to fibrotic deposition, suggesting that continued TGFβ signaling through the myofibroblasts was needed to maintain the heart fibrotic response to a chronic, disease-causing cardiomyocyte-only stimulus.