Hypermobile Ehlers-Danlos syndrome (hEDS) is a common heritable connective tissue disorder that lacks a known genetic etiology. To identify genetic contributions to hEDS, whole exome sequencing was performed on families and a cohort of sporadic hEDS patients. A missense variant in Kallikrein-15 (KLK15 p. Gly226Asp), segregated with disease in two families and genetic burden analyses of 197 sporadic hEDS patients revealed enrichment of variants within the Kallikrein gene family. To validate pathogenicity, the variant identified in familial studies was used to generate knock-in mice. Consistent with our clinical cohort, Klk15G224D/+ mice displayed structural and functional connective tissue defects within multiple organ systems. These findings support Kallikrein gene variants in the pathogenesis of hEDS and represent an important step towards earlier diagnosis and better clinical outcomes.

While numerous approaches have been reported towards understanding single cell regulation, there is limited understanding of single cell production of extracellular matrix phenotypes. Collagens are major proteins of the extracellular microenvironment extensively used in basic cell culture, tissue engineering, and biomedical applications. However, identifying compositional regulation of collagen remains challenging. Here, we report the development of In vitro ExtraCellular Matrix Mass Spectrometry Imaging (ivECM-MSI) as a tool to rapidly and simultaneously define collagen subtypes from coatings and basic cell culture applications. The tool uses the mass spectrometry imaging platform with reference libraries to produce visual and numerical data types. The method is highly integrated with basic in vitro strategies as it may be used with conventional cell chambers on minimal numbers of cells and with minimal changes to biological experiments. Applications tested include semi-quantitation of collagen composition in culture coatings, time course collagen deposition, deposition altered by gene knockout, and changes induced by drug treatment. This approach provides new access to proteomic information on how cell types respond to and change the extracellular microenvironment and provides a holistic understanding of both the cell and extracellular response.

OBJECTIVES/GOALS: Myocardial interstitial fibrosis leads to high hemodynamic load resulting in heart failure (HFrEF). Previous studies show that treatment with a left ventricular assist device (LVAD) does not reduce fibrosis. We hypothesize that human cardiac fibroblasts are highly activated in HFrEF and remain unresponsive to hemodynamic unloading by LVAD. METHODS/STUDY POPULATION: Forty human subjects with HFrEF undergoing LVAD implantation were enrolled to provide a portion of myocardium routinely removed during LVAD placement. In addition, 7 biopsies previously collected from transplanted hearts with extended LVAD treatment were also evaluated (LVEX). RESULTS/ANTICIPATED RESULTS: Quantification of PSR-stained sections reveals a significant increase in collagen content in the HFrEF tissue (CVF = 2.8) compared to control tissues (CVF = 0.9) that remained elevated in LVEX hearts (CVF = 3.1). HCFs from LV biopsies were isolated and grown to confluence. HCFs from HFrEF patients and control HCFs were plated on substrates with stiffnesses reflective of normal myocardium (2kPa) or HFrEF myocardium (8kPa). Cells were collected at 4- and 7-day time points and levels of collagen I and alpha-smooth muscle actin were quantified by western blot analysis. Control HCFs were responsive to changes in substrate stiffness producing more Col I and a-SMA on 8kPa versus 2kPa, HCFs from HFrEF patients were unresponsive to changes in stiffness exhibiting no significant difference in protein production on 2 vs. 8kPa. DISCUSSION/SIGNIFICANCE: Our data suggests that HCFs isolated from the failing myocardium do not respond to changes in mechanical load and might contribute to persistent increases in fibrosis. These findings bring us one step closer to elucidating mechanisms behind fibrosis in HFrEF which could lead to targeted therapies to improve patient outcomes from LVAD support.

Background: Myocardial interstitial fibrosis is a common pathology in cardiomyopathies leading to ventricular dilation and increased hemodynamic load that result in heart failure with reduced ejection fraction (HFrEF). Previous research has shown that HFrEF patients treated with a left ventricular assist device (LVAD) undergo hemodynamic unloading resulting in at least partial cardiomyocyte recovery. However, evidence supports that these patients do not experience a regression of fibrosis and demonstrate, in some cases, a worsening of fibrosis after LVAD treatment. Purpose: We hypothesize that human cardiac fibroblasts (HCFs) are constitutively activated in HFrEF myocardium but remain unresponsive to hemodynamic unloading with LVAD placement. Methods and Results: Forty human subjects with HFrEF undergoing LVAD implantation were enrolled to provide a portion of myocardium routinely removed during LVAD placement. In addition, 7 biopsies previously collected from transplanted hearts with extended LVAD treatment were also evaluated (LVEX). Quantification of PSR stained sections revealed a significant increase in collagen content in the HFrEF tissue (Collagen volume fraction % (CVF) = 2.8±0.2) in comparison to control tissues (CVF = 0.9±0.2) that remained elevated in LVEX hearts (CVF = 3.1±0.3). HCFs derived from biopsies received at LVAD placement were isolated and grown to confluence. HCFs from HFrEF patients and control HCFs from healthy donors were then plated on substrates with mechanical stiffnesses reflective of either normal myocardium (2kPa) or failing myocardium (8kPa). Cells were collected at 4- and 7-day timepoints and levels of collagen I (Col I) and alpha-smooth muscle actin (α-SMA) were quantified through western blot analysis with β-actin as a loading control. Whereas control HCFs were responsive to changes in substrate stiffness producing more Col I and α-SMA on 8kPa versus 2kPa, HCFs from HFrEF patients were unresponsive to changes in stiffness exhibiting no significant difference in production on 2 vs. 8kPa. Conclusion: These data suggest that HCFs isolated from the failing myocardium do not respond to changes in mechanical load and hence, might contribute to persistent increases in fibrosis in failing and unloaded hearts.

Objective: We recently demonstrated regionalized myocardial fibrosis in patients undergoing surgical repair for Mitral Valve Prolapse. The location of fibrosis suggests a mechanically driven process wherein mechanical stress alters the phenotype of ventricular fibroblasts to promote an increase in tissue stiffness, eventually causing establishment of a fibrotic scar. We sought to better understand how mechanical stiffness affected cellular programs. Methods: We cultured primary human cardiac fibroblasts derived from the same patient on a mechanically stiff (TC Plastic, ~2GPa), as well as a mechanically soft (2kPa) substrate and then performed immunofluorescence, western blotting, RNA Seq, and ATAC Seq. Following differential analysis of omics data, we performed an integration analysis by filterinig differentially accessible chromatin to only include sites that fell within 10 kB of opened/closed chromatin. Finally, we performed a motif enrichment analysis using the online bioinformatics package Homer. Results: Immunofluorescence and western blotting revealed that cells cultured on a stiff substrate expressed canonical markers of fibroblast activation compared with soft substrates (alpha SMA, COL1A1, P<0.0001). RNA Sequencing revealed differential changes in 1615 genes in fibroblasts (FDR adjusted P<1.6*10 -6 ), several of which were subsequently validated using western blot. ATAC Seq identified 5855 regions across the genome which opened/closed in response to different mechanical environments in fibroblasts (adjusted P<0.05). Integrating these datasets revealed 803 Chromatin changes that fell within 10kb of differentially expressed genes, with AP1 and TEAD family transcription factors predicted to be master regulators of closing and opening chromatin, respectively. Proof-of-concept motif validation conducted on differentially expressed chromatin within intron 12 of the MYH10 gene demonstrated presence of a transcriptional repressor with evidence of JunD as a binding partner for this site. Conclusions: The mechanical environment plays a major role in cellular phenotype and regulates chromatin organization and expression of pro-fibrotic genes through changes in chromatin accessibility.

Cancer is the second most common cause of death in the United States, accounting for 602,350 deaths in 2020. Cancer-related death rates have declined by 27% over the past two decades, partially due to the identification of novel anti-cancer drugs. Despite improvements in cancer treatment, newly approved oncology drugs are associated with increased toxicity risk. These toxicities may be mitigated by pharmacokinetic optimization and reductions in off-target interactions. As such, there is a need for early-stage implementation of pharmacokinetic (PK) prediction tools. Several PK prediction platforms exist, including pkCSM, SuperCypsPred, Pred-hERG, Similarity Ensemble Approach (SEA), and SwissADME. These tools can be used in screening hits, allowing for the selection of compounds were reduced toxicity and/or risk of attrition. In this short commentary, we used PK prediction tools in the optimization of mitogen activated extracellular signal-related kinase kinase 1 (MEK1) inhibitors. In doing so, we identified MEK1 inhibitors with retained activity and optimized predictive PK properties, devoid of hERG inhibition. These data support the use of publicly available PK prediction platforms in early-stage drug discovery to design safer drugs.

Introduction: Regionalized myocardial fibrosis exists in the inferobasal myocardium of patients undergoing surgical repair for Mitral Valve Prolapse, and supports a process in which cells within mechanically stressed myocardial regions alter their phenotype in order to increase tissue stiffness, eventually causing establishment of a fibrotic scar. How cells behave in different fibromechanical environments is not currently well understood. Hypothesis: Integrating RNA Seq and ATAC Seq datasets will identify mechanosensitive regulatory pathways in cardiac fibroblasts. Methods: Primary human cardiac fibroblasts (HCFs) were cultured on stiff (TC Plastic, ~2GPa), as well as soft (2kPa) substrates coated with type 1 collagen. Following 48 hours in serum-free conditions, HCFs were lysed and subjected to immunofluorescence (IF), Western blotting (WB), RNA Seq, and ATAC Seq. Differential analysis of RNA Seq, and ATAC Seq data was performed, followed by an integration analysis which linked differentially expressed genes to chromatin that opened/closed within 10 kB. Finally, motif enrichment analysis was performed using the online bioinformatics package Homer. Results: IF and WB revealed that HCFs cultured on a stiff substrate expressed markers of fibroblast activation (alpha SMA, P<0.0001), as well as increased amounts of Connexin 43 (P<0.0001). Additionally, there were higher numbers of Ki-67 positive cells (7.2% vs 2.1% P=0.001). RNA Seq revealed differential changes in 4324 genes (FDR adjusted P<0.05), several of which were subsequently validated using western blots. ATAC Seq identified 5855 chromatin regions which opened/closed in response to different mechanical environments in fibroblasts (adjusted P<0.05). Integrating RNA Seq and ATAC Seq datasets revealed 803 Chromatin changes that fell within 10kb of differentially expressed genes, with a predicted enrichment of TEAD binding sites in differentially opened chromatin (P=1.0*10-40). Conclusions: The mechanical environment plays a major role in cellular phenotype and directly regulates chromatin organization and expression of pro-fibrotic genes, with a possible role for TEAD as a driver of fibroblast activation.

Mitral valve prolapse (MVP) is a common cardiac valve disease that often progresses to serious secondary complications requiring surgery. MVP manifests as extracellular matrix disorganization and biomechanically incompetent tissues in the adult setting. However, MVP has recently been shown to have a developmental basis, as multiple causal genes expressed during embryonic development have been identified. Disease phenotypes have been observed in mouse models with human MVP mutations as early as birth. This study focuses on the developmental function of DCHS1, one of the first genes to be shown as causal in multiple families with non-syndromic MVP. By using various biochemical techniques as well as mouse and cell culture models, we demonstrate a unique link between DCHS1-based cell adhesions and the septin-actin cytoskeleton through interactions with cytoplasmic protein Lix1-Like (LIX1L). This DCHS1-LIX1L-SEPT9 axis interacts with and promotes filamentous actin organization to direct cell-ECM alignment and valve tissue shape.

BACKGROUND Trametinib is a MEK1 (mitogen-activated extracellular signal-related kinase kinase 1) inhibitor used in the treatment of BRAF (rapid accelerated fibrosarcoma B-type)-mutated metastatic melanoma. Roughly 11% of patients develop cardiomyopathy following long-term trametinib exposure. Although described clinically, the molecular landscape of trametinib cardiotoxicity has not been characterized. OBJECTIVES The aim of this study was to test the hypothesis that trametinib promotes widespread transcriptomic and cellular changes consistent with oxidative stress and impairs cardiac function. METHODS Mice were treated with trametinib (1 mg/kg/d). Echocardiography was performed pre- and post-treatment. Gross, histopathologic, and biochemical assessments were performed to probe for molecular and cellular changes. Human cardiac organoids were used as an in vitro measurement of cardiotoxicity and recovery. RESULTS Long-term administration of trametinib was associated with significant reductions in survival and left ventricular ejection fraction. Histologic analyses of the heart revealed myocardial vacuolization and calcification in 28% of animals. Bulk RNA sequencing identified 435 differentially expressed genes and 116 differential signaling pathways following trametinib treatment. Upstream gene analysis predicted interleukin-6 as a regulator of 17 relevant differentially expressed genes, suggestive of PI3K/AKT and JAK/STAT activation, which was subsequently validated. Trametinib hearts displayed elevated markers of oxidative stress, myofibrillar degeneration, an 11-fold down-regulation of the apelin receptor, and connexin-43 mislocalization. To confirm the direct cardiotoxic effects of trametinib, human cardiac organoids were treated for 6 days, followed by a 6-day media-only recovery. Trametinib-treated organoids exhibited reductions in diameter and contractility, followed by partial recovery with removal of treatment. CONCLUSIONS These data describe pathologic changes observed in trametinib cardiotoxicity, supporting the exploration of drug holidays and alternative pharmacologic strategies for disease prevention. (c) 2022 Published by Elsevier on behalf of the American College of Cardiology Foundation. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction: Mitral valve prolapse (MVP) is one of the most common forms of cardiac valve disease, affecting 1 in 40 humans and ~70% of small breed dogs. There are no effective nonsurgical treatments for MVP and therapeutic efforts have been hindered due to an incomplete understanding of its fundamental causes. Recent studies by our group have described the genetic basis for non-syndromic MVP and have provided new insights into molecular processes that underlie the disease. We have used this genetic information to identify druggable targets that can provide a non-surgical option for humans. In particular, we have identified the MEK/ERK pathway as the major disease initiating pathway and prolonged hyperactivation of MEK/ERK drives disease progression and severity. Hypothesis: Pharmacological blockade of MEK/ERK activities will sufficiently arrest the disease pathway and maintain the valves in a sub-clinical condition. Methods and Results: The FDA-approved MEK1 inhibitor Trametinib was administered to Dzip1 S24R/+ mice; a disease model that phenocopies human MVP. Preliminary results suggest safety concerns in mice associated with chronic administration of Trametinib (1 mg/kg/day). Animals demonstrated signs of cardiotoxicity on echocardiography, including left atrial enlargement; a reduction in E/A wave intensity; left ventricular wall movement abnormalities; prolonged isovolumetric relaxation time, and a reduced ejection fraction. As such, our lab has focused our efforts on identifying novel MEK1 inhibitors with a favorable safety profile. Utilizing a machine learning based workflow, we have identified highly selective MEK1 inhibitors with retained activity (<100nM IC50 values); high gastrointestinal absorption (>85%); favorable predicted LD50 values (>1,000 mg/kg), and devoid of major cytochrome interactions. Conclusions: MEK1 may serve as a druggable target in the treatment of mitral valve prolapse; however, currently available MEK inhibitors demonstrate limiting toxicities. Future studies will involve in vivo administration of our novel compounds to Dzip1 S24R/+ mice. We envision results from this study will help guide the development of systemic or targeted therapies for the use in human patients with mitral valve prolapse.