New York Presbyterian-Brooklyn Methodist Hospital's Quality Improvement Project Targeting High A1C Levels.

Quality Improvement Success Stories are published by the American Diabetes Association in collaboration with the American College of Physicians and the National Diabetes Education Program. This series is intended to highlight best practices and strategies from programs and clinics that have successfully improved the quality of care for people with diabetes or related conditions. Each article in the series is reviewed and follows a standard format developed by the editors of Clinical Diabetes. The following article describes a Brooklyn, NY, hospital's initiative to reduce high A1C rates among its patients.

Efficient in vivo genome editing prevents hypertrophic cardiomyopathy in mice.

Dominant missense pathogenic variants in cardiac myosin heavy chain cause hypertrophic cardiomyopathy (HCM), a currently incurable disorder that increases risk for stroke, heart failure and sudden cardiac death. In this study, we assessed two different genetic therapies-an adenine base editor (ABE8e) and a potent Cas9 nuclease delivered by AAV9-to prevent disease in mice carrying the heterozygous HCM pathogenic variant myosin R403Q. One dose of dual-AAV9 vectors, each carrying one half of RNA-guided ABE8e, corrected the pathogenic variant in ≥70% of ventricular cardiomyocytes and maintained durable, normal cardiac structure and function. An additional dose provided more editing in the atria but also increased bystander editing. AAV9 delivery of RNA-guided Cas9 nuclease effectively inactivated the pathogenic allele, albeit with dose-dependent toxicities, necessitating a narrow therapeutic window to maintain health. These preclinical studies demonstrate considerable potential for single-dose genetic therapies to correct or silence pathogenic variants and prevent the development of HCM.

Ablation of lysophosphatidic acid receptor 1 attenuates hypertrophic cardiomyopathy in a mouse model.

Myocardial fibrosis is a key pathologic feature of hypertrophic cardiomyopathy (HCM). However, the fibrotic pathways activated by HCM-causing sarcomere protein gene mutations are poorly defined. Because lysophosphatidic acid is a mediator of fibrosis in multiple organs and diseases, we tested the role of the lysophosphatidic acid pathway in HCM. Lysphosphatidic acid receptor 1 (LPAR1), a cell surface receptor, is required for lysophosphatidic acid mediation of fibrosis. We bred HCM mice carrying a pathogenic myosin heavy-chain variant (403+/-) with Lpar1-ablated mice to create mice carrying both genetic changes (403+/- LPAR1 -/-) and assessed development of cardiac hypertrophy and fibrosis. Compared with 403+/- LPAR1WT, 403+/- LPAR1 -/- mice developed significantly less hypertrophy and fibrosis. Single-nucleus RNA sequencing of left ventricular tissue demonstrated that Lpar1 was predominantly expressed by lymphatic endothelial cells (LECs) and cardiac fibroblasts. Lpar1 ablation reduced the population of LECs, confirmed by immunofluorescence staining of the LEC markers Lyve1 and Ccl21a and, by in situ hybridization, for Reln and Ccl21a. Lpar1 ablation also altered the distribution of fibroblast cell states. FB1 and FB2 fibroblasts decreased while FB0 and FB3 fibroblasts increased. Our findings indicate that Lpar1 is expressed predominantly by LECs and fibroblasts in the heart and is required for development of hypertrophy and fibrosis in an HCM mouse model. LPAR1 antagonism, including agents in clinical trials for other fibrotic diseases, may be beneficial for HCM.

LAMP2 Cardiomyopathy: Consequences of Impaired Autophagy in the Heart.

Background Human mutations in the X-linked lysosome-associated membrane protein-2 (LAMP2) gene can cause a multisystem Danon disease or a primary cardiomyopathy characterized by massive hypertrophy, conduction system abnormalities, and malignant ventricular arrhythmias. We introduced an in-frame LAMP2 gene exon 6 deletion mutation (denoted L2Δ6) causing human cardiomyopathy, into mouse LAMP2 gene, to elucidate its consequences on cardiomyocyte biology. This mutation results in in-frame deletion of 41 amino acids, compatible with presence of some defective LAMP2 protein. Methods and Results Left ventricular tissues from L2Δ6 and wild-type mice had equivalent amounts of LAMP2 RNA, but a significantly lower level of LAMP2 protein. By 20 weeks of age male mutant mice developed left ventricular hypertrophy which was followed by left ventricular dilatation and reduced systolic function. Cardiac electrophysiology and isolated cardiomyocyte studies demonstrated ventricular arrhythmia, conduction disturbances, abnormal calcium transients and increased sensitivity to catecholamines. Myocardial fibrosis was strikingly increased in 40-week-old L2Δ6 mice, recapitulating findings of human LAMP2 cardiomyopathy. Immunofluorescence and transmission electron microscopy identified mislocalization of lysosomes and accumulation of autophagosomes between sarcomeres, causing profound morphological changes disrupting the cellular ultrastructure. Transcription profile and protein expression analyses of L2Δ6 hearts showed significantly increased expression of genes encoding activators and protein components of autophagy, hypertrophy, and apoptosis. Conclusions We suggest that impaired autophagy results in cardiac hypertrophy and profound transcriptional reactions that impacted metabolism, calcium homeostasis, and cell survival. These responses define the molecular pathways that underlie the pathology and aberrant electrophysiology in cardiomyopathy of Danon disease.

Cardiomyocyte Proliferative Capacity Is Restricted in Mice With Lmna Mutation.

LMNA is one of the leading causative genes of genetically inherited dilated cardiomyopathy (DCM). Unlike most DCM-causative genes, which encode sarcomeric or sarcomere-related proteins, LMNA encodes nuclear envelope proteins, lamin A and C, and does not directly associate with contractile function. However, a mutation in this gene could lead to the development of DCM. The molecular mechanism of how LMNA mutation contributes to DCM development remains largely unclear and yet to be elucidated. The objective of this study was to clarify the mechanism of developing DCM caused by LMNA mutation. Methods and Results: We assessed cardiomyocyte phenotypes and characteristics focusing on cell cycle activity in mice with Lmna mutation. Both cell number and cell size were reduced, cardiomyocytes were immature, and cell cycle activity was retarded in Lmna mutant mice at both 5 weeks and 2 years of age. RNA-sequencing and pathway analysis revealed "proliferation of cells" had the most substantial impact on Lmna mutant mice. Cdkn1a, which encodes the cell cycle regulating protein p21, was strongly upregulated in Lmna mutants, and upregulation of p21 was confirmed by Western blot and immunostaining. DNA damage, which is known to upregulate Cdkn1a, was more abundantly detected in Lmna mutant mice. To assess the proliferative capacity of cardiomyocytes, the apex of the neonate mouse heart was resected, and recovery from the insult was observed. A restricted cardiomyocyte proliferating capacity after resecting the apex of the heart was observed in Lmna mutant mice. Conclusions: Our results strongly suggest that loss of lamin function contributes to impaired cell proliferation through cell cycle defects. The inadequate inborn or responsive cell proliferation capacity plays an essential role in developing DCM with LMNA mutation.

Prehospital use of a modified HEART Pathway and point-of-care troponin to predict cardiovascular events.

CLINICAL TRIAL REGISTRATION: clinicaltrials.gov (NCT02709135).

GATA6 mutations in hiPSCs inform mechanisms for maldevelopment of the heart, pancreas, and diaphragm.

Damaging GATA6 variants cause cardiac outflow tract defects, sometimes with pancreatic and diaphragmic malformations. To define molecular mechanisms for these diverse developmental defects, we studied transcriptional and epigenetic responses to GATA6 loss of function (LoF) and missense variants during cardiomyocyte differentiation of isogenic human induced pluripotent stem cells. We show that GATA6 is a pioneer factor in cardiac development, regulating SMYD1 that activates HAND2, and KDR that with HAND2 orchestrates outflow tract formation. LoF variants perturbed cardiac genes and also endoderm lineage genes that direct PDX1 expression and pancreatic development. Remarkably, an exon 4 GATA6 missense variant, highly associated with extra-cardiac malformations, caused ectopic pioneer activities, profoundly diminishing GATA4, FOXA1/2, and PDX1 expression and increasing normal retinoic acid signaling that promotes diaphragm development. These aberrant epigenetic and transcriptional signatures illuminate the molecular mechanisms for cardiovascular malformations, pancreas and diaphragm dysgenesis that arise in patients with distinct GATA6 variants.

BET bromodomain proteins regulate transcriptional reprogramming in genetic dilated cardiomyopathy.

The bromodomain and extraterminal (BET) family comprises epigenetic reader proteins that are important regulators of inflammatory and hypertrophic gene expression in the heart. We previously identified the activation of proinflammatory gene networks as a key early driver of dilated cardiomyopathy (DCM) in transgenic mice expressing a mutant form of phospholamban (PLNR9C) - a genetic cause of DCM in humans. We hypothesized that BETs coactivate this inflammatory process, representing a critical node in the progression of DCM. To test this hypothesis, we treated PLNR9C or age-matched WT mice longitudinally with the small molecule BET bromodomain inhibitor JQ1 or vehicle. BET inhibition abrogated adverse cardiac remodeling, reduced cardiac fibrosis, and prolonged survival in PLNR9C mice by inhibiting expression of proinflammatory gene networks at all stages of disease. Specifically, JQ1 had profound effects on proinflammatory gene network expression in cardiac fibroblasts, while having little effect on gene expression in cardiomyocytes. Cardiac fibroblast proliferation was also substantially reduced by JQ1. Mechanistically, we demonstrated that BRD4 serves as a direct and essential regulator of NF-κB-mediated proinflammatory gene expression in cardiac fibroblasts. Suppressing proinflammatory gene expression via BET bromodomain inhibition could be a novel therapeutic strategy for chronic DCM in humans.

CRISPR/Cas9-Mediated Fluorescent Tagging of Endogenous Proteins in Human Pluripotent Stem Cells.

Human induced pluripotent stem cells (hiPSCs) can be used to mass produce surrogates of human tissues, enabling new advances in drug screening, disease modeling, and cell therapy. Recent developments in clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing technology use homology-directed repair (HDR) to efficiently generate custom hiPSC lines harboring a variety of genomic insertions and deletions. Thus, hiPSCs that encode an endogenous protein fused to a fluorescent reporter protein can be rapidly created by employing CRISPR/Cas9 genome editing, enhancing HDR efficiency and optimizing homology arm length. These fluorescently tagged hiPSCs can be used to visualize protein function and dynamics in real time as cells proliferate and differentiate. Given that nearly any intracellular protein can be fluorescently tagged, this system serves as a powerful tool to facilitate new discoveries across many biological disciplines. In this unit, we present protocols for the design, generation, and monoclonal expansion of genetically customized hiPSCs encoding fluorescently tagged endogenous proteins. © 2018 by John Wiley & Sons, Inc.

Molecular profiling of dilated cardiomyopathy that progresses to heart failure.

Dilated cardiomyopathy (DCM) is defined by progressive functional and structural changes. We performed RNA-seq at different stages of disease to define molecular signaling in the progression from pre-DCM hearts to DCM and overt heart failure (HF) using a genetic model of DCM (phospholamban missense mutation, PLNR9C/+). Pre-DCM hearts were phenotypically normal yet displayed proliferation of nonmyocytes (59% relative increase vs. WT, P = 8 × 10-4) and activation of proinflammatory signaling with notable cardiomyocyte-specific induction of a subset of profibrotic cytokines including TGFß2 and TGFß3. These changes progressed through DCM and HF, resulting in substantial fibrosis (17.6% of left ventricle [LV] vs. WT, P = 6 × 10-33). Cardiomyocytes displayed a marked shift in metabolic gene transcription: downregulation of aerobic respiration and subsequent upregulation of glucose utilization, changes coincident with attenuated expression of PPARα and PPARγ coactivators -1α (PGC1α) and -1ß, and increased expression of the metabolic regulator T-box transcription factor 15 (Tbx15). Comparing DCM transcriptional profiles with those in hypertrophic cardiomyopathy (HCM) revealed similar and distinct molecular mechanisms. Our data suggest that cardiomyocyte-specific cytokine expression, early fibroblast activation, and the shift in metabolic gene expression are hallmarks of cardiomyopathy progression. Notably, key components of these profibrotic and metabolic networks were disease specific and distinguish DCM from HCM.

Rational design of efficient modular cells.

The modular cell design principle is formulated to devise modular (chassis) cells. These cells can be assembled with exchangeable production modules in a plug-and-play fashion to build microbial cell factories for efficient combinatorial biosynthesis of novel molecules, requiring minimal iterative strain optimization steps. A modular cell is designed to be auxotrophic, containing core metabolic pathways that are necessary but insufficient to support cell growth and maintenance. To be functional, it must tightly couple with an exchangeable production module containing auxiliary metabolic pathways that not only complement cell growth but also enhance production of targeted molecules. We developed a MODCELL (modular cell) framework based on metabolic pathway analysis to implement the modular cell design principle. MODCELL identifies genetic modifications and requirements to construct modular cell candidates and their associated exchangeable production modules. By defining the degree of similarity and coupling metrics, MODCELL can evaluate which exchangeable production module(s) can be tightly coupled with a modular cell candidate. We first demonstrated how MODCELL works in a step-by-step manner for example metabolic networks, and then applied it to design modular Escherichia coli cells for efficient combinatorial biosynthesis of five alcohols (ethanol, propanol, isopropanol, butanol and isobutanol) and five butyrate esters (ethyl butyrate, propyl butyrate, isopropyl butyrate, butyl butyrate and isobutyl butyrate) from pentose sugars (arabinose and xylose) and hexose sugars (glucose, mannose, and galactose) under anaerobic conditions. We identified three modular cells, MODCELL1, MODCELL2 and MODCELL3, that can couple well with Group 1 of modules (ethanol, isobutanol, butanol, ethyl butyrate, isobutyl butyrate, butyl butyrate), Group 2 (isopropanol, isopropyl butyrate), and Group 3 (propanol, isopropanol), respectively. We validated the design of MODCELL1 for anaerobic production of ethanol, butanol, and ethyl butyrate using experimental data available in literature.

The shock index as a predictor of vasopressor use in emergency department patients with severe sepsis.

INTRODUCTION: Severe sepsis is a leading cause of non-coronary death in hospitals across the United States. Early identification and risk stratification in the emergency department (ED) is difficult because there is limited ability to predict escalation of care. In this study we evaluated if a sustained shock index (SI) elevation in the ED was a predictor of short-term cardiovascular collapse, defined as vasopressor dependence within 72 hours of initial presentation. METHODS: Retrospective dual-centered cross-sectional study using patients identified in the Yale-New Haven Hospital Emergency Medicine sepsis registry. RESULTS: We included 295 patients in the study with 47.5% (n=140) having a sustained SI elevation in the ED. Among patients with a sustained SI elevation, 38.6% (54 of 140) required vasopressors within 72 hours of ED admission contrasted to 11.6% (18 of 155) without a sustained SI elevation (p=0.0001; multivariate modeling OR 4.42 with 95% confidence intervals 2.28-8.55) . In the SI elevation group the mean number of organ failures was 4.0 ± 2.1 contrasted to 3.2 ± 1.6 in the non-SI elevation group (p=0.0001). CONCLUSION: ED patients with severe sepsis and a sustained SI elevation appear to have higher rates of short-term vasopressor use, and a greater number of organ failures contrasted to patients without a sustained SI elevation. An elevated SI may be a useful modality to identify patients with severe sepsis at risk for disease escalation and cardiovascular collapse.

5'RNA-Seq identifies Fhl1 as a genetic modifier in cardiomyopathy.

The transcriptome is subject to multiple changes during pathogenesis, including the use of alternate 5' start-sites that can affect transcription levels and output. Current RNA sequencing techniques can assess mRNA levels, but do not robustly detect changes in 5' start-site use. Here, we developed a transcriptome sequencing strategy that detects genome-wide changes in start-site usage (5'RNA-Seq) and applied this methodology to identify regulatory events that occur in hypertrophic cardiomyopathy (HCM). Compared with transcripts from WT mice, 92 genes had altered start-site usage in a mouse model of HCM, including four-and-a-half LIM domains protein 1 (Fhl1). HCM-induced altered transcriptional regulation of Fhl1 resulted in robust myocyte expression of a distinct protein isoform, a response that was conserved in humans with genetic or acquired cardiomyopathies. Genetic ablation of Fhl1 in HCM mice was deleterious, which suggests that Fhl1 transcriptional changes provide salutary effects on stressed myocytes in this disease. Because Fhl1 is a chromosome X-encoded gene, stress-induced changes in its transcription may contribute to gender differences in the clinical severity of HCM. Our findings indicate that 5'RNA-Seq has the potential to identify genome-wide changes in 5' start-site usage that are associated with pathogenic phenotypes.

Select G-protein-coupled receptors modulate agonist-induced signaling via a ROCK, LIMK, and ß-arrestin 1 pathway.

G-protein-coupled receptors (GPCRs) are typically present in a basal, inactive state but, when bound to an agonist, activate downstream signaling cascades. In studying arrestin regulation of opioid receptors in dorsal root ganglia (DRG) neurons, we find that agonists of delta opioid receptors (δORs) activate cofilin through Rho-associated coiled-coil-containing protein kinase (ROCK), LIM domain kinase (LIMK), and ß-arrestin 1 (ß-arr1) to regulate actin polymerization. This controls receptor function, as assessed by agonist-induced inhibition of voltage-dependent Ca(2+) channels in DRGs. Agonists of opioid-receptor-like receptors (ORL1) similarly influence the function of this receptor through ROCK, LIMK, and ß-arr1. Functional evidence of this cascade was demonstrated in vivo, where the behavioral effects of δOR or ORL1 agonists were enhanced in the absence of ß-arr1 or prevented by inhibiting ROCK. This pathway allows δOR and ORL1 agonists to rapidly regulate receptor function.

Surfactant protein-C chromatin-bound green fluorescence protein reporter mice reveal heterogeneity of surfactant protein C-expressing lung cells.

The regeneration of alveolar epithelial cells is a critical aspect of alveolar reorganization after lung injury. Although alveolar Type II (AT2) cells have been described as progenitor cells for alveolar epithelia, more remains to be understood about how their progenitor cell properties are regulated. A nuclear, chromatin-bound green fluorescence protein reporter (H2B-GFP) was driven from the murine surfactant protein-C (SPC) promoter to generate SPC H2B-GFP transgenic mice. The SPC H2B-GFP allele allowed the FACS-based enrichment and gene expression profiling of AT2 cells. Approximately 97% of AT2 cells were GFP-labeled on Postnatal Day 1, and the percentage of GFP-labeled AT2 cells decreased to approximately 63% at Postnatal Week 8. Isolated young adult SPC H2B-GFP(+) cells displayed proliferation, differentiation, and self-renewal capacity in the presence of lung fibroblasts in a Matrigel-based three-dimensional culture system. Heterogeneity within the GFP(+) population was revealed, because cells with distinct alveolar and bronchiolar gene expression arose in three-dimensional cultures. CD74, a surface marker highly enriched on GFP(+) cells, was identified as a positive selection marker, providing 3-fold enrichment for AT2 cells. In vivo, GFP expression was induced within other epithelial cell types during maturation of the distal lung. The utility of the SPC H2B-GFP murine model for the identification of AT2 cells was greatest in early postnatal lungs and more limited with age, when some discordance between SPC and GFP expression was observed. In adult mice, this allele may allow for the enrichment and future characterization of other SPC-expressing alveolar and bronchiolar cells, including putative stem/progenitor cell populations.

Social cognitive theory as a framework for considering Farm to School programming.

BACKGROUND: Farm to School (FTS) programs are designed, in part, to improve childhood health and nutrition and may be implemented as a strategy to prevent childhood obesity. FTS programs have largely emerged out of grassroots efforts, and theory has not explicitly guided program development or implementation. This research considers the effectiveness of social cognitive theory (SCT) as a framework for FTS programming. METHODS: In 2010, a survey was administered to 632 elementary schoolchildren in Vermont. Six indices were developed from 46 variables that measured personal characteristics and experiences with regard to fruits and vegetables, as described in the SCT. RESULTS: These indices were the basis for cluster analysis, which identified three distinct clusters. Bivariate analysis showed significant differences among the clusters in the children's likelihood of meeting the Dietary Guidelines for Americans (DGA). The significant differences observed among the clusters suggest that SCT is an appropriate framework within which FTS interventions may be considered. CONCLUSIONS: The findings show that there are distinct food-related attitudes and behaviors that differ widely by the SCT informed clusters and that can be used to inform FTS programs.

Impaired lymphatic contraction associated with immunosuppression.

To trigger an effective immune response, antigen and antigen-presenting cells travel to the lymph nodes via collecting lymphatic vessels. However, our understanding of the regulation of collecting lymphatic vessel function and lymph transport is limited. To dissect the molecular control of lymphatic function, we developed a unique mouse model that allows intravital imaging of autonomous lymphatic vessel contraction. Using this method, we demonstrated that endothelial nitric oxide synthase (eNOS) in lymphatic endothelial cells is required for robust lymphatic contractions under physiological conditions. By contrast, under inflammatory conditions, inducible NOS (iNOS)-expressing CD11b(+)Gr-1(+) cells attenuate lymphatic contraction. This inhibition of lymphatic contraction was associated with a reduction in the response to antigen in a model of immune-induced multiple sclerosis. These results suggest the suppression of lymphatic function by the CD11b(+)Gr-1(+) cells as a potential mechanism of self-protection from autoreactive responses during on-going inflammation. The central role for nitric oxide also suggests that other diseases such as cancer and infection may also mediate lymphatic contraction and thus immune response. Our unique method allows the study of lymphatic function and its molecular regulation during inflammation, lymphedema, and lymphatic metastasis.

Rapamycin reverses hypertrophic cardiomyopathy in a mouse model of LEOPARD syndrome-associated PTPN11 mutation.

LEOPARD syndrome (LS) is an autosomal dominant "RASopathy" that manifests with congenital heart disease. Nearly all cases of LS are caused by catalytically inactivating mutations in the protein tyrosine phosphatase (PTP), non-receptor type 11 (PTPN11) gene that encodes the SH2 domain-containing PTP-2 (SHP2). RASopathies typically affect components of the RAS/MAPK pathway, yet it remains unclear how PTPN11 mutations alter cellular signaling to produce LS phenotypes. We therefore generated knockin mice harboring the Ptpn11 mutation Y279C, one of the most common LS alleles. Ptpn11(Y279C/+) (LS/+) mice recapitulated the human disorder, with short stature, craniofacial dysmorphia, and morphologic, histologic, echocardiographic, and molecular evidence of hypertrophic cardiomyopathy (HCM). Heart and/or cardiomyocyte lysates from LS/+ mice showed enhanced binding of Shp2 to Irs1, decreased Shp2 catalytic activity, and abrogated agonist-evoked Erk/Mapk signaling. LS/+ mice also exhibited increased basal and agonist-induced Akt and mTor activity. The cardiac defects in LS/+ mice were completely reversed by treatment with rapamycin, an inhibitor of mTOR. Our results demonstrate that LS mutations have dominant-negative effects in vivo, identify enhanced mTOR activity as critical for causing LS-associated HCM, and suggest that TOR inhibitors be considered for treatment of HCM in LS patients.

Activation of multiple signaling pathways causes developmental defects in mice with a Noonan syndrome­associated Sos1 mutation.

Noonan syndrome (NS) is an autosomal dominant genetic disorder characterized by short stature, unique facial features, and congenital heart disease. About 10%-15% of individuals with NS have mutations in son of sevenless 1 (SOS1), which encodes a RAS and RAC guanine nucleotide exchange factor (GEF). To understand the role of SOS1 in the pathogenesis of NS, we generated mice with the NS-associated Sos1E846K gain-of-function mutation. Both heterozygous and homozygous mutant mice showed many NS-associated pheno-types, including growth delay, distinctive facial dysmorphia, hematologic abnormalities, and cardiac defects. We found that the Ras/MAPK pathway as well as Rac and Stat3 were activated in the mutant hearts. These data provide in vivo molecular and cellular evidence that Sos1 is a GEF for Rac under physiological conditions and suggest that Rac and Stat3 activation might contribute to NS phenotypes. Furthermore, prenatal administration of a MEK inhibitor ameliorated the embryonic lethality, cardiac defects, and NS features of the homozygous mutant mice, demonstrating that this signaling pathway might represent a promising therapeutic target for NS.