Abstract
Three-dimensional engineered cardiac tissue (ECT) using purified human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) has emerged as an appealing model system for the study of human cardiac biology and disease. A recent study reported widely-used metabolic (lactate) purification of monolayer hiPSC-CM cultures results in an ischemic cardiomyopathy-like phenotype compared to magnetic antibody-based cell sorting (MACS) purification, complicating the interpretation of studies using lactate-purified hiPSC-CMs. Herein, our objective was to determine if use of lactate relative to MACS-purified hiPSC-CMs impacts the properties of resulting hiPSC-ECTs. Therefore, hiPSC-CMs were differentiated and purified using either lactate-based media or MACS. Global proteomics revealed lactate-purified hiPSC-CMs displayed a differential phenotype over MACS hiPSC-CMs. hiPSC-CMs were then integrated into 3D hiPSC-ECTs and cultured for four weeks. Structurally, there was no significant difference in sarcomere length between lactate and MACS hiPSC-ECTs. Assessment of isometric twitch force and Ca2+ transients measurements revealed similar functional performance between purification methods. High-resolution mass spectrometry (MS)-based quantitative proteomics showed no significant difference in protein pathway expression or myofilament proteoforms. Taken together, this study demonstrates lactate- and MACS-purified hiPSC-CMs generate ECTs with comparable structural, functional, and proteomic features, and suggests lactate purification does not result in an irreversible change in hiPSC-CM phenotype.
Authors
Kalina J. Rossler, Willem J. De Lange, Morgan W. Mann, Timothy J. Aballo, Jake A. Melby, Jianhua Zhang, Gina Kim, Elizabeth F. Bayne, Yanlong Zhu, Emily T. Farrell, Timothy J. Kamp, J. Carter Ralphe, Ying Ge
Abstract
Ventricular arrhythmias (VAs) in heart failure are enhanced by sympathoexcitation. However, radiotracer studies of catecholamine uptake in failing human hearts demonstrate a proclivity for VAs in patients with reduced cardiac sympathetic innervation. We hypothesized that this counterintuitive finding is explained by heterogeneous loss of sympathetic nerves in the failing heart. In a murine model of dilated cardiomyopathy (DCM), delayed PET imaging of sympathetic nerve density using the catecholamine analog [11C]meta-Hydroxyephedrine ([11C]-mHED) demonstrated global hypoinnervation in ventricular myocardium. Although reduced, sympathetic innervation in two distinct DCM models invariably exhibited transmural (epicardial to endocardial) gradients, with the endocardium being devoid of sympathetic nerve fibers vs. controls. Further, the severity of transmural innervation gradients was correlated with VAs (r = 0.6, P < 0.05). Transmural innervation gradients were also identified in human left ventricular free wall samples from DCM vs controls. We investigated mechanisms underlying this relationship by in silico studies in 1-D, 2-D, and 3-D models of failing and normal human hearts, finding that arrhythmogenesis increased as heterogeneity in sympathetic innervation worsened. Specifically, both DCM-induced myocyte electrical remodeling and spatially inhomogeneous innervation gradients synergistically worsened arrhythmogenesis. Thus, heterogeneous innervation gradients in DCM promoted arrhythmogenesis. Restoration of homogeneous sympathetic innervation in the failing heart may reduce VAs.
Authors
Al-Hassan J. Dajani, Michael B. Liu, Michael A. Olaopa, Lucian Cao, Carla Valenzuela Ripoll, Timothy J. Davis, Megan D. Poston, Elizabeth H. Smith, Jaime Contreras, Marissa Pennino, Christopher M. Waldmann, Donald B. Hoover, Jason T. Lee, Patrick Y. Jay, Ali Javaheri, Roger Slavik, Zhilin Qu, Olujimi A. Ajijola
Abstract
Background: Cardiorenal syndrome (CRS)—renal injury during heart failure (HF)—is linked to higher morbidity. Whether circulating extracellular vesicles (EVs) and their RNA cargo directly impact its pathogenesis remains unclear. Methods: We investigated the role of circulating EVs from patients with CRS on renal epithelial/endothelial cells using a microfluidic kidney-on-chip model (KOC). The small RNA cargo of circulating EVs was regressed against serum creatinine to prioritize subsets of functionally relevant EV miRNAs and their mRNA targets investigated using in silico pathway analysis, human genetics, and interrogation of expression in the KOC model and in renal tissue. The functional effects of EV-RNAs on kidney epithelial cells were experimentally validated.Results: Renal epithelial and endothelial cells in the KOC model exhibited uptake of EVs from HF patients. HF-CRS EVs led to higher expression of renal injury markers (IL18, LCN2, HAVCR1) relative to non-CRS EVs. 15 EV-miRNAs were associated with creatinine, targeting 1143 gene targets specifying pathways relevant to renal injury, including TGF beta and AMPK signaling. We observed directionally consistent changes in the expression of TGF beta pathway members (BMP6, FST, TIMP3) in the KOC model exposed to CRS EVs, which were validated in epithelial cells treated with corresponding inhibitors and mimics of miRNAs. A similar trend was observed in renal tissue with kidney injury. Mendelian randomization suggested a role for FST in renal function. Conclusion: Plasma EVs in CRS patients elicit adverse transcriptional and phenotypic responses in a KOC model by regulating biologically relevant pathways, suggesting a role for EVs in CRS.
Authors
Emeli Chatterjee, Rodosthenis S. Rodosthenous, Ville J. Kujala, Priyanka Gokulnath, Michail Spanos, H. Immo Lehmann, Getulio P de Oliveira-Jr, Mingjian Shi, Tyne W. Miller-Fleming, Guoping Li, Ionita Ghiran, Katia Karalis, JoAnn Lindenfeld, Jonathan D. Mosley, Emily S. Lau, Jennifer E. Ho, Quanhu Sheng, Ravi Shah, Saumya Das
Abstract
BACKGROUND. Oxidized ApoB (oxLDL) and other oxidation-modified lipoproteins (OMLs), such as oxidized ApoA-I (oxHDL), are known pro-atherogenic factors. However, OMLs prognostic value for assessing high-risk coronary plaques by coronary computed tomography angiography (CCTA) has not been fully evaluated. METHODS. In a prospective, observational study, 306 participants with known cardiovascular disease (CVD) had extensive lipoprotein profiling, including plasma OMLs and HDL function measured. Proteomics analysis was performed on oxHDL isolated by anti-oxApoA-I antibody. Atherosclerotic plaque assessment was accomplished by quantitative CCTA (QAngio, Medis). RESULTS. Patients were predominantly white, overweight males (58.5%) on statin therapy (43.5%). Significant increases in LDL-C, ApoB, LDL-TG, sdLDL-C (P<0.001 for all), and TGs (P=0.03) were observed in high oxLDL group, accompanied by less efficient HDL function. High oxLDL was associated with necrotic (NB) (β=0.20; P<0.0001) and fibro-fatty (FFB) burdens (β=0.15; P=0.001) after multivariate adjustment. Low oxHDL had a significant reverse association with these plaque characteristics. Plasma oxHDL levels better predicted NB and FFB after adjustment (2.22, 1.27-3.88 and 2.80, 1.71-4.58) (ORs, 95% CIs) compared to oxLDL and HDL-C. Interestingly, oxHDL was associated with fibrous burden (FB) change over 3.3 years of follow-up (rho=0.535; P=0.033), when compared to oxLDL. Finally, combined Met(136) monooxidation and Trp(132) dioxidation of HDL showed the most evident association with CAC score (r=0.786; P<0.001) and FB (r=0.539; P=0.012) in high oxHDL, whereas Met(136) monooxidation significantly associated with high-risk plaque in low oxHDL. CONCLUSION. Our findings suggest that the investigated OMLs are associated with high-risk coronary plaque features and progression over time in CVD patients. TRIAL REGISTRATION. URL: https://www.clinicaltrials.gov. Unique identifier: NCT01621594. FUNDING. This work was supported by the National Heart, Lung and Blood Institute (NHLBI) at the National Institutes of Health Intramural Research Program. The funding sources had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.
Authors
Alexander V. Sorokin, Christin G. Hong, Angel M. Aponte, Elizabeth M. Florida, Jingrong Tang, Nidhi Patel, Irina N. Baranova, Haiou Li, Philip M. Parel, Vicky Chen, Sierra R. Wilson, Emily L. Ongstad, Anna Collén, Martin P. Playford, Thomas L. Eggerman, Marcus Y. Chen, Kazuhiko Kotani, Alexander V. Bocharov, Alan T. Remaley
Abstract
Diabetic cardiomyopathy, an increasingly global epidemic and a major cause of heart failure with preserved ejection fraction (HFpEF), is associated with hyperglycemia, insulin resistance, and intra-cardiomyocyte calcium mishandling. Here we identify that, in db/db mice with type 2 diabetes induced HFpEF, abnormal remodeling of cardiomyocyte transverse-tubule microdomains occurs with downregulation of the membrane scaffolding protein cardiac bridging integrator 1 (cBIN1). Transduction of cBIN1 by AAV9 gene therapy can restore transverse-tubule microdomains to normalize intracellular distribution of calcium handling proteins and, surprisingly, glucose transporter 4 (GLUT4). Cardiac proteomics revealed that AAV9-cBIN1 normalizes components of calcium handling and GLUT4 translocation machineries. Functional studies further identified that AAV9-cBIN1 normalizes insulin-dependent glucose uptake in diabetic cardiomyocytes. Phenotypically, AAV9-cBIN1 rescues cardiac lusitropy, improves exercise intolerance, and ameliorates hyperglycemia in diabetic mice. Restoration of transverse-tubule microdomains can improve cardiac function in the setting of diabetic cardiomyopathy, and also improve systemic glycemic control.
Authors
Jing Li, Bradley Richmond, Ahmad A. Cluntun, Ryan Bia, Maureen A. Walsh, Kikuyo Shaw, J. David Symons, Sarah Franklin, Jared Rutter, Katsuhiko Funai, Robin M. Shaw, TingTing Hong
Abstract
We investigated the extent, biologic characterization, phenotypic specificity, and possible regulation of a β1-adrenergic receptor–linked (β1-AR–linked) gene signaling network (β1-GSN) involved in left ventricular (LV) eccentric pathologic remodeling. A 430-member β1-GSN was identified by mRNA expression in transgenic mice overexpressing human β1-ARs or from literature curation, which exhibited opposite directional behavior in interventricular septum endomyocardial biopsies taken from patients with beta-blocker–treated, reverse remodeled dilated cardiomyopathies. With reverse remodeling, the major biologic categories and percentage of the dominant directional change were as follows: metabolic (19.3%, 81% upregulated); gene regulation (14.9%, 78% upregulated); extracellular matrix/fibrosis (9.1%, 92% downregulated); and cell homeostasis (13.3%, 60% upregulated). Regarding the comparison of β1-GSN categories with expression from 19,243 nonnetwork genes, phenotypic selection for major β1-GSN categories was exhibited for LV end systolic volume (contractility measure), ejection fraction (remodeling index), and pulmonary wedge pressure (wall tension surrogate), beginning at 3 months and persisting to study completion at 12 months. In addition, 121 lncRNAs were identified as possibly involved in cis-acting regulation of β1-GSN members. We conclude that an extensive 430-member gene network downstream from the β1-AR is involved in pathologic ventricular remodeling, with metabolic genes as the most prevalent category.
Authors
Philip D. Tatman, David P. Kao, Kathryn C. Chatfield, Ian A. Carroll, Jessica A. Wagner, Eric R. Jonas, Carmen C. Sucharov, J. David Port, Brian D. Lowes, Wayne A. Minobe, Sophia P. Huebler, Anis Karimpour-Fard, Erin M. Rodriguez, Stephen B. Liggett, Michael R. Bristow
Abstract
Pediatric cardiomyopathy (CM) represents a group of rare, severe disorders that affect the myocardium. To date, the etiology and mechanisms underlying pediatric CM are incompletely understood, hampering accurate diagnosis and individualized therapy development. Here, bi-allelic variants in the highly conserved flightless-1 (FLII) gene were identified in three families with idiopathic early-onset dilated CM. We demonstrate that patient-specific FLII variants, when brought into the zebrafish genome using CRISPR/Cas9 genome editing, result in the manifestation of key aspects of morphological and functional abnormalities of the heart, as observed in our patients. Importantly, using these genetic animal models, complemented with in-depth loss-of-function studies, we provide insights into the function of Flii during ventricular chamber morphogenesis in vivo, including myofibril organization and cardiomyocyte cell adhesion, as well as trabeculation. In addition, we identify Flii function to be important for the regulation of Notch and Hippo signaling, crucial pathways associated with cardiac morphogenesis and function. Taken together, our data provide experimental evidence for a role for FLII in the pathogenesis of pediatric CM, and report bi-allelic variants as a novel genetic cause of pediatric CM.
Authors
Claudine W.B. Ruijmbeek, Filomena Housley, Hafiza Idrees, Michael P. Housley, Jenny Pestel, Leonie Keller, Jason Kuan Han Lai, Herma C. van der Linde, Rob Willemsen, Janett Piesker, Zuhair N. Al-Hassnan, Abdulrahman Almesned, Michiel Dalinghaus, Lisa M. van den Bersselaar, Marjon A. van Slegtenhorst, Federico Tessadori, Jeroen Bakkers, Tjakko J. van Ham, Didier Y.R. Stainier, Judith M.A. Verhagen, Sven Reischauer
Abstract
Metabolic mechanisms underlying the heterogeneity of major adverse cardiovascular events (MACE) risk in individuals with type 2 diabetes mellitus (T2D) remain unclear. We hypothesized that circulating metabolites reflecting mitochondrial dysfunction predict incident MACE in T2D. Targeted mass-spectrometry profiling of 60 metabolites was performed on baseline plasma from TECOS (discovery) and EXSCEL (validation) trial biomarker substudy cohorts. A principal components analysis metabolite factor comprised of medium-chain acylcarnitines was associated with MACE in TECOS and validated in EXSCEL, with higher levels associated with higher MACE risk. Meta-analysis showed that long-chain acylcarnitines and dicarboxylacylcarnitines were also associated with MACE. Metabolites remained associated with MACE in multivariate models and favorably changed with exenatide therapy. A third cohort (CATHGEN) with T2D assessed whether these metabolites improved discriminative capability multivariate for MACE; nine metabolites (medium- and long-chain acylcarnitines and one dicarboxylacylcarnitine) were associated with time-to-MACE in CATHGEN. Addition of these metabolites to clinical models minimally improved the discriminative capability for MACE but did significantly down reclassify risk. Thus, metabolites reporting on dysregulated mitochondrial fatty acid oxidation are higher in individuals with T2D who experience subsequent MACE. These biomarkers may improve CV risk prediction models, be therapy responsive, and highlight emerging risk mechanisms.
Authors
Jessica A. Regan, Robert J. Mentz, Maggie Nguyen, Jennifer B. Green, Lauren K. Truby, Olga Ilkayeva, Christopher Newgard, John B. Buse, Harald Sourij, C. David Sjöström, Naveed Sattar, Robert W. McGarrah, Yinggan Zheng, Darren K. McGuire, Eberhard Standl, Paul Armstrong, Eric Peterson, Adrian Hernandez, Rury R. Holman, Svati H. Shah
Abstract
Rationale: Femoral atherosclerotic plaques are less inflammatory than carotid plaques histologically, but limited cell-level data exist regarding comparative immune landscapes and polarization at these sites. Objectives: We investigated intraplaque leukocyte phenotypes and transcriptional polarization in 49 total patients undergoing femoral (N=23) or carotid (N=26) endarterectomy using single-cell ribonucleic acid sequencing (scRNA-seq; N=13), flow cytometry (N=24), and immunohistochemistry (N=12). Findings: Comparative scRNA-seq of CD45 positive-selected leukocytes from femoral (N=9; 35265 cells) and carotid (N=4; 30655 cells) plaque revealed distinct transcriptional profiles. Inflammatory foam cell-like macrophages and monocytes comprised 2.5- to 4-fold higher proportions of myeloid cells in carotid plaques, whereas non-inflammatory foam cell-like macrophages and LYVE1-overexpressing resident-like macrophages comprised 3.5- to 9-fold higher proportions of myeloid cells in femoral plaque (p<0.001 for all). A significant comparative excess of CCR2+ macrophages in carotid versus femoral plaque was observed by flow cytometry in a separate validation cohort. B cells were more prevalent and exhibited a comparatively anti-inflammatory profile in femoral plaque, whereas cytotoxic CD8+ T cells were more prevalent in carotid plaque. Conclusion: Human femoral plaques exhibit distinct macrophage profiles and diminished CD8+ T cell populations compared with carotid plaques. Experimental models elucidating determinants of plaque site-specific cell polarization cues are warranted.
Authors
Joshua Slysz, Arjun Sinha, Matthew DeBerge, Shalini Singh, Harris Avgousti, Inhyeok Lee, Kristofor Glinton, Reina Nagasaka, Prarthana J. Dalal, Shaina J. Alexandria, Ching Man Wai, Ricardo Tellez, Mariavittoria Vescovo, Ashwin Sunderraj, Xinkun Wang, Matthew J. Schipma, Ryan K Sisk, Rishab Gulati, Jenifer Vallejo, Ryosuke Saigusa, Donald M. Lloyd-Jones, Jon Lomasney, Samuel E. Weinberg, Karen J. Ho, Klaus Ley, Chiara Giannarelli, Edward B. Thorp, Matthew J. Feinstein
Abstract
Morrbid is a new identified leukocyte-specific long noncoding RNA (lncRNA). However, the expression and biological functions of Morrbid in cardiomyocytes and in heart disease are currently unclear. The study is to determine the roles of cardiac Morrbid in acute myocardial infarction (AMI) and to identify the potential cellular and molecular mechanisms involved. We found that both human and mouse cardiomyocytes could express a significant amount of Morrbid and its expression was increased in cardiomyocytes with hypoxia or oxidative stress, and in mouse hearts with AMI. Overexpression of Morrbid reduced the myocardial infarct size and cardiac dysfunction, whereas the infarct size and cardiac dysfunction were deteriorated in cardiomyocyte-specific Morrbid knockout (Morrbidfl/fl/Myh6-Cre) mice. We identified that Morrbid had a protective effect against hypoxia or H2O2-induce apoptosis, which was also confirmed in vivo in mouse hearts after AMI. We further discovered that serpine1 was a direct target gene of Morrbid, which was involved in Morrbid-mediated protective effect on cardiomyocytes. In summary, we have found for the first time that the cardiac Morrbid is a stress-enhanced lncRNA, which protects hearts from AMI via anti-apoptosis through its target gene serpine1. Morrbid may be a novel promising therapeutic target for ischemic heart diseases such as AMI.
Authors
Yang Yu, Haiqiong Yang, Qiuting Li, Nianhui Ding, Jiali Gao, Gan Qiao, Jianguo Feng, Xin Zhang, Jianming Wu, Yajun Yu, Xiangyu Zhou, Xiaobin Wang, Chunxiang Zhang
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