Description of Research Expertise:
Current Areas of Active Research in the Margulies Lab
The Margulies Lab examines mechanisms of myocardial remodeling to identify targets for therapeutic interventions. Many of our inquiries are initiated by multilevel examinations of explanted human heart tissues obtained at the time of transplantation or organ donation to permit insights into both the heterogeneity of myocardial adaptations to disease and identification of dominant mechanisms and responses. The Margulies lab has a longstanding focus on load-dependent myocardial remodeling with current studies focusing on mechanisms of load-dependent maturation, mechanical memory and pathological myocardial hypertrophy. In addition, the lab is actively engaged in studies examining regulation of contractility by microtubules and other cytoskeletal elements, cardiotoxicity associated with VEGF-anti-angiogenic tyrosine kinase inhibitors, disease-associated shifts in cardiac metabolism, mechanisms of inherited cardiomyopathies, advanced myocardial phenotyping using digital pathology and machine learning techniques, and integrated genomic inquiries designed to identify molecular mechanisms of myocardial pathology. Increasingly, complementary use of primary human cardiomyocytes, iPSC-derived cardiomyocytes and engineered cardiac microtissues are employed as versatile tools to balance human relevance and mechanistic clarity to advance these inquiries. Our work also includes, strategic patient-based proof-of-concept studies.
Studies of load-dependent myocardial cell adaptations
have employed these materials to both two- (2D) and three-dimensional (3D) culture formats. In conventional 2D culture formats, the high degree of temporal control provided by these new tunable elastomers are proving ideal for studies of “mechanical memory”. Recognizing shortcomings of 2D formats for inducing cardiac cell maturation or modeling pathological processes, we have increasingly employed a 3D heterocellular model in which cardiomyocytes and fibroblasts self-assemble into contracting cardiac microtissues (CMT) mounted on flexible cantilevers. These cantilevers both provide biomechanical input and report force generation in real time. In this format, application of our tunable elastomers allow control of in vitro afterload to study processes of load-dependent myocardial maturation, pathologic hypertrophy during sustained biomechanical stress, hypertrophy regression and the molecular dynamics transducing these processes. The use of cardiac myocytes derived from human induced pluripotent stem cells (Hu-iPSC-CM), molecular reporters and advances in gene editing provides diverse unprecedented opportunities to rigorously study these fundamental processes in human cardiac cells. (Collaboration with Prosser, Musunuru, Turner laboratories)
Novel Contractility Modulation in Heart Failure
There is an unmet clinical need for agents that safely improve impaired contraction and relaxation observed in most patients with heart failure (HF). At present, all agents that augment contractility increase adverse outcomes and are considered palliative. In this context, the Prosser and Margulies labs have recently demonstrated that the microtubule network (MTN) within each cardiac myocyte acts as a viscous restraint that impedes contraction and slows relaxation in HF. The stiffness and abundance of the MTN is regulated by the ratio of the detyrosinated and tyrosinated forms of α-tubulin (dT/T). More detyrosination, as we have observed in HF, is associated with greater stiffness impairment of both contraction and relaxation and more pronounced improvement in contraction and relaxation in response manipulations that destabilize microtubules or decrease dT/T2. Accordingly, our labs are actively addressing timely and strategic questions that are relevant to clinical translation of our recent findings including: 1) what causes increased MTN density in HF?; 2) does sustained targeting of the enzymes regulating dT/T3 improve cardiac contractility and relaxation without detrimental effects?; 3) Do interventions that decrease dT/T improve contractility without increasing myocardial oxygen consumption and/or ATP utilization?; and 4) Are there detrimental non-cardiac effects of sustained decreases in dT/T that necessitate myocardial targeting via gene therapy. These inquiries involve utilization of several core capabilities in the Prosser and Margulies laboratories including: super-resolution live-cell microscopy, advanced primary human cardiomyocytes mechanical characterization, and an engineered myocardial microtissue model derived from human induced pluripotent stem cells, and murine models currently being developed. (Collaboration with Prosser laboratory)
Cardiotoxicity of Anti-Angiogenic Tyrosine Kinase Inhibitors
Cancer is becoming curable or treatable as a chronic disease, largely as a result of the rapid expansion of anti-cancer agents, however, treatment-related cardiovascular (CV) disease is emerging as an important contributor to morbidity and mortality in cancer patients. For example, anti-angiogenic tyrosine kinase inhibitors (AA-TKIs), like sunitinib, are the mainstay of first-line therapies in several cancer types, but are associated with substantial CV toxicity including hypertension in at least half of all participants and left ventricular dysfunction in 10 to 15%. To address the question of whether hypertension accentuates the cardiotoxicity of sunitinib or simply unmasks it, we recently employed the CMT model described above to demonstrate that culturing tissues on stiff mountings (cantilevers), to mimic in vivo hypertension, augments sensitivity to sunitinib-induced cardiomyocyte toxicity.[Truitt 2018, JACC BTS in press] Cardiotoxicity was based on caspase activation (a marker of programmed cell death pathway activation), force generation, spontaneous beating frequency or mitochondrial membrane potential. Increased in vitro afterload augmented cardiotoxicity in engineered CMTs made with either neonatal rat cardiac myocytes or human cardiac myocytes. In the human CMTs, increased in vitro afterload was necessary for sunitinib-induced apoptosis at clinically relevant exposure concentrations. These findings indicate that increased afterload contributes to sunitinib cardiotoxicity and provide key mechanistic data and scientific rationale for translating the findings to patients with hypertension induced by AA-TKIs. (Collaboration with Ky laboratory)
Disease-Associated Shifts in Cardiac Metabolism
The normal human heart consumes 6 kg of ATP daily 30 times its own weight and fatty acids (FAs) are the predominant energetic substrate for the heart with about 30% of ATP generation coming from carbohydrates. As HF progresses, there is a quantitative switch from a predominance of FA utilization to the more carbohydrates. In advanced HF, insulin resistance develops and deficits in both FA and glucose metabolism become evident. In severely failing human hearts, we recently reported that there is an unexpected up-regulation in Ketone metabolism. In murine models, complementary findings in Dan Kelly’s lab have confirmed this HF stage-dependent progression of myocardial substrate utilization in HF while indicating that increased burning of ketones in advanced heart failure is adaptive. Ongoing studies in our laboratory are using primary human cardiomyocytes to study substrate preferences, identify the factors regulating these preferences and determine the physiological of substrate compositions better adapted to the metabolic remodeling associated with advanced heart failure. Using our 3D CMT model, we are also exploring whether manipulation of metabolic pathways can drive myocardial tissue maturation and whether the phenotype of CMTs derived from Hu-iPSC-CM can mimic the increased fat oxidation and storage of lipids as observed in diabetic hearts. These studies are adapt methods for characterizing labeled substrate oxidation and mass spectrometry-based stable isotope incorporation in the CMT model. We plan to evaluate whether sustained substrate alterations and/or induction of insulin resistance increase toxic lipid species, oxidative products of fatty acid oxidation or mitochondrial dysfunction within the cardiomyocytes of CMTs, compared with human heart samples. Finally, by increasing cantilever stiffness after CMTs have formed, we will examine whether increased in vitro afterload exacerbates the pathological functional CMT phenotype, as occurs in patients with diabetes and hypertension. (Collaboration with Kelly, Arany, Lazar and Musunuru laboratories)
Targeting Mechanisms of Inherited Cardiomyopathy
Inherited cardiomyopathies are a major cause of heart disease in all age groups and affect both patients and their families. The identification of “cardiomyopathy genes” has raised expectations for full understanding of disease mechanisms and treatments precisely targeting these mechanisms. However, our understanding of causes of divergent impacts of cardiomyopathy genes remains elusive. In this context, a cadre of Penn CVI investigators is exceptionally well-positioned to identify the mechanisms through which “disease-enabling” mutations contribute to inherited cardiomyopathies and develop strategies to mitigate the pathological processes involved. We have a unique ability to thoroughly characterize a patient’s disease mutation in multiple model systems in parallel including primary human cardiac myocytes, patient-specific induced pluripotent stem cell (iPSC)-derived cardiac myocytes, engineered heart tissues and mouse models. These model systems are empowered by a rich clinical infrastructure, bioresources and state-of-the-art technical expertise in several key domains. Our investigative team, versatile models and expertise provide unprecedented opportunities to understand the cellular basis of genetically enabled cardiomyopathies. In proof-of-concept studies we have performed both physiological and ultrastructural phenotyping of primary myocytes from a Penn heart transplant recipient with a LAMIN A/C We have already begun to improve, validate and ultimately exploit our engineered cell and tissue models via direct comparison with primary cardiac myocytes from the individuals whose diseases we are modeling. (Collaboration with Owens, Musunuru, Arany, Jain, Prosser and Wallace laboratories)
Enhanced Diagnostics via Myocardial Digital Pathology and Advanced Machine Learning
In recent years, advanced machine learning techniques have been applied to rapidly widening array of complex processes to gain new insights into underlying patterns that have previously escaped detection. For example, deep learning algorithms enabling facial recognition technology have been applied to advanced analysis of histological sections to identify features of previously unappreciated diagnostic and prognostic value. Recognizing that “digital pathology” successful in Oncology has had no application in Cardiology, we initiated a series of studies to address this opportunity. In one study, we performed a first-in-heart deep learning neural network classification of failing vs. non-failing hearts based on histologic samples from full thickness biopsies taken at the time of heart explant or VAD placement. This deep algorithm achieved a sensitivity of 99% and specificity of 94%, far exceeding the performance of two expert pathologists. Building on this result, we are now applying this approach to computerized-assisted detection and grading of cardiac allograft rejection, an area where trained pathologists exhibit wide diagnostic variability. We are also applying these techniques in an ongoing clinical trial in which we are comparing electrogram-targeted myocardial biopsies with “untargeted” fluroscopically-guided biopsies in patients with new-onset nonischemic cardiomyopathy. By using artificial intelligence and machine-based learning algorithms to analyze heart biopsy specimens, we intend to discover novel histopathological features that will permit more refined diagnostics and perhaps better targeted therapeutics.
Functional Genomics of Failing Human Myocardium
The Margulies and Cappola Labs recently led a large integrated genomic analysis deigned to identify common genetic variants that influence myocardial gene expression in normal and failing human hearts. This effort included combined whole-genome SNP genotyping and whole-genome myocardial transcriptional profiling of over 700 cases, including 380 with RNA sequencing. The results of the expression quantitative trait loci (eQTL) analysis were coupled with a meta-analysis of cardiac genome-wide association (GWAS) studies to prioritize loci that influence HF risk. Through collaboration with extramural investigators, this effort has been further enriched by analyses of epigenetic marks and DNA methylation from the same cases to provide additional insights into the factors regulating myocardial gene expression. Application of the above digital pathology techniques, advanced proteomic profiling and queries of published studies provide further opportunities to identify mechanisms of molecular regulation in the diseased myocardium and potential therapeutic targets suitable for further exploration. Ongoing studies have been exploring the functional biology of newly implicated genes and pathways. In one such analysis, we have identified several human myocardial eQTLs that have already been shown to influence the degree of cardiac hypertrophy during sustained pressure overload in mice. We are using our CMT model to demonstrate that molecular manipulation of these prioritized pro-hypertrophic targets can alter the hypertrophic response in Hu-iPSC-CMs to sustained increases in cantilever stiffness.
Corbin EA, Vite A, Peyster EG, Bhoopalam M, Brandimarto J, Wang X, Bennett AI, Clark AT, Cheng X, Turner KT, Musunuru K, Margulies KB: Tunable and Reversible Substrate Stiffness Reveals a Dynamic Mechanosensitivity of Cardiomyocytes ACS Appl Mater Interfaces 11 (23): 20603-20614,2019.
Morley MP, Wang X, Hu R, Brandimarto J, Tucker NR, Felix JF, Smith NL, van der Harst P, Ellinor PT, Margulies KB, Musunuru K, Cappola TP: Cardioprotective Effects of MTSS1 Enhancer Variants Circulation 139 (17): 2073-2076,2019.
Xiong Y, Bedi KC, Berritt S, Brooks TG, Attipoe BK, Wang K, Margulies KB, Field J: Targeting MRTF/SRF in CAP2-dependent dilated cardiomyopathy delays disease onset JCI Insight : 2019.
Moyes AJ, Chu SM, Aubdool AA, Dukinfield MS, Margulies KB, Bedi KC, Hodivala-Dilke K, Baliga RS, Hobbs AJ: C-type natriuretic peptide co-ordinates cardiac structure and function Eur Heart J : 2019.
Blackstone EH, Chang HL, Rajeswaran J, Parides MK, Ishwaran H, Li L, Ehrlinger J, Gelijns AC, Moskowitz AJ, Argenziano M, DeRose JJ Jr, Couderc JP, Balda D, Dagenais F, Mack MJ, Ailawadi G, Smith PK, Acker MA, O'Gara PT, Gillinov AM; Cardiothoracic Surgical Trials Network Investigators: Biatrial maze procedure versus pulmonary vein isolation for atrial fibrillation during mitral valve surgery: New analytical approaches and end points J Thorac Cardiovasc Surg 157 (1): 234-243,2019.
Sharma A, Ambrosy AP, DeVore AD, Margulies KB, McNulty SE, Mentz RJ, Hernandez AF, Michael Felker G, Cooper LB, Lala A, Vader J, Groake JD, Borlaug BA, Velazquez EJ: Liraglutide and weight loss among patients with advanced heart failure and a reduced ejection fraction: insights from the FIGHT trial ESC Heart Fail : 2018 .
Chen CY, Caporizzo MA, Bedi K, Vite A, Bogush AI, Robison P, Heffler JG, Salomon AK, Kelly NA, Babu A, Morley MP, Margulies KB, Prosser BL: Suppression of detyrosinated microtubules improves cardiomyocyte function in human heart failure Nat Med : 2018 .
Nirschl JJ, Janowczyk A, Peyster EG, Frank R, Margulies KB, Feldman MD, Madabhushi A: A deep-learning classifier identifies patients with clinical heart failure using whole-slide images of H&E tissue PLoS One 3;13 (4): 2018 .
Baggio LL, Yusta B, Mulvihill EE, Cao X, Streutker CJ, Butany J, Cappola TP, Margulies KB, Drucker DJ: GLP-1 receptor expression within the human heart Endocrinology : 2018 .
Birati EY, Hanff TC, Maldonado D, Grandin EW, Kennel PJ, Mazurek JA, Vorovich E, Seigerman M, Howard JLL, Acker MA, Naka Y, Wald J, Goldberg LR, Jessup M, Atluri P, Margulies KB, Schulze PC, Rame JE: Predicting Long Term Outcome in Patients Treated With Continuous Flow Left Ventricular Assist Device: The Penn-Columbia Risk Score J Am Heart Assoc : 2018 .
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