Neutrophil extracellular traps (NETs) promote inflammation and atherosclerosis progression. NETs are increased in diabetes and impair the resolution of inflammation during wound healing. Atherosclerosis resolution, a process resembling wound healing, is also impaired in diabetes. Thus, we hypothesized that NETs impede atherosclerosis resolution in diabetes by increasing plaque inflammation. Indeed, transcriptomic profiling of plaque macrophages from NET positive and negative areas in low-density lipoprotein receptor-deficient (Ldlr-/-) mice revealed inflammasome and glycolysis pathway upregulation, indicating a heightened inflammatory phenotype. We found that NETs decline during atherosclerosis resolution, which was induced by reducing hyperlipidemia in non-diabetic mice, but they persist in diabetes, exacerbating macrophage inflammation and impairing resolution. In diabetic mice deoxyribonuclease 1 (DNase1) treatment reduced plaque NETs content and macrophage inflammation, promoting atherosclerosis resolution after lipid-lowering. Given that humans with diabetes also exhibit impaired atherosclerosis resolution with lipid-lowering, these data suggest that NETs contribute to the increased cardiovascular disease risk in this population and are a potential therapeutic target.
Tatjana Josefs, Tessa J. Barrett, Emily J. Brown, Alexandra Quezada, Xiaoyun Wu, Maud Voisin, Jaume Amengual, Edward A. Fisher
Clonal hematopoiesis of indeterminate potential is prevalent in elderly individuals and associated with increased risks of all-cause mortality and cardiovascular disease. However, mouse models to study the dynamics of clonal hematopoiesis and its consequences on the cardiovascular system under homeostatic conditions are lacking. We employed a model of clonal hematopoiesis using adoptive transfer of unfractionated ten-eleven translocation 2 (Tet2)-deficient bone marrow cells into non-irradiated mice. Consistent with age-related clonal hematopoiesis observed in humans, these mice displayed a progressive expansion of Tet2-deficient cells in multiple hematopoietic stem and progenitor cell fractions and blood cell linages. The expansion of the Tet2 mutant fraction was also observed in bone marrow-derived CCR2+ myeloid cell populations within the heart, but there was negligible impact on the yolk sac-derived CCR2– cardiac-resident macrophage population. Transcriptome profiling revealed an enhanced inflammatory signature in the donor-derived macrophages isolated from the heart. Mice receiving Tet2-deficient bone marrow cells spontaneously developed age-related cardiac dysfunction characterized by greater hypertrophy and fibrosis. Altogether we show that Tet2-deficient hematopoiesis contributes to cardiac dysfunction in a non-conditioned setting that faithfully models the human clonal hematopoiesis in unperturbed bone marrow. Our data support clinical findings that clonal hematopoiesis per se may contribute to diminished health span.
Ying Wang, Soichi Sano, Yoshimitsu Yura, Zhonghe Ke, Miho Sano, Kosei Oshima, Hayato Ogawa, Keita Horitani, Kyung-Duk Min, Emiri Miura-Yura, Anupreet Kour, Megan A. Evans, María A. Zuriaga, Karen K. Hirschi, Jose J. Fuster, Eric M. Pietras, Kenneth Walsh
The mitochondrial calcium uniporter is widely accepted as the primary route of rapid calcium entry into mitochondria, where increases in matrix calcium contribute to bioenergetics but also mitochondrial permeability and cell death. Hence, regulation of uniporter activity is critical to mitochondrial homeostasis. The uniporter subunit EMRE is known to be an essential regulator of the channel-forming protein MCU in cell culture, but EMRE’s impact on organismal physiology is less understood. Here we characterize a novel mouse model of EMRE deletion and show that EMRE is indeed required for mitochondrial calcium uniporter function in vivo. EMRE–/– mice are born less frequently; however, the mice which are born are viable, healthy, and do not manifest overt metabolic impairment, at rest or with exercise. Finally, to investigate the role of EMRE in disease processes, we examine the effects of EMRE deletion in a muscular dystrophy model associated with mitochondrial calcium overload.
Julia C. Liu, Nicole C. Syder, Nima S. Ghorashi, Thomas B. Willingham, Randi J. Parks, Junhui Sun, Maria M. Fergusson, Jie Liu, Kira M. Holmström, Sara Menazza, Danielle A. Springer, Chengyu Liu, Brian Glancy, Toren Finkel, Elizabeth Murphy
Current models of B lymphocyte biology posit that B cells continuously recirculate between lymphoid organs without accumulating in peripheral healthy tissues. Nevertheless, B lymphocytes are one of the most prevalent leukocyte populations in the naive murine heart. To investigate this apparent inconsistency in the literature, we conducted a systematic analysis of myocardial B cell ontogeny, trafficking dynamics, histology, and gene expression patterns. We found that myocardial B cells represent a subpopulation of circulating B cells that make close contact with the microvascular endothelium of the heart and arrest their transit as they pass through the heart. The vast majority (> 95%) of myocardial B cells remain intravascular, whereas few (< 5%) myocardial B cells cross the endothelium into myocardial tissue. Analyses of mice with B cell deficiency or depletion indicated that B cells modulate the myocardial leukocyte pool composition. Analysis of B cell deficient animals suggested that B cells modulate myocardial growth and contractility. These results transform our current understanding of B cell recirculation in the naive state and reveal a previously unknown relationship between B cells and myocardial physiology. Further work will be needed to assess the relevance of these findings to other organs.
Luigi Adamo, Cibele Rocha-Resende, Chieh-Yu Lin, Sarah Evans, Jesse W. Williams, Hao Dun, Wenjun Li, Cedric Mpoy, Prabhakar Andhey, Buck E. Rogers, Kory Lavine, Daniel Kreisel, Maxim N. Artyomov, Gwendalyn J. Randolph, Douglas Mann
Mutations in cardiac myosin binding protein (MyBP-C, encoded by MYBPC3) are the most common cause of hypertrophic cardiomyopathy (HCM). Most MYBPC3 mutations result in premature termination codons (PTCs) that cause RNA degradation and a reduction of MyBP-C in HCM patient hearts. However, a reduction in MyBP-C has not been consistently observed in MYBPC3 mutant induced pluripotent stell cell cardiomyocytes (iPSCMs). To determine early MYBPC3 mutation effects, we utilized both patient and genome-engineered iPSCMs. iPSCMs with frameshift mutations were compared to iPSCMs with MYBPC3 promoter and translational start site deletions, revealing that allelic loss of function is the primary inciting consequence of mutations that cause PTCs. Despite a reduction in wild type mRNA in all heterozygous iPSCMs, no reduction in MyBP-C protein was observed, indicating protein-level compensation through a previously uncharacterized mechanism. Although homozygous mutant iPSCMs exhibited contractile dysregulation, heterozygous mutant iPSCMs had normal contractile function in the context of compensated MyBP-C levels. Agnostic RNA-seq analysis revealed differential expression in protein chaperone genes as the only dysregulated gene set. To determine how MYBPC3 mutant iPSCMs achieve compensated MyBP-C levels, sarcomeric protein synthesis and degradation were measured with stable isotope-labeling. Heterozygous mutant iPSCMs showed reduced MyBP-C synthesis rates but with a corresponding reduction in MyBP-C degradation. These findings indicate that cardiomyocytes have an innate capacity to attain normal MyBP-C stoichiometry despite MYBPC3 allelic loss of function due to truncating mutations. Modulating MyBP-C degradation to maintain MyBP-C protein levels may be a novel treatment approach upstream of contractile dysfunction for HCM patients.
Adam S Helms, Vi T. Tang, Thomas S. O'Leary, Sabrina Friedline, Mick Wauchope, Akul Arora, Aaron H Wasserman, Eric D Smith, Lap Man Lee, Xiaoquan Wen, Jordan A. Shavit, Allen P Liu, Michael J Previs, Sharlene M. Day
Chronic sympathoexcitation is implicated in ventricular arrhythmogenesis (VAs) following myocardial infarction (MI), but the critical neural pathways involved are not well understood. Cardiac adrenergic function is partly regulated by sympathetic afferent reflexes, transduced by spinal afferent fibers expressing the TRPV1 channel. The role of chronic TRPV1 afferent signaling in VAs is not known. We hypothesized that persistent TRPV1 afferent neurotransmission promotes VAs after MI. Using epicardial Resiniferatoxin (RTX) to deplete cardiac TRPV1-expressing fibers, we dissected the role of this neural circuit in VAs after chronic MI in a porcine model. We examined the underlying mechanisms using molecular approaches, immunohistochemistry, in vitro and in vivo cardiac electrophysiology, and simultaneous cardio-neural mapping. Epicardial RTX depleted cardiac TRPV1 afferent fibers and abolished functional responses to TRPV1 agonists. Ventricular tachycardia/fibrillation (VT/VF) was readily inducible in MI subjects by programmed electrical stimulation or cesium chloride administration, however, TRPV1 afferent depletion prevented VT/VF induced by either method. Mechanistically, TRPV1 afferent depletion neither altered cardiomyocyte action potentials and calcium transients; nor the expression of ion channels and calcium handling proteins. However, it attenuated fibrosis and mitigated electrical instability in the scar-border zone. In vivo recordings of cardiovascular-related stellate ganglion neurons (SGNs) revealed that MI enhances SGN function and disrupts integrated neural processing. Depleting TRPV1 afferents normalized these processes. Taken together, these data indicate that after MI, TRPV1 afferent-induced adrenergic dysfunction promotes fibrosis, adverse cardiac remodeling, and worsens border zone electrical heterogeneity, resulting in electrically unstable ventricular myocardium. We propose targeting TRPV1-expressing afferent to reduce VT/VF following MI.
Koji Yoshie, Pradeep S. Rajendran, Louis Massoud, Janki Mistry, M. Amer Swid, Xiaohui Wu, Tamer Sallam, Rui Zhang, Joshua I. Goldhaber, Siamak Salavatian, Olujimi A. Ajijola
High-density lipoproteins (HDL) contain hundreds of lipid species and proteins and exert many potentially vasoprotective and anti-diabetogenic activities on cells. To resolve structure-function-disease relationships of HDL we characterized HDL of 51 healthy subjects and 98 patients with diabetes (T2DM), coronary heart disease (CHD), or both for protein and lipid composition as well as functionality in five cell types. The integration of 40 clinical characteristics, 34 NMR features, 182 proteins, 227 lipid species, and 12 functional read-outs by high-dimensional statistical modelling revealed first that CHD and T2DM are associated with different changes of HDL in size distribution, protein and lipid composition as well as function. Second, different cellular functions of HDL are weakly correlated with each other and determined by different structural components. Cholesterol efflux capacity was no proxy of other functions. Third, three novel determinants of HDL function were identified and validated by the use of artificially reconstituted HDL, namely the sphingadienine-based sphingomyelin SM 42:3 and glycosylphosphatidylinositol-phospholipase D1 for the ability of HDL to inhibit starvation induced apoptosis of human aortic endothelial cells and apolipoprotein F for the ability of HDL to promote maximal respiration of brown adipocytes.
Mathias Cardner, Mustafa Yalcinkaya, Sandra Goetze, Edlira Luca, Miroslav Balaz, Monika Hunjadi, Johannes Hartung, Andrej Shemet, Nicolle Kraenkel, Silvija Radosavljevic, Michaela Keel, Alaa Othman, Gergely Karsai, Thorsten Hornemann, Manfred Claassen, Gerhard Liebisch, Erick Carreira, Andreas Ritsch, Ulf Landmesser, Jan Krützfeldt, Christian Wolfrum, Bernd Wollscheid, Niko Beerenwinkel, Lucia Rohrer, Arnold von Eckardstein
The adult mammalian heart regenerates poorly after injury and, as a result, ischemic heart diseases are among the leading causes of death worldwide. The recovery of the injured heart is dependent on orchestrated repair processes including inflammation, fibrosis, cardiomyocyte survival, proliferation, and contraction properties that could be modulated in patients. In this work we designed an automated high-throughput screening system for small molecules that induce cardiomyocyte proliferation in vitro and identified the small molecule Chicago Sky Blue 6B (CSB). Following induced myocardial infarction, CSB treatment reduced scar size and improved heart function of adult mice. Mechanistically, we show that although initially identified using in vitro screening for cardiomyocyte proliferation, in the adult mouse CSB promotes heart repair through (i) inhibition of CaMKII signaling, which improves cardiomyocyte contractility; and (ii) inhibition of neutrophil and macrophage activation, which attenuates the acute inflammatory response, thereby contributing to reduced scarring. In summary, we identified CSB as a potential therapeutic agent that enhances cardiac repair and function by suppressing postinjury detrimental processes, with no evidence for cardiomyocyte renewal.
Oren Yifa, Karen Weisinger, Elad Bassat, Hanjun Li, David Kain, Haim Barr, Noga Kozer, Alexander Genzelinakh, Dana Rajchman, Tamar Eigler, Kfir Baruch Umansky, Daria Lendengolts, Ori Brener, Nenad Bursac, Eldad Tzahor
Aging is a major risk factor for cardiovascular disease. Although the impact of aging has been extensively studied, little is known regarding the aging processes in cells of the heart. Here we analyzed the transcriptomes of hearts of 12-week-old and 18-month-old mice by single-nucleus RNA-sequencing. Among all cell types, aged fibroblasts showed most significant differential gene expression, increased RNA dynamics, and network entropy. Aged fibroblasts exhibited significantly changed expression patterns of inflammatory, extracellular matrix organization angiogenesis, and osteogenic genes. Functional analyses indicated deterioration of paracrine signatures between fibroblasts and endothelial cells in old hearts. Aged heart-derived fibroblasts had impaired endothelial cell angiogenesis and autophagy and augmented proinflammatory response. In particular, expression of Serpine1 and Serpine2 were significantly increased and secreted by old fibroblasts to exert antiangiogenic effects on endothelial cells, an effect that could be significantly prevented by using neutralizing antibodies. Moreover, we found an enlarged subpopulation of aged fibroblasts expressing osteoblast genes in the epicardial layer associated with increased calcification. Taken together this study provides system-wide insights and identifies molecular changes of aging cardiac fibroblasts, which may contribute to declined heart function.
Ramon Vidal, Julian Uwe Gabriel Wagner, Caroline Braeuning, Cornelius Fischer, Ralph Patrick, Lukas Tombor, Marion Muhly-Reinholz, David John, Magdalena Kliem, Thomas Conrad, Nuno Guimarães-Camboa, Richard Harvey, Stefanie Dimmeler, Sascha Sauer
Mutations in B cell lymphoma 2–associated athanogene 3 (BAG3) are recurrently associated with dilated cardiomyopathy (DCM) and muscular dystrophy. Using isogenic genome-edited human induced pluripotent stem cell–derived cardiomyocytes (iPSC-CMs), we examined how a DCM-causing BAG3 mutation (R477H), as well as complete loss of BAG3 (KO), impacts myofibrillar organization and chaperone networks. Although unchanged at baseline, fiber length and alignment declined markedly in R477H and KO iPSC-CMs following proteasome inhibition. RNA sequencing revealed extensive baseline changes in chaperone- and stress response protein–encoding genes, and protein levels of key BAG3 binding partners were perturbed. Molecular dynamics simulations of the BAG3-HSC70 complex predicted a partial disengagement by the R477H mutation. In line with this, BAG3-R477H bound less HSC70 than BAG3-WT in coimmunoprecipitation assays. Finally, myofibrillar disarray triggered by proteasome inhibition in R477H cells was mitigated by overexpression of the stress response protein heat shock factor 1 (HSF1). These studies reveal the importance of BAG3 in coordinating protein quality control subsystem usage within the cardiomyocyte and suggest that augmenting HSF1 activity might be beneficial as a means to mitigate proteostatic stress in the context of BAG3-associated DCM.
Chris McDermott-Roe, Wenjian Lv, Tania Maximova, Shogo Wada, John Bukowy, Maribel Marquez, Shuping Lai, Amarda Shehu, Ivor Benjamin, Aron Geurts, Kiran Musunuru
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