Arrhythmogenic cardiomyopathy (AC) is a genetic disease causing arrhythmia and sudden cardiac death with only symptomatic therapy available at present. Mutations of desmosomal proteins, including desmoglein-2 (Dsg2) and plakoglobin (Pg), are the major cause of AC and have been shown to lead to impaired gap junction function. Recent data indicated the involvement of anti-Dsg2 autoantibodies in AC pathogenesis. We applied a peptide to stabilize Dsg2 binding similar to a translational approach to pemphigus, which is caused by anti-desmoglein autoantibodies. We provide evidence that stabilization of Dsg2 binding by a linking peptide (Dsg2-LP) is efficient to rescue arrhythmia in an AC mouse model immediately upon perfusion. Dsg2-LP, designed to cross-link Dsg2 molecules in proximity to the known binding pocket, stabilized Dsg2-mediated interactions on the surface of living cardiomyocytes as revealed by atomic force microscopy and induced Dsg2 oligomerization. Moreover, Dsg2-LP rescued disrupted cohesion induced by siRNA-mediated Pg or Dsg2 depletion or l-tryptophan, which was applied to impair overall cadherin binding. Dsg2-LP rescued connexin-43 mislocalization and conduction irregularities in response to impaired cardiomyocyte cohesion. These results demonstrate that stabilization of Dsg2 binding by Dsg2-LP can serve as a novel approach to treat arrhythmia in patients with AC.
Camilla Schinner, Bernd Markus Erber, Sunil Yeruva, Angela Schlipp, Vera Rötzer, Ellen Kempf, Sebastian Kant, Rudolf E. Leube, Thomas D. Mueller, Jens Waschke
Doxorubicin (DOX), a chemotherapeutic agent, induces a cardiotoxicity referred to as doxorubicin-induced cardiomyopathy (DIC). This cardiotoxicity often limits chemotherapy for malignancies and is associated with poor prognosis. However, the molecular mechanism underlying this cardiotoxicity is yet to be fully elucidated. Here, we show that DOX downregulated glutathione peroxidase 4 (GPx4) and induced excessive lipid peroxidation through DOX-Fe2+ complex in mitochondria, leading to mitochondria-dependent ferroptosis; we also show that mitochondria-dependent ferroptosis is a major cause of DOX cardiotoxicity. In DIC mice, the left ventricular ejection fraction was significantly impaired, and fibrosis and TUNEL+ cells were induced at day 14. Additionally, GPx4, an endogenous regulator of ferroptosis, was downregulated, accompanied by the accumulation of lipid peroxides, especially in mitochondria. These cardiac impairments were ameliorated in GPx4 Tg mice and exacerbated in GPx4 heterodeletion mice. In cultured cardiomyocytes, GPx4 overexpression or iron chelation targeting Fe2+ in mitochondria prevented DOX-induced ferroptosis, demonstrating that DOX triggered ferroptosis in mitochondria. Furthermore, concomitant inhibition of ferroptosis and apoptosis with ferrostatin-1 and zVAD-FMK fully prevented DOX-induced cardiomyocyte death. Our findings suggest that mitochondria-dependent ferroptosis plays a key role in progression of DIC and that ferroptosis is the major form of regulated cell death in DOX cardiotoxicity.
Tomonori Tadokoro, Masataka Ikeda, Tomomi Ide, Hiroko Deguchi, Soichiro Ikeda, Kosuke Okabe, Akihito Ishikita, Shouji Matsushima, Tomoko Koumura, Ken-ichi Yamada, Hirotaka Imai, Hiroyuki Tsutsui
B-type natriuretic peptide (BNP) is secreted by ventricular cardiomyocytes in response to various types of cardiac stress and has been used as a heart failure marker. In septic patients, increased BNP suggests poor prognosis; however, no causal link has been established. Among various effects, BNP decreases systemic vascular resistance and increases natriuresis that leads to lower blood pressure. We previously observed that JNK inhibition corrects cardiac dysfunction and suppresses cardiac BNP mRNA in endotoxemia. In this study, we investigated the transcriptional mechanism that regulates BNP expression and the involvement of plasma BNP in causing septic hypotension. Our in vitro and in vivo findings confirmed that activation of JNK signaling increases BNP expression in sepsis via direct binding of c-Jun in activating protein–1 (AP-1) regulatory elements of the Nppb promoter. Accordingly, genetic ablation of BNP, as well as treatment with a potentially novel neutralizing anti-BNP monoclonal antibody (19B3) or suppression of its expression via administration of JNK inhibitor SP600125 improved cardiac output, stabilized blood pressure, and improved survival in mice with polymicrobial sepsis. Therefore, inhibition of JNK signaling or BNP in sepsis appears to stabilize blood pressure and improve survival.
Matthew Hoffman, Ioannis D. Kyriazis, Alexandra Dimitriou, Santosh K. Mishra, Walter J. Koch, Konstantinos Drosatos
Atrial fibrillation (AF) alters atrial-cardiomyocyte (ACM) Ca2+-handling, promoting ectopic-beat formation. Here, we examined the effects of AF-associated remodeling on Ca2+-related action-potential (AP) dynamics and consequences for AF-susceptibility. AF was maintained electrically (x1 week) in dogs by right-atrial (RA) tachypacing. ACMs isolated from AF-dogs showed increased Ca2+-release refractoriness, spontaneous Ca2+-spark frequency and cycle-length (CL) threshold for Ca2+ and APD alternans versus controls. Similarly, AF increased the in-situ CL-threshold for Ca2+/APD-alternans and spatial dispersion in Ca2+-release recovery kinetics, leading to spatially-discordant alternans associated with reentrant rotor formation and susceptibility to AF induction/maintenance. The clinically-available agent dantrolene reduced Ca2+-leak and CL-threshold for Ca2+/APD-alternans in both ACMs and AF-dog RA, while suppressing AF-susceptibility; caffeine increased Ca2+-leak, CL-threshold for Ca2+/APD-alternans in control-dog ACMs and RA-tissues. In vivo, the atrial repolarization alternans CL-threshold was increased in AF vs control, as was AF-vulnerability. Intravenous dantrolene restored repolarization alternans-threshold and reduced AF-vulnerability. Immunoblots showed significantly reduced expression of total and phosphorylated ryanodine-receptors and calsequestrin in AF, along with unchanged phospholamban/SERCA expression. Thus, in addition to promoting spontaneous ectopy, AF-induced Ca2+-handling abnormalities favor AF-occurrence by enhancing vulnerability to repolarization-alternans, thereby promoting the initiation and maintenance of reentrant activity; the clinically-available compound dantrolene provides a lead-molecule to target this mechanism.
Tao Liu, Feng Xiong, Xiao-yan Qi, Jiening Xiao, Louis Villeneuve, Issam Abu-Taha, Dobromir Dobrev, Congxin Huang, Stanley Nattel
Pressure overload (PO) cardiac hypertrophy and heart failure are associated with generalized insulin resistance and hyperinsulinemia, which may exacerbate left ventricular (LV) remodeling. While PO activates insulin receptor tyrosine kinase activity that is transduced by insulin receptor substrate 1 (IRS1), the present study tested the hypothesis that IRS1 and IRS2 have divergent effects on PO-induced LV remodeling. We therefore subjected mice with cardiomyocyte-restricted deficiency of IRS1 (CIRS1KO) or IRS2 (CIRS2KO) to PO induced by transverse aortic constriction (TAC). In WT mice, TAC-induced LV hypertrophy was associated with hyperactivation of IRS1 and Akt1, but not IRS2 and Akt2. CIRS1KO hearts were resistant to cardiac hypertrophy and heart failure in concert with attenuated Akt1 activation. In contrast, CIRS2KO hearts following TAC developed more severe LV dysfunction than WT controls, and this was prevented by haploinsufficiency of Akt1. Failing human hearts exhibited isoform-specific IRS1 and Akt1 activation, while IRS2 and Akt2 activation were unchanged. Kinomic profiling identified IRS1 as a potential regulator of cardioprotective protein kinase G–mediated signaling. In addition, gene expression profiling revealed that IRS1 signaling may promote a proinflammatory response following PO. Together, these data identify IRS1 and Akt1 as critical signaling nodes that mediate LV remodeling in both mice and humans.
Christian Riehle, Eric T. Weatherford, Adam R. Wende, Bharat P. Jaishy, Alec W. Seei, Nicholas S. McCarty, Monika Rech, Qian Shi, Gopireddy R. Reddy, William J. Kutschke, Karen Oliveira, Karla Maria Pires, Joshua C. Anderson, Nikolaos A. Diakos, Robert M. Weiss, Morris F. White, Stavros G. Drakos, Yang K. Xiang, E. Dale Abel
Heart failure (HF) remains a grievous illness with poor prognosis even with optimal care. The apelin receptor (APJ) counteracts the pressor effect of angiotensin II, attenuates ischemic injury and has the potential to be a novel target to treat HF. Intravenous administration of apelin improves cardiac function acutely in HF patients. However, its short half-life restricts its use to infusion therapy. To identify a longer acting APJ agonist, we conducted a medicinal chemistry campaign leading to the discovery of potent small-molecule APJ agonists with comparable activity to apelin by mimicking the C-terminal portion of apelin-13. Acute infusion increased systolic function and reduced systemic vascular resistance in two rat models of impaired cardiac function. Similar results were obtained in an anesthetized but not a conscious canine HF model. Chronic oral dosing in a rat myocardial infarction model reduced myocardial collagen content and improved diastolic function to a similar extent as losartan, a RAS antagonist standard of care therapy, but lacked additivity with co-administration. Collectively, this work demonstrates the feasibility of developing clinical viable potent small molecule agonists that mimic the endogenous APJ ligand with more favorable drug-like properties and highlight potential limitations for APJ agonism for this indication.
Brandon Ason, Yinhong Chen, Qi Guo, Kimberly M Hoagland, Ray W. Chui, Mark Fielden, Weston Sutherland, Rhonda Chen, Ying Zhang, Shirley Mihardja, Xiaochuan Ma, Xun Li, Yaping Sun, Dongming Liu, Khanh Nguyen, Jinghong Wang, Ning Li, Sridharan Rajamani, Yusheng Qu, BaoXi Gao, Andrea Boden, Vishnu Chintalgattu, Jim R. Turk, Joyce C. Y. Chan, Liaoyuan A. Hu, Paul Dransfield, Jonathan B. Houze, Jing Man Wong, Ji Ma, Vatee Pattaropong, Murielle M. Veniant, Hugo M Vargas, Gayathri Swaminath, Aarif Khakoo
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
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