Mutations in nuclear envelope proteins (NEPs) cause devastating genetic diseases, known as envelopathies, that primarily affect the heart and skeletal muscle. A mutation in the NEP LEM domain–containing protein 2 (LEMD2) causes severe cardiomyopathy in humans. However, the roles of LEMD2 in the heart and the pathological mechanisms responsible for its association with cardiac disease are unknown. We generated knockin (KI) mice carrying the human c.T38>G Lemd2 mutation, which causes a missense amino acid exchange (p.L13>R) in the LEM domain of the protein. These mice represent a preclinical model that phenocopies the human disease, as they developed severe dilated cardiomyopathy and cardiac fibrosis leading to premature death. At the cellular level, KI/KI cardiomyocytes exhibited disorganization of the transcriptionally silent heterochromatin associated with the nuclear envelope. Moreover, mice with cardiac-specific deletion of Lemd2 also died shortly after birth due to heart abnormalities. Cardiomyocytes lacking Lemd2 displayed nuclear envelope deformations and extensive DNA damage and apoptosis linked to p53 activation. Importantly, cardiomyocyte-specific Lemd2 gene therapy via adeno-associated virus rescued cardiac function in KI/KI mice. Together, our results reveal the essentiality of LEMD2 for genome stability and cardiac function and unveil its mechanistic association with human disease.
Xurde M. Caravia, Andres Ramirez-Martinez, Peiheng Gan, Feng Wang, John R. McAnally, Lin Xu, Rhonda Bassel-Duby, Ning Liu, Eric N. Olson
People with kidney disease are disproportionately affected by atherosclerosis for unclear reasons. Soluble urokinase plasminogen activator receptor (suPAR) is an immune-derived mediator of kidney disease, levels of which are strongly associated with cardiovascular outcomes. We assessed suPAR’s pathogenic involvement in atherosclerosis using epidemiologic, genetic, and experimental approaches. We found serum suPAR levels to be predictive of coronary artery calcification and cardiovascular events in 5,406 participants without known coronary disease. In a genome-wide association meta-analysis including over 25,000 individuals, we identified a missense variant in the PLAUR gene (rs4760) confirmed experimentally to lead to higher suPAR levels. Mendelian randomization analysis in the UK Biobank using rs4760 indicated a causal association between genetically predicted suPAR levels and atherosclerotic phenotypes. In an experimental model of atherosclerosis, Pcsk9 transfection in mice over-expressing suPAR (suPARTg) led to substantially increased atherosclerotic plaques with necrotic cores and macrophage infiltration compared to wild-type mice, despite similar cholesterol levels. Pre-atherosclerosis, aortas of suPARTg mice excreted higher levels of CCL2 and had higher monocyte counts compared to wild-type aortas. Aortic and circulating suPARTg monocytes exhibited a pro-inflammatory profile and enhanced chemotaxis. These findings characterize suPAR as a pathogenic factor for atherosclerosis acting at least partially through modulation of monocyte function.
George Hindy, Daniel J. Tyrrell, Alexi Vasbinder, Changli Wei, Feriel Presswalla, Hui Wang, Pennelope K. Blakely, Ayse Bilge Ozel, Sarah E. Graham, Grace H. Holton, Joseph Dowsett, Akl C. Fahed, Kingsley-Michael Amadi, Grace K. Erne, Annika Tekumulla, Anis Ismail, Christopher Launius, Nona Sotoodehnia, James S. Pankow, Lise Wegner Thørner, Christian Erikstrup, Ole Birger Pedersen, Karina Banasik, Søren Brunak, Henrik Ullum, Jesper Eugen-Olsen, Sisse Rye Ostrowski, Mary E. Haas, Jonas B. Nielsen, Luca A. Lotta, Gunnar Engström, Olle Melander, Marju Orho-Melander, Lili Zhao, Venkatesh L. Murthy, David J. Pinsky, Cristen J. Willer, Susan R. Heckbert, Jochen Reiser, Daniel R. Goldstein, Karl C. Desch, Salim S. Hayek
Sudden cardiac death (SCD) in patients with heart failure (HF) is allied with an imbalance in reduction and oxidation (redox) signaling in cardiomyocytes; however, the basic pathways and mechanisms governing redox homeostasis in cardiomyocytes are not fully understood. Here, we show that cytochrome b5 reductase 3 (CYB5R3), an enzyme known to regulate redox signaling in erythrocytes and vascular cells, is essential for cardiomyocyte function. Using a conditional cardiomyocyte-specific CYB5R3-knockout mouse, we discovered that deletion of CYB5R3 in male, but not female, adult cardiomyocytes causes cardiac hypertrophy, bradycardia, and SCD. The increase in SCD in CYB5R3-KO mice is associated with calcium mishandling, ventricular fibrillation, and cardiomyocyte hypertrophy. Molecular studies reveal that CYB5R3-KO hearts display decreased adenosine triphosphate (ATP), increased oxidative stress, suppressed coenzyme Q levels, and hemoprotein dysregulation. Finally, from a translational perspective, we reveal that the high-frequency missense genetic variant rs1800457, which translates into a CYB5R3 T117S partial loss-of-function protein, associates with decreased event-free survival (~20%) in Black persons with HF with reduced ejection fraction (HFrEF). Together, these studies reveal a crucial role for CYB5R3 in cardiomyocyte redox biology and identify a genetic biomarker for persons of African ancestry that may potentially increase the risk of death from HFrEF.
Nolan T. Carew, Heidi M. Schmidt, Shuai Yuan, Joseph C. Galley, Robert Hall, Helene M. Altmann, Scott A. Hahn, Megan P. Miller, Katherine C. Wood, Bethann Gabris, Margaret C. Stapleton, Sean Hartwick, Marco Fazzari, Yijen L. Wu, Mohamed Trebak, Brett A. Kaufman, Charles F. McTiernan, Francisco J. Schopfer, Placido Navas, Patrick H. Thibodeau, Dennis M. McNamara, Guy Salama, Adam C. Straub
Increasing evidences advocate for an important function of T cells in controlling immune homeostasis and pathogenesis after myocardial infarction (MI), although the underlying molecular mechanisms remain elusive. In this study, a broad analysis of immune markers in 283 patients revealed a significant CD69 overexpression on Treg cells after MI. Our results in mice showed that CD69 expression on Treg cells increased survival after left-anterior-descending coronary artery (LAD)-ligation. Cd69-/- mice developed strong IL-17+ γδT cell responses after ischemia that increased myocardial inflammation and, consequently, worsened cardiac function. CD69+ Treg cells, by induction of AhR-dependent CD39 ectonucleotidase activity, induced apoptosis and decreased IL-17A production in γδT cells. Adoptive transfer of CD69+ Treg cells to Cd69-/- mice after LAD-ligation reduced IL-17+ γδT cell recruitment, thus increasing survival. Consistently, clinical data from two independent cohorts of patients indicated that increased CD69 expression in peripheral blood cells after acute MI was associated with a lower risk of re-hospitalization for heart failure (HF) after 2.5 years of follow-up. This result remained significant after adjustment for age, sex and traditional cardiac damage biomarkers. Our data highlight CD69 expression on Treg cells as a potential prognostic factor and a therapeutic option to prevent HF after MI.
Rafael Blanco-Domínguez, Hortensia de la Fuente, Cristina Rodríguez, Laura Martín-Aguado, Raquel Sánchez-Díaz, Rosa Jiménez-Alejandre, Iker Rodriguez-Arabaolaza, Andrea Curtabbi, Marcos M. Garcia-Guimaraes, Alberto Vera, Fernando Rivero, Javier Cuesta, Luis Jesus Jimenez-Borreguero, Alberto Cecconi, Albert Duran-Cambra, Manel Taurón, Judith Alonso, Héctor Bueno, María Villalba-Orero, José Antonio Enriquez, Simon C. Robson, Fernando Alfonso, Francisco Sánchez-Madrid, José Martínez-González, Pilar Martín
Accidental injury to the cardiac conduction system (CCS), a network of specialized cells embedded within the heart and indistinguishable from the surrounding heart muscle tissue, is a major complication in cardiac surgeries. Here, we addressed this unmet need by engineering targeted antibody-dye conjugates directed against CCS, allowing for the visualization of the CCS in vivo following a single intravenous injection in mice. These optical imaging tools showed high sensitivity, specificity, and resolution, with no adverse effects to CCS function. Further, with the goal of creating a viable prototype for human use, we generated a fully human monoclonal Fab, that similarly targets the CCS with high specificity. We demonstrate that, when conjugated to an alternative cargo, this Fab can also be used to modulate CCS biology in vivo providing a proof-of-principle for targeted cardiac therapeutics. Finally, in performing differential gene expression analyses of the entire murine CCS at single-cell resolution, we uncovered and validated a suite of additional cell surface markers that can be used to molecularly target the distinct subcomponents of the CCS, each prone to distinct life-threatening arrhythmias. These findings lay the foundation for translational approaches targeting the CCS for visualization and therapy in cardiothoracic surgery, cardiac imaging and arrhythmia management.
William R. Goodyer, Benjamin M. Beyersdorf, Lauren Duan, Nynke S. van den Berg, Sruthi Mantri, Francisco X. Galdos, Nazan Puluca, Jan W. Buikema, Soah Lee, Darren Salmi, Elise R. Robinson, Stephan Rogalla, Dillon P. Cogan, Chaitan Khosla, Eben L. Rosenthal, Sean M. Wu
Atrial fibrosis is an essential contributor to atrial fibrillation (AF). It remains unclear whether atrial endocardial endothelial cells (AEECs) that undergo endothelial-mesenchymal transition (EndMT) are among the sources of atrial fibroblasts. We studied human atria, TGF-β–treated human AEECs, cardiac-specific TGF-β–transgenic mice, and heart failure rabbits to identify the underlying mechanism of EndMT in atrial fibrosis. Using isolated AEECs, we found that miR-181b was induced in TGF-β–treated AEECs, which decreased semaphorin 3A (Sema3A) and increased EndMT markers, and these effects could be reversed by a miR-181b antagomir. Experiments in which Sema3A was increased by a peptide or decreased by a siRNA in AEECs revealed a mechanistic link between Sema3A and LIM-kinase 1/phosphorylated cofilin (LIMK/p-cofilin) signaling and suggested that Sema3A is upstream of LIMK in regulating actin remodeling through p-cofilin. Administration of the miR-181b antagomir or recombinant Sema3A to TGF-β–transgenic mice evoked increased Sema3A, reduced EndMT markers, and significantly decreased atrial fibrosis and AF vulnerability. Our study provides a mechanistic link between the induction of EndMT by TGF-β via miR-181b/Sema3A/LIMK/p-cofilin signaling to atrial fibrosis. Blocking miR-181b and increasing Sema3A are potential strategies for AF therapeutic intervention.
Ying-Ju Lai, Feng-Chun Tsai, Gwo-Jyh Chang, Shang-Hung Chang, Chung-Chi Huang, Wei-Jan Chen, Yung-Hsin Yeh
The discovery of microRNAs and their role in diseases was a breakthrough that inspired research into microRNAs as drug targets. Cardiovascular diseases are an area in which limitations of conventional pharmacotherapy are highly apparent and where microRNA-based drugs have appreciably progressed into preclinical and clinical testing. In this Review, we summarize the current state of microRNAs as therapeutic targets in the cardiovascular system. We report recent advances in the identification and characterization of microRNAs, their manipulation and clinical translation, and discuss challenges and perspectives toward clinical application.
Bernhard Laggerbauer, Stefan Engelhardt
In hypertrophied and failing hearts, fuel metabolism is reprogrammed to increase glucose metabolism, especially glycolysis. This metabolic shift favors biosynthetic function at the expense of ATP production. Mechanisms responsible for the switch are poorly understood. We found that inhibitory factor 1 of the mitochondrial FoF1-ATP synthase (ATPIF1), a protein known to inhibit ATP hydrolysis by the reverse function of ATP synthase during ischemia, was significantly upregulated in pathological cardiac hypertrophy induced by pressure overload, myocardial infarction, or α-adrenergic stimulation. Chemical cross-linking mass spectrometry analysis of hearts hypertrophied by pressure overload suggested that increased expression of ATPIF1 promoted the formation of FoF1-ATP synthase nonproductive tetramer. Using ATPIF1 gain- and loss-of-function cell models, we demonstrated that stalled electron flow due to impaired ATP synthase activity triggered mitochondrial ROS generation, which stabilized HIF1α, leading to transcriptional activation of glycolysis. Cardiac-specific deletion of ATPIF1 in mice prevented the metabolic switch and protected against the pathological remodeling during chronic stress. These results uncover a function of ATPIF1 in nonischemic hearts, which gives FoF1-ATP synthase a critical role in metabolic rewiring during the pathological remodeling of the heart.
Bo Zhou, Arianne Caudal, Xiaoting Tang, Juan D. Chavez, Timothy S. McMillen, Andrew Keller, Outi Villet, Mingyue Zhao, Yaxin Liu, Julia Ritterhoff, Pei Wang, Stephen C. Kolwicz Jr., Wang Wang, James E. Bruce, Rong Tian
Passive stiffness of the heart is determined largely by extracellular matrix and titin, which functions as a molecular spring within sarcomeres. Titin stiffening is associated with the development of diastolic dysfunction (DD), while augmented titin compliance appears to impair systolic performance in dilated cardiomyopathy. We found that myofibril stiffness was elevated in mice lacking histone deacetylase 6 (HDAC6). Cultured adult murine ventricular myocytes treated with a selective HDAC6 inhibitor also exhibited increased myofibril stiffness. Conversely, HDAC6 overexpression in cardiomyocytes led to decreased myofibril stiffness, as did ex vivo treatment of mouse, rat, and human myofibrils with recombinant HDAC6. Modulation of myofibril stiffness by HDAC6 was dependent on 282 amino acids encompassing a portion of the PEVK element of titin. HDAC6 colocalized with Z-disks, and proteomics analysis suggested that HDAC6 functions as a sarcomeric protein deacetylase. Finally, increased myofibril stiffness in HDAC6-deficient mice was associated with exacerbated DD in response to hypertension or aging. These findings define a role for a deacetylase in the control of myofibril function and myocardial passive stiffness, suggest that reversible acetylation alters titin compliance, and reveal the potential of targeting HDAC6 to manipulate the elastic properties of the heart to treat cardiac diseases.
Ying-Hsi Lin, Jennifer L. Major, Tim Liebner, Zaynab Hourani, Joshua G. Travers, Sara A. Wennersten, Korey R. Haefner, Maria A. Cavasin, Cortney E. Wilson, Mark Y. Jeong, Yu Han, Michael Gotthardt, Scott K. Ferguson, Amrut V. Ambardekar, Maggie P.Y. Lam, Chunaram Choudhary, Henk L. Granzier, Kathleen C. Woulfe, Timothy A. McKinsey
Subendothelial macrophage internalization of modified lipids and foam cell formation are hallmarks of atherosclerosis. Deubiquitinating enzymes (DUBs) are involved in various cellular activities; however, their role in foam cell formation is not fully understood. Here, using a loss-of-function lipid accumulation screening, we identified ubiquitin-specific peptidase 9 X-linked (USP9X) as a factor that suppressed lipid uptake in macrophages. We found that USP9X expression in lesional macrophages was reduced during atherosclerosis development in both humans and rodents. Atherosclerotic lesions from macrophage USP9X-deficient mice showed increased macrophage infiltration, lipid deposition, and necrotic core content than control apolipoprotein E-knockout (Apoe-/-) mice. Additionally, loss-of-function USP9X exacerbated lipid uptake, foam cell formation and inflammatory responses in macrophages. Mechanistically, the class A1 scavenger receptor (SR-A1) was identified as a USP9X substrate that removed the K63 polyubiquitin chain at the K27 site. Genetic or pharmacological inhibition of USP9X increased SR-A1 cell surface internalization following binding of oxidized low-density lipoprotein (ox-LDL). The K27R mutation of SR-A1 dramatically attenuated basal and USP9X knockdown-induced ox-LDL uptake. Moreover, blocking binding of USP9X to SR-A1 with a cell-penetrating peptide exacerbated foam cell formation and atherosclerosis. In this study, we identified macrophage USP9X as a beneficial regulator of atherosclerosis and revealed the specific mechanisms for the development of potential therapeutic strategies for atherosclerosis.
Biqing Wang, Xuening Tang, Liu Yao, Yuxin Wang, Zhipeng Chen, Mengqi Li, Naishi Wu, Dawei Wu, Xiangchen Dai, Hongfeng Jiang, Ding Ai