Cardiology
Abstract
Mono-ADP-ribosylation of an (arginine) protein catalyzed by ADP-ribosyltransferase 1 (ART1) — i.e., transfer of ADP-ribose from NAD to arginine — is reversed by ADP-ribosylarginine hydrolase 1 (ARH1) cleavage of the ADP-ribose–arginine bond. ARH1-deficient mice developed cardiomyopathy with myocardial fibrosis, decreased myocardial function under dobutamine stress, and increased susceptibility to ischemia/reperfusion injury. The membrane repair protein TRIM72 was identified as a substrate for ART1 and ARH1; ADP-ribosylated TRIM72 levels were greater in ARH1-deficient mice following ischemia/reperfusion injury. To understand better the role of TRIM72 and ADP-ribosylation, we used C2C12 myocytes. ARH1 knockdown in C2C12 myocytes increased ADP-ribosylation of TRIM72 and delayed wound healing in a scratch assay. Mutant TRIM72 (R207K, R260K) that is not ADP-ribosylated interfered with assembly of TRIM72 repair complexes at a site of laser-induced injury. The regulatory enzymes ART1 and ARH1 and their substrate TRIM72 were found in multiple complexes, which were coimmunoprecipitated from mouse heart lysates. In addition, the mono-ADP-ribosylation inhibitors vitamin K1 and novobiocin inhibited oligomerization of TRIM72, the mechanism by which TRIM72 is recruited to the site of injury. We propose that a mono-ADP-ribosylation cycle involving recruitment of TRIM72 and other regulatory factors to sites of membrane damage is critical for membrane repair and wound healing following myocardial injury.
Authors
Hiroko Ishiwata-Endo, Jiro Kato, Akihiko Tonouchi, Youn Wook Chung, Junhui Sun, Linda A. Stevens, Jianfeng Zhu, Angel M. Aponte, Danielle A. Springer, Hong San, Kazuyo Takeda, Zu-Xi Yu, Victoria Hoffmann, Elizabeth Murphy, Joel Moss
Abstract
The mitochondrial Ca2+ uniporter (MCU) complex mediates acute mitochondrial Ca2+ influx. In skeletal muscle, MCU links Ca2+ signaling to energy production by directly enhancing the activity of key metabolic enzymes in the mitochondria. Here, we examined the role of MCU in skeletal muscle development and metabolic function by generating mouse models for the targeted deletion of Mcu in embryonic, postnatal, and adult skeletal muscle. Loss of Mcu did not affect muscle growth and maturation or otherwise cause pathology. Skeletal muscle–specific deletion of Mcu in mice also did not affect myofiber intracellular Ca2+ handling, but it did inhibit acute mitochondrial Ca2+ influx and mitochondrial respiration stimulated by Ca2+, resulting in reduced acute exercise performance in mice. However, loss of Mcu also resulted in enhanced muscle performance under conditions of fatigue, with a preferential shift toward fatty acid metabolism, resulting in reduced body fat with aging. Together, these results demonstrate that MCU-mediated mitochondrial Ca2+ regulation underlies skeletal muscle fuel selection at baseline and under enhanced physiological demands, which affects total homeostatic metabolism.
Authors
Jennifer Q. Kwong, Jiuzhou Huo, Michael J. Bround, Justin G. Boyer, Jennifer A. Schwanekamp, Nasab Ghazal, Joshua T. Maxwell, Young C. Jang, Zaza Khuchua, Kevin Shi, Donald M. Bers, Jennifer Davis, Jeffery D. Molkentin
Abstract
The mechanisms of J wave syndrome (JWS) are incompletely understood. Here, we showed that the concomitant activation of small-conductance calcium-activated potassium (SK) current (IKAS) and inhibition of sodium current by cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine (CyPPA) recapitulate the phenotypes of JWS in Langendorff-perfused rabbit hearts. CyPPA induced significant J wave elevation and frequent spontaneous ventricular fibrillation (SVF), as well as sinus bradycardia, atrioventricular block, and intraventricular conduction delay. IKAS activation by CyPPA resulted in heterogeneous shortening of action potential (AP) duration (APD) and repolarization alternans. CyPPA inhibited cardiac sodium current (INa) and decelerated AP upstroke and intracellular calcium transient. SVFs were typically triggered by short-coupled premature ventricular contractions, initiated with phase 2 reentry and originated more frequently from the right than the left ventricles. Subsequent IKAS blockade by apamin reduced J wave elevation and eliminated SVF. β-Adrenergic stimulation was antiarrhythmic in CyPPA-induced electrical storm. Like CyPPA, hypothermia (32.0°C) also induced J wave elevation and SVF. It facilitated negative calcium-voltage coupling and phase 2 repolarization alternans with spatial and electromechanical discordance, which were ameliorated by apamin. These findings suggest that IKAS activation contributes to the development of JWS in rabbit ventricles.
Authors
Mu Chen, Dong-Zhu Xu, Adonis Z. Wu, Shuai Guo, Juyi Wan, Dechun Yin, Shien-Fong Lin, Zhenhui Chen, Michael Rubart-von der Lohe, Thomas H. Everett IV, Zhilin Qu, James N. Weiss, Peng-Sheng Chen
Abstract
The precise mechanisms by which oxidative stress (OS) causes atrial fibrillation (AF) are not known. Since AF frequently originates in the posterior left atrium (PLA), we hypothesized that OS, via calmodulin-dependent protein kinase II (CaMKII) signaling, creates a fertile substrate in the PLA for triggered activity and reentry. In a canine heart failure (HF) model, OS generation and oxidized-CaMKII–induced (Ox-CaMKII–induced) RyR2 and Nav1.5 signaling were increased preferentially in the PLA (compared with left atrial appendage). Triggered Ca2+ waves (TCWs) in HF PLA myocytes were particularly sensitive to acute ROS inhibition. Computational modeling confirmed a direct relationship between OS/CaMKII signaling and TCW generation. CaMKII phosphorylated Nav1.5 (CaMKII-p-Nav1.5 [S571]) was located preferentially at the intercalated disc (ID), being nearly absent at the lateral membrane. Furthermore, a decrease in ankyrin-G (AnkG) in HF led to patchy dropout of CaMKII-p-Nav1.5 at the ID, causing its distribution to become spatially heterogeneous; this corresponded to preferential slowing and inhomogeneity of conduction noted in the HF PLA. Computational modeling illustrated how conduction slowing (e.g., due to increase in CaMKII-p-Nav1.5) interacts with fibrosis to cause reentry in the PLA. We conclude that OS via CaMKII leads to substrate for triggered activity and reentry in HF PLA by mechanisms independent of but complementary to fibrosis.
Authors
Shin Yoo, Gary Aistrup, Yohannes Shiferaw, Jason Ng, Peter J. Mohler, Thomas J. Hund, Trent Waugh, Suzanne Browne, Georg Gussak, Mehul Gilani, Bradley P. Knight, Rod Passman, Jeffrey J. Goldberger, J. Andrew Wasserstrom, Rishi Arora
Abstract
Fibrosis is a major contributor to organ disease for which no specific therapy is available. MicroRNA-21 (miR-21) has been implicated in the fibrogenetic response, and inhibitors of miR-21 are currently undergoing clinical trials. Here, we explore how miR-21 inhibition may attenuate fibrosis using a proteomics approach. Transfection of miR-21 mimic or inhibitor in murine cardiac fibroblasts revealed limited effects on extracellular matrix (ECM) protein secretion. Similarly, miR-21–null mouse hearts showed an unaltered ECM composition. Thus, we searched for additional explanations as to how miR-21 might regulate fibrosis. In plasma samples from the community-based Bruneck Study, we found a marked correlation of miR-21 levels with several platelet-derived profibrotic factors, including TGF-β1. Pharmacological miR-21 inhibition with an antagomiR reduced the platelet release of TGF-β1 in mice. Mechanistically, Wiskott-Aldrich syndrome protein, a negative regulator of platelet TGF-β1 secretion, was identified as a direct target of miR-21. miR-21–null mice had lower platelet and leukocyte counts compared with littermate controls but higher megakaryocyte numbers in the bone marrow. Thus, to our knowledge this study reports a previously unrecognized effect of miR-21 inhibition on platelets. The effect of antagomiR-21 treatment on platelet TGF-β1 release, in particular, may contribute to the antifibrotic effects of miR-21 inhibitors.
Authors
Temo Barwari, Seda Eminaga, Ursula Mayr, Ruifang Lu, Paul C. Armstrong, Melissa V. Chan, Mahnaz Sahraei, Marta Fernández-Fuertes, Thomas Moreau, Javier Barallobre-Barreiro, Marc Lynch, Xiaoke Yin, Christian Schulte, Ferheen Baig, Raimund Pechlaner, Sarah R. Langley, Anna Zampetaki, Peter Santer, Martin Weger, Roberto Plasenzotti, Markus Schosserer, Johannes Grillari, Stefan Kiechl, Johann Willeit, Ajay M. Shah, Cedric Ghevaert, Timothy D. Warner, Carlos Fernández-Hernando, Yajaira Suárez, Manuel Mayr
Abstract
Patients with diabetes are at significantly higher risk of developing heart failure. Increases in advanced glycation end products are a proposed pathophysiological link, but their impact and mechanism remain incompletely understood. Methylglyoxal (MG) is a glycolysis byproduct, elevated in diabetes, and modifies arginine and lysine residues. We show that left ventricular myofilament from patients with diabetes and heart failure (dbHF) exhibited increased MG modifications compared with nonfailing controls (NF) or heart failure patients without diabetes. In skinned NF human and mouse cardiomyocytes, acute MG treatment depressed both calcium sensitivity and maximal calcium-activated force in a dose-dependent manner. Importantly, dbHF myocytes were resistant to myofilament functional changes from MG treatment, indicating that myofilaments from dbHF patients already had depressed function arising from MG modifications. In human dbHF and MG-treated mice, mass spectrometry identified increased MG modifications on actin and myosin. Cosedimentation and in vitro motility assays indicate that MG modifications on actin and myosin independently depress calcium sensitivity, and mechanistically, the functional consequence requires actin/myosin interaction with thin-filament regulatory proteins. MG modification of the myofilament may represent a critical mechanism by which diabetes induces heart failure, as well as a therapeutic target to avoid the development of or ameliorate heart failure in these patients.
Authors
Maria Papadaki, Ronald J. Holewinski, Samantha Beck Previs, Thomas G. Martin, Marisa J. Stachowski, Amy Li, Cheavar A. Blair, Christine S. Moravec, Jennifer E. Van Eyk, Kenneth S. Campbell, David M. Warshaw, Jonathan A. Kirk
Abstract
BACKGROUND. Because injury is universal in organ transplantation, heart transplant endomyocardial biopsies present an opportunity to explore response to injury in heart parenchyma. Histology has limited ability to assess injury, potentially confusing it with rejection, whereas molecular changes have potential to distinguish injury from rejection. Building on previous studies of transcripts associated with T cell–mediated rejection (TCMR) and antibody-mediated rejection (ABMR), we explored transcripts reflecting injury. METHODS. Microarray data from 889 prospectively collected endomyocardial biopsies from 454 transplant recipients at 14 centers were subjected to unsupervised principal component analysis and archetypal analysis to detect variation not explained by rejection. The resulting principal component and archetype scores were then examined for their transcript, transcript set, and pathway associations and compared to the histology diagnoses and left ventricular function. RESULTS. Rejection was reflected by principal components PC1 and PC2, and by archetype scores S2TCMR, and S3ABMR, with S1normal indicating normalness. PC3 and a new archetype score, S4injury, identified unexplained variation correlating with expression of transcripts inducible in injury models, many expressed in macrophages and associated with inflammation in pathway analysis. S4injury scores were high in recent transplants, reflecting donation-implantation injury, and both S4injury and S2TCMR were associated with reduced left ventricular ejection fraction. CONCLUSION. Assessment of injury is necessary for accurate estimates of rejection and for understanding heart transplant phenotypes. Biopsies with molecular injury but no molecular rejection were often misdiagnosed rejection by histology. TRAIL REGISTRATION. ClinicalTrials.gov NCT02670408 FUNDING. Roche Organ Transplant Research Foundation, the University of Alberta Hospital Foundation, and Alberta Health Services.
Authors
Philip F. Halloran, Jeff Reeve, Arezu Z. Aliabadi, Martin Cadeiras, Marisa G. Crespo-Leiro, Mario Deng, Eugene C. Depasquale, Johannes Goekler, Xavier Jouven, Daniel H. Kim, Jon Kobashigawa, Alexandre Loupy, Peter Macdonald, Luciano Potena, Andreas Zuckermann, Michael D. Parkes
Abstract
Sudden death is the most common mode of exodus in patients with heart failure and preserved ejection fraction (HFpEF). Cardiosphere-derived cells (CDCs) reduce inflammation and fibrosis in a rat model of HFpEF, improving diastolic function and prolonging survival. We tested the hypothesis that CDCs decrease ventricular arrhythmias (VAs) and thereby possibly contribute to prolonged survival. Dahl salt-sensitive rats were fed a high-salt diet to induce HFpEF. Allogeneic rat CDCs (or phosphate-buffered saline as placebo) were injected in rats with echo-verified HFpEF. CDC-injected HFpEF rats were less prone to VA induction by programmed electrical stimulation. Action potential duration (APD) was shortened, and APD homogeneity was increased by CDC injection. Transient outward potassium current density was upregulated in cardiomyocytes from CDC rats relative to placebo, as were the underlying transcript (Kcnd3) and protein (Kv4.3) levels. Fibrosis was attenuated in CDC-treated hearts, and survival was increased. Sudden death risk also trended down, albeit nonsignificantly. CDC therapy decreased VA in HFpEF rats by shortening APD, improving APD homogeneity, and decreasing fibrosis. Unlike other stem/progenitor cells, which often exacerbate arrhythmias, CDCs reverse electrical remodeling and suppress arrhythmogenesis in HFpEF.
Authors
Jae Hyung Cho, Peter J. Kilfoil, Rui Zhang, Ryan E. Solymani, Catherine Bresee, Elliot M. Kang, Kristin Luther, Russell G. Rogers, Geoffrey de Couto, Joshua I. Goldhaber, Eduardo Marbán, Eugenio Cingolani
Abstract
Cardiac Nav1.5 and Kir2.1–2.3 channels generate Na (INa) and inward rectifier K (IK1) currents, respectively. The functional INa and IK1 interplay is reinforced by the positive and reciprocal modulation between Nav15 and Kir2.1/2.2 channels to strengthen the control of ventricular excitability. Loss-of-function mutations in the SCN5A gene, which encodes Nav1.5 channels, underlie several inherited arrhythmogenic syndromes, including Brugada syndrome (BrS). We investigated whether the presence of BrS-associated mutations alters IK1 density concomitantly with INa density. Results obtained using mouse models of SCN5A haploinsufficiency, and the overexpression of native and mutated Nav1.5 channels in expression systems — rat ventricular cardiomyocytes and human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) — demonstrated that endoplasmic reticulum (ER) trafficking–defective Nav1.5 channels significantly decreased IK1, since they did not positively modulate Kir2.1/2.2 channels. Moreover, Golgi trafficking–defective Nav1.5 mutants produced a dominant negative effect on Kir2.1/2.2 and thus an additional IK1 reduction. Moreover, ER trafficking–defective Nav1.5 channels can be partially rescued by Kir2.1/2.2 channels through an unconventional secretory route that involves Golgi reassembly stacking proteins (GRASPs). Therefore, cardiac excitability would be greatly affected in subjects harboring Nav1.5 mutations with Golgi trafficking defects, since these mutants can concomitantly trap Kir2.1/2.2 channels, thus unexpectedly decreasing IK1 in addition to INa.
Authors
Marta Pérez-Hernández, Marcos Matamoros, Silvia Alfayate, Paloma Nieto-Marín, Raquel G. Utrilla, David Tinaquero, Raquel de Andrés, Teresa Crespo, Daniela Ponce-Balbuena, B. Cicero Willis, Eric N. Jiménez-Vazquez, Guadalupe Guerrero-Serna, Andre M. da Rocha, Katherine Campbell, Todd J. Herron, F. Javier Díez-Guerra, Juan Tamargo, José Jalife, Ricardo Caballero, Eva Delpón
Abstract
Stroke triggers a complex inflammatory process in which the balance between pro- and antiinflammatory mediators is critical for the development of the brain infarct. However, systemic changes may also occur in parallel with brain inflammation. Here we demonstrate that administration of recombinant IL-33, a recently described member of the IL-1 superfamily of cytokines, promotes Th2-type effects following focal ischemic stroke, resulting in increased plasma levels of Th2-type cytokines and fewer proinflammatory (3-nitrotyrosine+F4/80+) microglia/macrophages in the brain. These effects of IL-33 were associated with reduced infarct size, fewer activated microglia and infiltrating cytotoxic (natural killer–like) T cells, and more IL-10–expressing regulatory T cells. Despite these neuroprotective effects, mice treated with IL-33 displayed exacerbated post-stroke lung bacterial infection in association with greater functional deficits and mortality at 24 hours. Supplementary antibiotics (gentamicin and ampicillin) mitigated these systemic effects of IL-33 after stroke. Our findings highlight the complex nature of the inflammatory mechanisms differentially activated in the brain and periphery during the acute phase after ischemic stroke. The data indicate that a Th2-promoting agent can provide neuroprotection without adverse systemic effects when given in combination with antibiotics.
Authors
Shenpeng R. Zhang, Marius Piepke, Hannah X. Chu, Brad R.S. Broughton, Raymond Shim, Connie H.Y. Wong, Seyoung Lee, Megan A. Evans, Antony Vinh, Samy Sakkal, Thiruma V. Arumugam, Tim Magnus, Samuel Huber, Mathias Gelderblom, Grant R. Drummond, Christopher G. Sobey, Hyun Ah Kim
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