Genetic factors undoubtedly affect the development of congenital heart disease (CHD), but still remain ill-defined. We sought to identify genetic risk factors associated with CHD and to accomplish functional analysis of single nucleotide polymorphisms (SNP)-carrying genes. We performed a genome-wide association study of 4,034 Caucasian CHD patients and 8,486 healthy controls. One SNP on chromosome 5q22.2 reached genome-wide significance across all CHD phenotypes and was also indicative for septal defects. One region on chromosome 20p12.1 pointing to the MACROD2 locus identified four highly significant SNPs in patients with transposition of the great arteries (TGA). Three highly significant risk variants on chromosome 17q21.32 within the GOSR2 locus were detected in patients with anomalies of thoracic arteries and veins (ATAV). Genetic variants associated with ATAV are suggested to influence expression of WNT3, and variant rs870142 related to septal defects is proposed to influence expression of MSX1. The expression of all four genes was analyzed during cardiac differentiation of human and murine induced pluripotent stem cells in vitro and by single-cell RNAseq analyses of developing murine and human hearts. Our data show that MACROD2, GOSR2, WNT3 and MSX1 play an essential functional role in heart development at the embryonic and newborn stage.
Harald Lahm, Meiwen Jia, Martina Dreßen, Felix F. M. Wirth, Nazan Puluca, Ralf Gilsbach, Bernard Keavney, Julie Cleuziou, Nicole Beck, Olga Bondareva, Elda Dzilic, Melchior Burri, Karl C. König, Johannes A. Ziegelmüller, Claudia Abou-Ajram, Irina Neb, Zhong Zhang, Stefanie A. Doppler, Elisa Mastantuono, Peter Lichtner, Gertrud Eckstein, Jürgen Hörer, Peter Ewert, James R. Priest, Lutz Hein, Rüdiger Lange, Thomas Meitinger, Heather J. Cordell, Bertram Müller-Myhsok, Markus Krane
Diabetes mellitus (DM) and atrial fibrillation (AF) are major unsolved public health problems, and diabetes is an independent risk factor for AF. However, the mechanism(s) underlying this clinical association is unknown. Reactive oxygen species (ROS) and protein O-GlcNAcylation (OGN) are increased in diabetic hearts, and calmodulin kinase II (CaMKII) is a proarrhythmic signal that may be activated by ROS (ox-CaMKII) and OGN (OGN-CaMKII). We induced type 1 (T1D) and type 2 DM (T2D) in a portfolio of genetic mouse models capable of dissecting the role of ROS and OGN at CaMKII and global OGN in diabetic AF. Here we show that T1D and T2D significantly increased AF, and this increase required CaMKII and OGN. T1D and T2D both require ox-CaMKII to increase AF, however we did not detect OGN-CaMKII nor a role for OGN-CaMKII in diabetic AF. Collectively, our data affirm CaMKII as a critical proarrhythmic signal in diabetic AF, and suggest ROS primarily promotes AF by ox-CaMKII, while OGN promotes AF by a CaMKII-independent mechanism(s). These results provide new and unanticipated insights into the mechanisms for increased AF in DM, and suggest potential benefits for future CaMKII and OGN targeted therapies.
Olurotimi O. Mesubi, Adam G. Rokita, Neha Abrol, Yuejin Wu, Biyi Chen, Qinchuan Wang, Jonathan M. Granger, Anthony Tucker-Bartley, Elizabeth D. Luczak, Kevin R Murphy, Priya Umapathi, Partha S. Banerjee, Tatiana Boronina, Robert N. Cole, Lars S. Maier, Xander H.T. Wehrens, Joel L. Pomerantz, Long-Sheng Song, Rexford Ahima, Gerald W. Hart, Natasha E. Zachara, Mark E. Anderson
Background:While mitochondria play an important role in innate immunity, the relationship between mitochondrial dysfunction and inflammation in heart failure (HF) is poorly understood. In this study we aimed to investigate the mechanistic link between mitochondrial dysfunction and inflammatory activation in peripheral blood mononuclear cells (PBMCs), and the potential anti-inflammatory effect of boosting NAD level.Methods:We compared the PBMC mitochondrial respiration of 19 hospitalized Stage D HF patients with 19 healthy participants. We then created an in vitro model of sterile inflammation by treating healthy PBMC with MitoDAMP (Mitochondrial Damage-Associated Molecular Patterns) isolated from human heart tissue. Lastly, we enrolled Stage D HF patients and sampled their blood before and after taking 5-9 days of oral nicotinamide riboside, an NAD precursor.Results:We demonstrated that HF is associated with both reduced respiratory capacity and elevated proinflammatory cytokine gene expressions. In our in vitro model, MitoDAMP-treated PBMCs secreted IL-6 that impaired mitochondrial respiration by reducing Complex I activity. Last, oral NR administration enhanced PBMC respiration and reduced proinflammatory cytokine gene expression in 4 HF subjects.Conclusion:These findings suggest that systemic inflammation in HF patients is causally linked to mitochondrial function of the PBMC. Increasing NAD levels may have the potential to improve mitochondrial respiration and attenuate proinflammatory activation of PBMC in HF. FundingThis study is funded by NIH R21 HL126209 (to RT and KO), NIH R01 HL144937 (to KO and RT) and University of Washington ITHS Catalyst Award (to DDW). Both BZ (18POST33990352) and DDW (18POST34030098) are funded by the AHA Postdoctoral Fellowships.
Bo Zhou, Dennis D. Wang, Yanhua Qiu, Sophia Airhart, Yaxin Liu, April Stempien-Otero, Kevin D. O’Brien, Rong Tian.
Oxidant stress can contribute to health and disease. Here we show that invertebrates and vertebrates share a common stereospecific redox pathway that protects against pathological responses to stress, at the cost of reduced physiological performance, by constraining Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity. MICAL1, a methionine monooxygenase thought to exclusively target actin, and MSRB, a methionine reductase, control the stereospecific redox status of M308, a highly conserved residue in the calmodulin-binding (CaM-binding) domain of CaMKII. Oxidized or mutant M308 (M308V) decreased CaM binding and CaMKII activity, while absence of MICAL1 in mice caused cardiac arrhythmias and premature death due to CaMKII hyperactivation. Mimicking the effects of M308 oxidation decreased fight-or-flight responses in mice, strikingly impaired heart function in Drosophila melanogaster, and caused disease protection in human induced pluripotent stem cell–derived cardiomyocytes with catecholaminergic polymorphic ventricular tachycardia, a CaMKII-sensitive genetic arrhythmia syndrome. Our studies identify a stereospecific redox pathway that regulates cardiac physiological and pathological responses to stress across species.
Klitos Konstantinidis, Vassilios J. Bezzerides, Lo Lai, Holly M. Isbell, An-Chi Wei, Yuejin Wu, Meera C. Viswanathan, Ian D. Blum, Jonathan M. Granger, Danielle Heims-Waldron, Donghui Zhang, Elizabeth D. Luczak, Kevin R. Murphy, Fujian Lu, Daniel H. Gratz, Bruno Manta, Qiang Wang, Qinchuan Wang, Alex L. Kolodkin, Vadim N. Gladyshev, Thomas J. Hund, William T. Pu, Mark N. Wu, Anthony Cammarato, Mario A. Bianchet, Madeline A. Shea, Rodney L. Levine, Mark E. Anderson
In mammalian heart, left ventricle (LV) rapidly becomes more dominant in size and function over right ventricle (RV) after birth. The molecular regulators responsible for this chamber specific differential growth are largely unknown. We found the cardiomyocytes in neonatal mouse RV had lower proliferation, more apoptosis and smaller sizes comparing to the LV. Such chamber specific growth pattern was associated with a selective activation of p38 MAPK activity in the RV and simultaneous inactivation in the LV. Cardiomyocyte-specific deletion of both mapk14 and mapk11 genes in mice results in loss of p38 MAP kinase expression and activity in the neonatal heart. Inactivation of p38 activity led to marked increase in myocytes proliferation and hypertrophy but diminished myocyte apoptosis, specifically in the RV. Consequently, the p38 inactivated hearts showed RV specific enlargement postnatally, progressing to pulmonary hypertension and right heart failure at adult stage. Chamber-specific p38 activity was associated with differential expression of dual-specific phosphatases (DUSPs) in neonatal hearts, including Dusp26. Unbiased transcriptome analysis revealed IRE1/XBP mediated gene regulation contributed to p38 MAPK dependent regulation of neonatal myocyte proliferation and binucleation. These findings establish an obligatory role of DUSP-p38-IRE1 signaling in myocytes for chamber specific growth in postnatal heart.
Tomohiro Yokota, Jin LI, Jijun Huang, Zhaojun Xiong, Qing Zhang, Tracey W. Chan, Yichen Ding, Christoph D. Rau, Kevin Sung, Shuxun Ren, Rajan P. Kulkarni, Tzung Hsiai, Xinshu Xiao, Marlin Touma, Susumu Minamisawa, Yibin Wang
Connexin 43 (Cx43) gap junctions provide intercellular coupling which ensures rapid action potential propagation and synchronized heart contraction. Altered Cx43 localization and reduced gap junction coupling occur in failing hearts, contributing to ventricular arrhythmias and sudden cardiac death. Recent reports have found that an internally translated Cx43 isoform, GJA1-20k, is an auxiliary subunit for the trafficking of Cx43 in heterologous expression systems. Here, we have created a mouse model by using CRISPR technology to mutate a single internal translation initiation site in Cx43 (M213L mutation), which generates full length Cx43 but not GJA1-20k. We find that GJA1M213L/M213L mice have severely abnormal electrocardiograms despite preserved contractile function, reduced total Cx43, reduced gap junctions, and die suddenly at two to four weeks of age. Heterozygous GJA1M213L/WT mice survive to adulthood with increased ventricular ectopy. Biochemical experiments indicate that cytoplasmic Cx43 has a half life that is 50% shorter than membrane associated Cx43. Without GJA1-20k, poorly trafficked Cx43 is degraded. The data support that GJA1-20k, an endogenous entity translated independently of Cx43, is critical for Cx43 gap junction trafficking, maintenance of Cx43 protein, and normal electrical function of the mammalian heart.
Shaohua Xiao, Daisuke Shimura, Rachel Baum, Diana M. Hernandez, Sosse Agvanian, Yoshiko Nagaoka, Makoto Katsumata, Paul D. Lampe, Andre G. Kleber, TingTing Hong, Robin M. Shaw
Mutation in the LMNA gene, encoding Lamin A/C, cause a diverse group of diseases called laminopathies. Cardiac involvement is the major cause of death and manifests as dilated cardiomyopathy (DCM), heart failure, arrhythmias, and sudden death. There is no specific therapy for LMNA-associated cardiomyopathy. We report that deletion of Lmna in cardiac myocytes in mice leads to severe cardiac dysfunction, conduction defect, ventricular arrhythmias, fibrosis, apoptosis, and premature death within 4 weeks. The phenotype is similar to LMNA-associated cardiomyopathy in humans. RNA sequencing, performed prior to the onset of cardiac dysfunction, led to identification of 2,338 differentially expressed genes (DEGs) in Lmna-deleted cardiac myocytes. DEGs predicted activation of bromodomain-containing protein 4 (BRD4), a regulator of chromatin-associated proteins and transcription factors, which was confirmed by complementary approaches, including chromatin immunoprecipitation-sequencing. Daily injection of JQ1, a specific BET bromodomain inhibitor partially reversed the DEGs, including those encoding secretome, improved cardiac function, abrogated cardiac arrhythmias, fibrosis, and apoptosis, and prolonged the median survival time by 2-fold in the myocyte-specific Lmna-deleted mice. The findings highlight the important role of LMNA in cardiac myocyte and identify BET bromodomain inhibition as a potential therapeutic target in LMNA-associated cardiomyopathy, for which there is no specific effective therapy.
Gaelle Auguste, Leila Rouhi, Scot J. Matkovich, Cristian Coarfa, Matthew J. Robertson, Grazyna Czernuszewicz, Priyatansh Gurha, Ali J. Marian
Although autophagy is generally protective, uncontrolled or excessive activation of autophagy can be detrimental. However, it is often difficult to distinguish death by autophagy from death with autophagy, and whether autophagy contributes to death in cardiomyocytes (CMs) is still controversial. Excessive activation of autophagy induces a morphologically and biochemically defined form of cell death termed autosis. Whether autosis is involved in tissue injury induced under pathologically relevant conditions is poorly understood. In the present study, myocardial ischemia/reperfusion (I/R) induced autosis in CMs, as evidenced by cell death with numerous vacuoles and perinuclear spaces, and depleted intracellular membranes. Autosis was observed frequently after 6 hours of reperfusion, accompanied by upregulation of Rubicon, attenuation of autophagic flux, and marked accumulation of autophagosomes. Genetic downregulation of Rubicon inhibited autosis and reduced I/R injury, whereas stimulation of autosis during the late phase of I/R with Tat–Beclin 1 exacerbated injury. Suppression of autosis by ouabain, a cardiac glycoside, in humanized Na+,K+-ATPase–knockin mice reduced I/R injury. Taken together, these results demonstrate that autosis is significantly involved in I/R injury in the heart and triggered by dysregulated accumulation of autophagosomes due to upregulation of Rubicon.
Jihoon Nah, Peiyong Zhai, Chun-Yang Huang, Álvaro F. Fernández, Satvik Mareedu, Beth Levine, Junichi Sadoshima
Salt inducible kinases (SIKs) are key regulators of cellular metabolism and growth, but their role in cardiomyocyte plasticity and heart failure pathogenesis remains unknown. Here, we showed that loss of SIK1 kinase activity protected against adverse cardiac remodeling and heart failure pathogenesis in rodent models and human iPSC-derived cardiomyocytes. We found that SIK1 phosphorylated and stabilized histone deacetylase 7 (HDAC7) protein during cardiac stress, an event that is required for pathologic cardiomyocyte remodeling. Gain- and loss-of-function studies of HDAC7 in cultured cardiomyocytes implicated HDAC7 as a pro-hypertrophic signaling effector that can induce c-Myc expression, indicating a functional departure from the canonical MEF2 co-repressor function of class IIa HDACs. Taken together, our findings reveal what we believe to be a previously unrecognized role for a SIK1-HDAC7 axis in regulating cardiac stress responses and implicate this pathway as a potential target in human heart failure.
Austin Hsu, Qiming Duan, Sarah McMahon, Yu Huang, Sarah A.B. Wood, Nathanael S. Gray, Biao Wang, Benoit G. Bruneau, Saptarsi M. Haldar
Arterial cardiovascular events are the leading cause of death in patients with JAK2V617F myeloproliferative neoplasms (MPN). However, their mechanisms are poorly understood. The high prevalence of myocardial infarction without significant coronary stenosis or atherosclerosis in patients with MPN suggests that vascular function is altered. Consequences of JAK2V617F mutation on vascular reactivity are unknown. We observe here increased responses to vasoconstrictors in arteries from Jak2V617F mice, resulting from disturbed endothelial nitric oxide pathway and increased endothelial oxidative stress. This response was reproduced in wild-type mice by circulating microvesicles isolated from patients carrying JAK2V617F and by erythrocyte-derived microvesicles from transgenic mice. Microvesicles of other cellular origins had no effect. This effect was observed ex vivo on isolated aortas, but also in vivo on femoral arteries. Proteomic analysis of microvesicles derived from JAK2V617F erythrocytes identified increased expression of myeloperoxidase as the likely mechanism accounting for microvesicles effect. Myeloperoxidase inhibition in microvesicles derived from JAK2V617F erythrocytes supressed their effect on oxidative stress. Antioxidants, such as simvastatin and N-acetyl-cysteine, improved arterial dysfunction in Jak2V617F mice. In conclusion, JAK2V617F MPN are characterized by exacerbated vasoconstrictor responses resulting from increased endothelial oxidative stress caused by circulating erythrocyte-derived microvesicles. Simvastatin appears as promising therapeutic strategy in this setting.
Johanne Poisson, Marion Tanguy, Hortense Davy, Fatoumata Camara, Marie-Belle El Mdawar, Marouane Kheloufi, Tracy Dagher, Cécile Devue, Juliette Lasselin, Aurelie Plessier, Salma Merchant, Olivier Blanc-Brude, Michele Souyri, Nathalie Mougenot, Florent Dingli, Damarys Loew, Stephane N. Hatem, Chloe James, Jean-Luc Villeval, Chantal M. Boulanger, Pierre-Emmanuel Rautou