Cardiology
Abstract
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an arrhythmia syndrome due to gene mutations that render RYR2 calcium release channels hyperactive, causing spontaneous calcium release and delayed afterdepolarizations (DADs). What remains unknown is the cellular source of ventricular arrhythmia triggered by DADs – Purkinje cells in the conduction system or ventricular cardiomyocytes in the working myocardium. To answer this question, we used a genetic approach in mice to knock out cardiac calsequestrin either in Purkinje cells or in ventricular cardiomyocytes. Total loss of calsequestrin in the heart causes a severe CPVT phenotype in mice and humans. We found that loss of calsequestrin only in ventricular myocytes produced a full-blown CPVT phenotype, whereas mice with loss of calsequestrin only in Purkinje cells were comparable to wild-type mice. Subendocardial chemical ablation or restoration of calsequestrin expression in subendocardial cardiomyocytes neighboring Purkinje cells was sufficient to protect against catecholamine-induced arrhythmias. In silico modeling demonstrated that DADs in ventricular myocardium can trigger full action potentials in the Purkinje fiber, but not vice versa. Hence, ectopic beats in CPVT are likely generated at the Purkinje-myocardial junction via a heretofore unrecognized tissue mechanism, whereby DADs in the ventricular myocardium trigger full action potentials in adjacent Purkinje cells.
Authors
Daniel J. Blackwell, Michela Faggioni, Matthew J. Wleklinski, Nieves Gomez-Hurtado, Raghav Venkataraman, Chelsea E. Gibbs, Franz J. Baudenbacher, Shiaoching Gong, Glenn I. Fishman, Patrick M. Boyle, Karl Pfeifer, Bjorn C. Knollmann
Abstract
Myosin heavy chain 7 (MYH7) is a major causative gene for hypertrophic cardiomyopathy, but the affected signaling pathways and therapeutics remain elusive. In this research, we identified ventricle myosin heavy chain like (vmhcl) as a zebrafish homolog of human MYH7, and we generated vmhcl frameshift mutants. We noted vmhcl-based embryonic cardiac dysfunction (VEC) in the vmhcl homozygous mutants and vmhcl-based adult cardiomyopathy (VAC) phenotypes in the vmhcl heterozygous mutants. Using the VEC model, we assessed 7 known cardiomyopathy signaling pathways pharmacologically and 11 candidate genes genetically via CRISPR/Cas9 genome editing technology based on microhomology-mediated end joining (MMEJ). Both studies converged on therapeutic benefits of mTOR or mitogen-activated protein kinase (MAPK) inhibition of VEC. While mTOR inhibition rescued the enlarged nuclear size of cardiomyocytes, MAPK inhibition restored the prolonged cell shape in the VEC model. The therapeutic effects of mTOR and MAPK inhibition were later validated in the VAC model. Together, vmhcl/myh7 loss of function is sufficient to induce cardiomyopathy in zebrafish. The VEC and VAC models in zebrafish are amenable to both efficient genetic and chemical genetic tools, offering a rapid in vivo platform for discovering candidate signaling pathways of MYH7 cardiomyopathy.
Authors
Haisong Bu, Yonghe Ding, Jiarong Li, Ping Zhu, Yu-Huan Shih, Mingmin Wang, Yuji Zhang, Xueying Lin, Xiaolei Xu
Abstract
Systemic hypoxia is characterized by peripheral vasodilation and pulmonary vasoconstriction. However, the system-wide mechanism for signaling hypoxia remains unknown. Accumulating evidence suggests that hemoglobin in RBCs may serve as an O2 sensor and O2-responsive NO signal transducer to regulate systemic and pulmonary vascular tone, but this remains unexamined at the integrated system level. One residue invariant in mammalian hemoglobins (Hb), β-globin Cys93 (βCys93), carries NO as vasorelaxant S-nitrosothiol (SNO) to autoregulate blood flow during oxygen delivery. βCys93Ala mutant mice thus exhibit systemic hypoxia despite transporting oxygen normally. Here we show that βCys93Ala mutant mice have reduced S-nitrosohemoglobin (SNO-Hb) at baseline and upon targeted SNO repletion, and that hypoxic vasodilation by RBCs is impaired in vitro and in vivo, recapitulating hypoxic pathophysiology. Notably, βCys93Ala mutant mice show marked impairment of hypoxic peripheral vasodilation and develop signs of pulmonary hypertension with age. Mutant mice also die prematurely with cor pulmonale (pulmonary hypertension with right ventricular dysfunction) when living under low oxygen. Altogether, we identify a major role for RBC-SNO in clinically-relevant vasodilatory responses attributed previously to endothelial NO. We conclude that SNO-Hb transduces the integrated, system-wide response to hypoxia in the mammalian respiratory cycle, expanding a core physiological principle.
Authors
Rongli Zhang, Alfred Hausladen, Zhaoxia Qian, Xudong Liao, Richard T. Premont, Jonathan S. Stamler
Abstract
Tetralogy of Fallot (TOF) is the most common cyanotic heart defect, yet the underlying genetic mechanisms remain poorly understood. Here, we performed whole genome sequencing analysis on 146 non-syndromic TOF parent-offspring trios of Chinese ethnicity. Comparison of de novo variants and recessive genotypes of this dataset to a European cohort identified both overlapping and novel gene loci, and revealed differential functional enrichment between cohorts. To assess the impact of these mutations on early cardiac development, we integrated single-cell and spatial transcriptomics of early human heart development with our genetic findings. We discovered that the candidate gene expression was enriched in the myogenic progenitors of the cardiac outflow tract. Moreover, subsets of the candidate genes were found in specific gene co-expression modules along cardiomyocyte differentiation trajectory. These integrative functional analyses help dissect the pathogenesis of TOF, revealing cellular hotspots in early heart development resulting in cardiac malformations.
Authors
Clara Sze Man Tang, Mimmi Mononen, Wai-Yee Lam, Sheng Chih Jin, Xuehan Zhuang, Maria-Mercè Garcia-Barcelo, Qiongfen Lin, Yujia Yang, Makoto Sahara, Elif Eroglu, Kenneth R. Chien, Haifa Hong, Paul K.H. Tam, Peter J. Gruber
Abstract
Acute cardiac injury is prevalent in critical COVID-19 and associated with increased mortality. Its etiology remains debated, as initially presumed causes--- myocarditis and cardiac necrosis--- have proven uncommon. To elucidate the pathophysiology of COVID-19-associated cardiac injury, we conducted a prospective study of the first 69 consecutive COVID-19 decedents at Columbia University Irving Medical Center in New York City. Of six acute cardiac histopathologic features, microthrombi was the most commonly detected amongst our cohort (n=48, 70%). We tested associations of cardiac microthrombi with biomarkers of inflammation, cardiac injury, and fibrinolysis and with in-hospital antiplatelet therapy, therapeutic anticoagulation, and corticosteroid treatment, while adjusting for multiple clinical factors, including COVID-19 therapies. Higher peak erythrocyte sedimentation rate and c-reactive protein were independently associated with increased odds of microthrombi, supporting an immunothrombotic etiology. Using single nuclei RNA-sequencing analysis on 3 patients with and 4 patients without cardiac microthrombi, we discovered an enrichment of pro-thrombotic/anti-fibrinolytic, extracellular matrix remodeling, and immune-potentiating signaling amongst cardiac fibroblasts in microthrombi-positive, relative to microthrombi-negative, COVID-19 hearts. Non-COVID-19 non-failing hearts were used as reference controls. Our study identifies a specific transcriptomic signature in cardiac fibroblasts as a salient feature of microthrombi-positive COVID-19 hearts. Our findings warrant further mechanistic study as cardiac fibroblasts may represent a potential therapeutic target for COVID-19-associated cardiac microthrombi.
Authors
Michael I. Brener, Michelle L. Hulke, Nobuaki Fukuma, Stephanie Golob, Robert S. Zilinyi, Zhipeng Zhou, Christos Tzimas, Ilaria Russo, Claire McGroder, Ryan D. Pfeiffer, Alexander Chong, Geping Zhang, Daniel Burkhoff, Martin B. Leon, Mathew S. Maurer, Jeffrey W. Moses, Anne-Catrin Uhlemann, Hanina Hibshoosh, Nir Uriel, Matthias J. Szabolcs, Björn Redfors, Charles C. Marboe, Matthew R. Baldwin, Nathan R. Tucker, Emily J. Tsai
Abstract
SNHG12, a long non-coding RNA (lncRNA) dysregulated in atherosclerosis, is known to be a key regulator of vascular senescence in endothelial cells (ECs). However, its role in angiogenesis and peripheral artery disease (PAD) has not been elucidated. Hindlimb ischemia studies using femoral artery ligation in mice showed that SNHG12 expression falls readily in the acute phase of the response to limb ischemia in gastrocnemius muscle and recovers to normal when blood flow recovery is restored to ischemic muscle, indicating that it likely plays a role in the angiogenic response to ischemia. Gain and loss of function studies demonstrated that SNHG12 regulated angiogenesis – SNHG12 deficiency reduced cell proliferation, migration, and endothelial sprouting, whereas overexpression promoted these angiogenic functions. We identified SNHG12 binding partners by proteomics that may contribute to its role in angiogenesis, including insulin growth factor 2 mRNA binding protein 3 (IGF2BP3/IMP3). RNA-seq profiling of SNHG12-deficient ECs showed effects on angiogenesis pathways and identified a strong effect on cell cycle regulation, which may be modulated by IGF2BP3/IMP3. Knockdown of SNHG12 in mice undergoing femoral artery ligation using injected gapmeRs decreased angiogenesis, an effect that was more pronounced in a model of insulin resistant db/db mice. RNA-seq profiling of the EC and non-EC compartments in these mice revealed a likely role of SNHG12-knockdown on Wnt, Notch, and angiopoietin signaling pathways. Together, these findings indicate that SNHG12 plays an important role in the angiogenic EC response to ischemia.
Authors
David A. Gross, Henry S. Cheng, Rulin Zhuang, Michael G. McCoy, Daniel Pérez-Cremades, Zachary Salyers, A.K.M. Khyrul Wara, Stefan Haemmig, Terence E. Ryan, Mark W. Feinberg
Abstract
Mechanistically driven therapies for atrial fibrillation (AF), the most common cardiac arrhythmia, are urgently needed, the development of which require improved understanding of the cellular signaling pathways that facilitate the structural and electrophysiological remodeling that occurs in the atria. Similar to humans, increased persistent Na+ current leads to the development of an atrial myopathy and spontaneous and long-lasting episodes of AF in mice. How increased persistent Na+ current causes both structural and electrophysiological remodeling in the atria is unknown. We cross-bred mice expressing human F1759A-NaV1.5 channels with mice expressing human mitochondrial catalase (mCAT). Increased expression of mitochondrial catalase attenuated mitochondrial and cellular reactive oxygen species (ROS), and the structural remodeling that was induced by persistent F1759A-Na+ current. Despite the heterogeneously prolonged atrial action potential, which was unaffected by the reduction in ROS, the incidence of both spontaneous AF and pacing-induced after-depolarizations and AF was substantially reduced. Expression of mitochondrial catalase markedly reduced persistent Na+ current induced ryanodine receptor oxidation and dysfunction. In summary, increased persistent Na+ current in atrial cardiomyocytes, which is observed in patients with AF, induces atrial enlargement, fibrosis, mitochondrial dysmorphology, early after-depolarizations and AF, all of which can be attenuated by resolving mitochondrial oxidative stress.
Authors
Uma Mahesh R. Avula, Haikel Dridi, Bi-xing Chen, Qi Yuan, Alexander N. Katchman, Steven R. Reiken, Amar D. Desai, Samantha Parsons, Haajra Baksh, Elaine Ma, Parmanand Dasrat, Ruiping Ji, Yejun Lin, Christine Sison, W. Jonathan Lederer, Humberto C. Joca, Christopher Ward, Maura Greiser, Andrew R. Marks, Steven O. Marx, Elaine Y. Wan
Abstract
Point mutations within sarcomeric proteins have been associated with altered function and cardiomyopathy development. Difficulties remain, however, in establishing the pathogenic potential of individual mutations, often limiting the use of genotype in management of affected families. To directly address this challenge, we utilized our all-atom computational model of the human full cardiac thin filament (CTF) to predict how sequence substitutions in CTF proteins might affect structure and dynamics on an atomistic level.Utilizing molecular dynamics (MD) calculations, we simulated 21 well-defined genetic pathogenic cardiac troponin T and tropomyosin variants to establish a baseline of pathogenic changes induced in computational observables. Computational results were verified via differential scanning calorimetry on a subset of variants to develop an experimental correlation. Calculations were performed on 9 independent variants of unknown significance (VUS) and results were compared to pathogenic variants to identify high resolution pathogenic signatures.Results for VUS were compared to the baseline set to determine induced structural and dynamic changes and potential variant reclassifications were proposed. This unbiased, high- resolution computational methodology can provide unique structural and dynamic information that can be incorporated into existing analyses to facilitate classification both for de novo variants and those where established approaches have provided conflicting information.
Authors
Allison B. Mason, Melissa L. Lynn, Anthony P. Baldo, Andrea E. Deranek, Jil C. Tardiff, Steven D. Schwartz
Abstract
The meager regenerative capacity of adult mammalian hearts appears to be driven by the proliferation of endogenous cardiomyocytes; thus, strategies targeting mechanisms of cardiomyocyte cell cycle regulation, such as the Hippo/Yes-associated protein (Hippo/Yap) pathway, could lead to the development of promising therapies for heart disease. The pharmacological product TT-10 increases cardiomyocyte proliferation by upregulating nuclear Yap levels. When intraperitoneal injections of TT-10 were administered to infarcted mouse hearts, the treatment promoted cardiomyocyte proliferation and was associated with declines in infarct size 1 week after administration, but cardiac function worsened at later time points. Here, we investigated whether encapsulating TT-10 into poly-lactic-co-glycolic acid nanoparticles (NPs) before administration could extend the duration of TT-10 delivery and improve the potency of TT-10 for myocardial repair. TT-10 was released from the TT-10–loaded NPs for up to 4 weeks in vitro, and intramyocardial injections of TT-10–delivered NPs stably improved cardiac function from week 1 to week 4 after administration to infarcted mouse hearts. TT-10–delivered NP treatment was also associated with significantly smaller infarcts at week 4, with increases in cardiomyocyte proliferation and nuclear Yap abundance and with declines in cardiomyocyte apoptosis. Thus, NP-mediated delivery appears to enhance both the potency and durability of TT-10 treatment for myocardial repair.
Authors
Wangping Chen, Danielle Pretorius, Yang Zhou, Yuji Nakada, Jinfu Yang, Jianyi Zhang
Abstract
Tregs play vital roles in suppressing atherogenesis. Pathological conditions reshape Tregs and increase Treg-weakening plasticity. It remains unclear how Tregs preserve their function and how Tregs switch into alternative phenotypes in the environment of atherosclerosis. In this study, we observed a great induction of CD4+Foxp3+ Tregs in the spleen and aorta of ApoE–/– mice, accompanied by a significant increase of plasma IL-35 levels. To determine if IL-35 devotes its role in the rise of Tregs, we generated IL-35 subunit P35–deficient (IL-35P35–deficient) mice on an ApoE–/– background and found Treg reduction in the spleen and aorta compared with ApoE–/– controls. In addition, our RNA sequencing data show the elevation of a set of chemokine receptor transcripts in the ApoE–/– Tregs, and we have validated higher CCR5 expression in ApoE–/– Tregs in the presence of IL-35 than in the absence of IL-35. Furthermore, we observed that CCR5+ Tregs in ApoE–/– have lower Treg-weakening AKT-mTOR signaling, higher expression of inhibitory checkpoint receptors TIGIT and PD-1, and higher expression of IL-10 compared with WT CCR5+ Tregs. In conclusion, IL-35 counteracts hyperlipidemia in maintaining Treg-suppressive function by increasing 3 CCR5-amplified mechanisms, including Treg migration, inhibition of Treg weakening AKT-mTOR signaling, and promotion of TIGIT and PD-1 signaling.
Authors
Ying Shao, William Y. Yang, Fatma Saaoud, Charles Drummer IV, Yu Sun, Keman Xu, Yifan Lu, Huimin Shan, Ethan M. Shevach, Xiaohua Jiang, Hong Wang, Xiaofeng Yang
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