Chronic inflammation is a hallmark of atherosclerosis and results from an imbalance between pro-inflammatory and pro-resolving signaling. The human GPR32 receptor, together with the ALX/FPR2 receptor, transduces biological actions of several pro-resolving mediators that stimulate resolution of inflammation. However, since no murine homologs of the human GPR32 exist, comprehensive in vivo studies are lacking. Using human atherosclerotic lesions from carotid endarterectomies and creating a transgenic mouse model expressing human GPR32 on a Fpr2×apolipoprotein E double KO background (hGPR32myc×Fpr2-/-×Apoe-/-), we investigated the role of GPR32 in atherosclerosis and self-limiting acute inflammation. GPR32 mRNA was reduced in human atherosclerotic lesions and correlated with the immune cell markers ARG1, NOS2 and FOXP3. Atherosclerotic lesions, necrotic core and aortic inflammation were reduced in hGPR32mycTg×Fpr2-/-×Apoe-/- transgenic mice as compared to Fpr2-/-×Apoe-/- non-transgenic littermates. In a zymosan induced peritonitis model, the hGPR32mycTg×Fpr2-/-×Apoe-/- transgenic mice had reduced inflammation at 4h and enhanced pro-resolving macrophage responses at 24h compared to non-transgenic littermates. The GPR32 agonist aspirin-triggered resolvin D1 (AT-RvD1) regulated leukocyte responses, including enhancing macrophage phagocytosis and intracellular signaling in hGPR32mycTg×Fpr2-/-×Apoe-/- transgenic mice but not in the Fpr2-/-×Apoe-/- non-transgenic littermates. Altogether these results provide the first evidence that GPR32 regulates resolution of inflammation and is atheroprotective in vivo.
Hildur Arnardottir, Silke Thul, Sven-Christian Pawelzik, Glykeria Karadimou, Gonzalo Artiach, Alessandro L. Gallina, Victoria Mysdotter, Miguel Carracedo, Laura Tarnawski, April S. Caravaca, Roland Baumgartner, Daniel F.J. Ketelhuth, Peder S. Olofsson, Gabrielle Paulsson-Berne, Göran K. Hansson, Magnus Bäck
The cardiac conduction system (CCS) ensures regular contractile function, and injury to any of its components can cause cardiac dysrhythmia. Although all cardiomyocytes (CMs) originate from common progenitors, the CCS is composed of biologically distinct cell types with unique functional and developmental characteristics. In contrast to ventricular cardiomyocytes, which continue to proliferate after birth, most CCS cells terminally exit the cell cycle during fetal development. Although the CCS should thus provide a poor substrate for postnatal injury repair, its regenerative capacity remains untested. Here, we describe a genetic system for ablating CMs that reside within the atrioventricular conduction system (AVCS). Adult mouse AVCS ablation resulted in regenerative failure characterized by persistent atrioventricular conduction defects and contractile dysfunction. In contrast, AVCS injury in neonatal mice led to recovery in a subset of these mice, thus providing evidence for CCS plasticity. Furthermore, CM proliferation did not appear to completely account for the observed functional recovery, suggesting that mechanisms regulating recovery from dysrhythmia are likely to be distinct from cardiac regeneration associated with ventricular injury. Taken together, we anticipate that our results will motivate further mechanistic studies of CCS plasticity and enable the exploration of rhythm restoration as an alternative therapeutic strategy.
Lin Wang, Minoti Bhakta, Antonio Fernandez-Perez, Nikhil V. Munshi
In recent decades, treatments for myocardial infarction (MI), such as stem and progenitor cell therapy, have attracted considerable scientific and clinical attention but failed to improve patient outcomes. These efforts indicate that more rigorous mechanistic and functional testing of potential MI therapies is required. Recent studies have suggested that augmenting post-MI lymphatic growth via VEGF-C administration improves cardiac function. However, the mechanisms underlying this proposed therapeutic approach remain vague and untested. To more rigorously test the role of lymphatic vessel growth after MI, we examined the post-MI cardiac function of mice in which lymphangiogenesis had been blocked genetically by pan-endothelial or lymphatic endothelial loss of the lymphangiogenic receptor VEGFR3 or global loss of the VEGF-C and VEGF-D ligands. The results obtained using all three genetic approaches were highly concordant and demonstrated that loss of lymphatic vessel growth did not impair left ventricular ejection fraction two weeks after MI in mice. We observed a trend toward excess fluid in the infarcted region of the left ventricle, but immune cell infiltration and clearance were unchanged with loss of expanded lymphatics. These studies refute the hypothesis that lymphangiogenesis contributes significantly to cardiac function after MI, and suggest that any effect of exogenous VEGF-C is likely to be mediated by non-lymphangiogenic mechanisms.
T.C. Stevenson Keller IV, Lillian Lim, Swapnil V. Shewale, Kendra McDaid, Ingrid Marti-Pamies, Alan T. Tang, Carl Wittig, Andrea A. Guerrero, Stephanie Sterling, N. Adrian Leu, marielle scherrer-crosbie, Phyllis A. Gimotty, Mark L. Kahn
Endothelial-mesenchymal transition (EndMT) is associated with various cardiovascular diseases and in particular with atherosclerosis and plaque instability. However, the molecular pathways that govern EndMT are poorly defined. Specifically, the role of epigenetic factors and histone deacetylases (HDACs) in controlling EndMT and the atherosclerotic plaque phenotype remains unclear. Here, we identified histone deacetylation, specifically that mediated by HDAC9 (a class IIa HDAC), as playing an important role in both EndMT and atherosclerosis. Using in vitro models, we found class IIa HDAC inhibition sustained the expression of endothelial proteins and mitigated the increase in mesenchymal proteins, effectively blocking EndMT. Similarly, ex vivo genetic knockout of Hdac9 in endothelial cells prevented EndMT and preserved a more endothelial-like phenotype. In vivo, atherosclerosis-prone mice with endothelial-specific Hdac9 knockout showed reduced EndMT and significantly reduced plaque area. Furthermore, these mice displayed a more favorable plaque phenotype, with reduced plaque lipid content and increased fibrous cap thickness. Together, these findings indicate that HDAC9 contributes to vascular pathology by promoting EndMT. Our study provides evidence for a pathological link among EndMT, HDAC9, and atherosclerosis and suggests that targeting of HDAC9 may be beneficial for plaque stabilization or slowing the progression of atherosclerotic disease.
Laura Lecce, Yang Xu, Bhargavi V’Gangula, Nirupama Chandel, Venu Pothula, Axelle Caudrillier, Maria Paola Santini, Valentina d’Escamard, Delaine K. Ceholski, Przemek A. Gorski, Lijiang Ma, Simon Koplev, Martin Mæng Bjørklund, Johan L.M. Björkegren, Manfred Boehm, Jacob Fog Bentzon, Valentin Fuster, Ha Won Kim, Neal L. Weintraub, Andrew H. Baker, Emily Bernstein, Jason C. Kovacic
Ischemic cardiomyopathy is associated with an increased risk of sudden death, activation of the unfolded protein response (UPR), and reductions in multiple cardiac ion channels. When activated, the protein kinase-like ER kinase (PERK) branch of the UPR reduces protein translation and abundance. We hypothesized that PERK inhibition could prevent ion channel downregulation and reduce arrhythmic risk after myocardial infarct (MI). MI induced by coronary artery ligation resulted in mice exhibited reduced ion channel levels, ventricular tachycardia (VT), and prolonged corrected intervals between the Q and T waves of the ECGs (QTc). Protein levels of major cardiac ion channels were decreased. MI cardiomyocytes showed significantly prolonged action potential duration and decreased maximum upstroke velocity. Cardiac-specific PERK knockout (PERKKO) reduced electrical remodeling in response to MI with shortened QTc intervals, less VT episodes, and higher survival rates (P<0.05 vs. MI). Pharmacological PERK inhibition had similar effects. In conclusion, activated PERK during MI contributed to arrhythmic risk by downregulation of select cardiac ion channels. PERK inhibition prevented these changes and reduced arrhythmic risk. These results suggest that ion channel downregulation during MI is a fundamental arrhythmic mechanism and maintaining ion channel levels is antiarrhythmic.
Man Liu, Hong Liu, Preethy Parthiban, Gyeoung-Jin Kang, Guangbin Shi, Feng Feng, Anyu Zhou, Lianzhi Gu, Courtney Karnopp, Elena G. Tolkacheva, Samuel C. Dudley
The start codon c.1A>G mutation in KLHL24, encoding ubiquitin-ligase KLHL24, results in the loss of 28 N-terminal amino acids (KLHL24-ΔN28) by skipping the initial start codon. In skin, KLHL24-ΔN28 leads to gain of function, excessively targeting intermediate filament keratin-14 for proteasomal degradation, ultimately causing epidermolysis bullosa simplex (EBS). The majority of these EBS-patients are also diagnosed with dilated cardiomyopathy (DCM), but the pathological mechanism in the heart is unknown. As desmin is the cardiac homologue of keratin-14, we hypothesized that KLHL24-ΔN28 leads to excessive degradation of desmin, resulting in DCM. Dynamically loaded engineered heart tissues (dyn-EHTs) were generated from human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes from two patients and three (non)familial controls. Ten-fold lower desmin protein levels were observed in patient-derived dyn-EHTs, in line with diminished desmin levels detected in patients’ explanted heart. This was accompanied by tissue dilatation, impaired mitochondrial function, decreased force values and increased cardiomyocyte stress. HEK293 transfection studies confirmed KLHL24-mediated desmin degradation. KLHL24 RNA interference or direct desmin overexpression recovered desmin protein levels, restoring morphology and function in patient-derived dyn-EHTs. To conclude, presence of KLHL24-ΔN28 in cardiomyocytes leads to excessive degradation of desmin, affecting tissue morphology and function, that can be prevented by restoring desmin protein levels.
Mathilde C.S.C. Vermeer, Maria C. Bolling, Jacqueline M. Bliley, Karla F. Arevalo Gomez, Mario G. Pavez-Giani, Duco Kramer, Pedro H. Romero-Herrera, B. Daan Westenbrink, Gilles F.H. Diercks, Maarten P. van den Berg, Adam W. Feinberg, Herman H. W. Silljé, Peter van der Meer
The inflammatory response after myocardial infarction (MI) is a precisely regulated process that greatly affects subsequent remodeling. Here, we show that basophil granulocytes infiltrated infarcted murine hearts, with a peak occurring between days 3 and 7. Antibody-mediated and genetic depletion of basophils deteriorated cardiac function and resulted in enhanced scar thinning after MI. Mechanistically, we found that basophil depletion was associated with a shift from reparative Ly6Clo macrophages toward increased numbers of inflammatory Ly6Chi monocytes in the infarcted myocardium. Restoration of basophils in basophil-deficient mice by adoptive transfer reversed this proinflammatory phenotype. Cellular alterations in the absence of basophils were accompanied by lower cardiac levels of IL-4 and IL-13, two major cytokines secreted by basophils. Mice with basophil-specific IL-4/IL-13 deficiency exhibited a similarly altered myeloid response with an increased fraction of Ly6Chi monocytes and aggravated cardiac function after MI. In contrast, IL-4 induction in basophils via administration of the glycoprotein IPSE/α-1 led to improved post-MI healing. These results in mice were corroborated by the finding that initially low counts of blood basophils in patients with acute MI were associated with a worse cardiac outcome after 1 year, characterized by a larger scar size. In conclusion, we show that basophils promoted tissue repair after MI by increasing cardiac IL-4 and IL-13 levels.
Florian Sicklinger, Ingmar Sören Meyer, Xue Li, Daniel Radtke, Severin Dicks, Moritz P. Kornadt, Christina Mertens, Julia K. Meier, Kory J. Lavine, Yunhang Zhang, Tim Christian Kuhn, Tobias Terzer, Jyoti Patel, Melanie Boerries, Gabriele Schramm, Norbert Frey, Hugo A. Katus, David Voehringer, Florian Leuschner
Vascular calcification (VC) predicts cardiovascular morbidity and mortality in chronic kidney disease (CKD). To date, the underlying mechanisms remain unclear. We detected leukocyte DNA N6-methyladenine (6mA) levels in CKD patients with or without aortic arch calcification. We used arteries from CKD mice infected with vascular smooth muscle cells (VSMCs)-targeted adeno-associated virus encoding alkB homolog 1 (Alkbh1) gene or Alkbh1 shRNA to evaluate features of calcification. We identified that leukocyte 6mA levels were significantly reduced as the severity of VC increased in CKD patients. Decreased 6mA demethylation resulted from the upregulation of ALKBH1. Here, ALKBH1 overexpression aggravated, whereas its depletion blunted VC progression and osteogenic reprogramming in vivo and in vitro. Mechanistically, ALKBH1-demethylated DNA 6mA modification could facilitate the binding of octamer-binding transcription factor 4 (Oct4) to bone morphogenetic protein 2 (BMP2) promoter and activate BMP2 transcription. This resulted in osteogenic reprogramming of VSMCs and subsequent VC progression. Either BMP2 or Oct4 depletion alleviated the pro-calcifying effects of ALKBH1. This suggests targeting ALKBH1 might be a therapeutic method to reduce the burden of VC in CKD.
Liu Ouyang, Xiaoyan Su, Wenxin Li, Liangqiu Tang, Mengbi Zhang, Yongjun Zhu, Changming Xie, Puhua Zhang, Jie Chen, Hui Huang
Intestinal farnesoid X receptor (FXR) signaling is involved in the development of obesity, fatty liver disease, and type 2 diabetes. However, the role of intestinal FXR in atherosclerosis and its potential as a target for clinical treatment have not been explored. The serum levels of fibroblast growth factor 19 (FGF19), which is encoded by an FXR target gene, were much higher in patients with hypercholesterolemia than in control subjects and were positively related to circulating ceramide levels, indicating a link between intestinal FXR, ceramide metabolism, and atherosclerosis. Among ApoE–/– mice fed a high-cholesterol diet (HCD), intestinal FXR deficiency (in FxrΔIE ApoE–/– mice) or direct FXR inhibition (via treatment with the FXR antagonist glycoursodeoxycholic acid [GUDCA]) decreased atherosclerosis and reduced the levels of circulating ceramides and cholesterol. Sphingomyelin phosphodiesterase 3 (SMPD3), which is involved in ceramide synthesis in the intestine, was identified as an FXR target gene. SMPD3 overexpression or C16:0 ceramide supplementation eliminated the improvements in atherosclerosis in FxrΔIE ApoE–/– mice. Administration of GUDCA or GW4869, an SMPD3 inhibitor, elicited therapeutic effects on established atherosclerosis in ApoE–/– mice by decreasing circulating ceramide levels. This study identified an intestinal FXR/SMPD3 axis that is a potential target for atherosclerosis therapy.
Qing Wu, Lulu Sun, Xiaomin Hu, Xuemei Wang, Feng Xu, Bo Chen, Xianyi Liang, Jialin Xia, Pengcheng Wang, Daisuke Aibara, Shaofei Zhang, Guangyi Zeng, Chuyu Yun, Yu Yan, Yicheng Zhu, Michael Bustin, Shuyang Zhang, Frank J. Gonzalez, Changtao Jiang
Dysregulated protein degradative pathways are increasingly recognized as mediators of human disease. This mechanism may have particular relevance to desmosomal proteins that play critical structural roles in both tissue architecture and cell-cell communication as destabilization/breakdown of the desmosomal proteome is a hallmark of genetic-based desmosomal-targeted diseases, such as the cardiac disease, arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C). However, no information exists on whether there are resident proteins that regulate desmosomal proteome homeostasis. Here we uncovered a cardiac COP9 desmosomal resident protein complex, composed of subunit 6 of the COP9 signalosome (CSN6), that enzymatically restricted neddylation and targeted desmosomal proteome degradation. CSN6 binding, localization, levels and function were impacted in hearts of classic mouse and human models of ARVD/C impacted by desmosomal loss and mutations, respectively. Loss of desmosomal proteome degradation control due to CSN6 loss and human desmosomal mutations destabilizing CSN6 were also sufficient to trigger ARVD/C in mice. We identified a desmosomal resident regulatory complex that restricted desmosomal proteome degradation and disease.
Yan Liang, Robert C. Lyon, Jason Pellman, William H. Bradford, Stephan Lange, Julius Bogomolovas, Nancy D. Dalton, Yusu Gu, Marcus Bobar, Mong-Hong Lee, Tomoo Iwakuma, Vishal Nigam, Angeliki Asimaki, Melvin Scheinman, Kirk L. Peterson, Farah Sheikh