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
Autophagy modulates lipid turnover, cell survival, inflammation and atherogenesis. Scavenger receptor class B type I (SR-BI) plays a crucial role in lysosome function. Here, we demonstrate that SR-BI regulates autophagy in atherosclerosis. SR-BI deletion attenuated lipid-induced expression of autophagy mediators in macrophages and atherosclerotic aortas. Consequently, SR-BI deletion resulted in 1.8- and 2.5-fold increases in foam cell formation and apoptosis, respectively, and increased oxidized LDL-induced inflammatory cytokine expression. Pharmacological activation of autophagy failed to reduce lipid content or apoptosis in Sr-b1-/- macrophages. SR-BI deletion reduced both basal and inducible levels of transcription factor EB (TFEB), a master regulator of autophagy, causing decreased expression of autophagy genes encoding VPS34 and Beclin-1. Notably, SR-BI regulated Tfeb expression by enhancing PPARα activation. Moreover, intracellular macrophage SR-BI localized to autophagosomes, where it formed cholesterol domains resulting in enhanced association of Barkor and recruitment of the VPS34/Beclin-1 complex. Thus, SR-BI deficiency led to lower VPS34 activity in macrophages and in atherosclerotic aortic tissues. Overexpression of Tfeb or Vps34 rescues the defective autophagy in Sr-b1-/- macrophages. Taken together, macrophage SR-BI regulates autophagy via Tfeb expression and recruitment of the VPS34/Beclin-1 complex, thus identifying previously unrecognized roles for SR-BI and novel targets for the treatment of atherosclerosis.
Huan Tao, Patricia G. Yancey, John L. Blakemore, Youmin Zhang, Lei Ding, W. Gray Jerome, Jonathan D. Brown, Kasey C. Vickers, MacRae F. Linton
Congenital heart disease is the most common type of birth defect, accounting for one-third of all congenital anomalies. Using whole-exome sequencing of 2718 patients with congenital heart disease and a search in GeneMatcher, we identified 30 patients from 21 unrelated families of different ancestries with biallelic phospholipase D1 (PLD1) variants who presented predominantly with congenital cardiac valve defects. We also associated recessive PLD1 variants with isolated neonatal cardiomyopathy. Furthermore, we established that p.I668F is a founder variant among Ashkenazi Jews (allele frequency of ~2%) and describe the phenotypic spectrum of PLD1-associated congenital heart defects. PLD1 missense variants were overrepresented in regions of the protein critical for catalytic activity, and, correspondingly, we observed a strong reduction in enzymatic activity for most of the mutant proteins in an enzymatic assay. Finally, we demonstrate that PLD1 inhibition decreased endothelial-mesenchymal transition, an established pivotal early step in valvulogenesis. In conclusion, our study provides a more detailed understanding of disease mechanisms and phenotypic expression associated with PLD1 loss of function.
Najim Lahrouchi, Alex V. Postma, Christian M. Salazar, Daniel M. De Laughter, Fleur Tjong, Lenka Piherová, Forrest Z. Bowling, Dominic Zimmerman, Elisabeth M. Lodder, Asaf Ta-Shma, Zeev Perles, Leander Beekman, Aho Ilgun, Quinn Gunst, Mariam Hababa, Doris Škorić-Milosavljević, Viktor Stránecký, Viktor Tomek, Peter de Knijff, Rick de Leeuw, Jamille Y. Robinson, Sabrina C. Burn, Hiba Mustafa, Matthew Ambrose, Timothy Moss, Jennifer Jacober, Dmitriy M. Niyazov, Barry Wolf, Katherine H. Kim, Sara Cherny, Andreas Rousounides, Aphrodite Aristidou-Kallika, George Tanteles, Bruel Ange-Line, Anne-Sophie Denommé-Pichon, Christine Francannet, Damara Ortiz, Monique C. Haak, Arend D. J. Ten Harkel, Gwendolyn T.R. Manten, Annemiek C. Dutman, Katelijne Bouman, Monia Magliozzi, Francesca Clementina Radio, Gijs W.E. Santen, Johanna C. Herkert, H. Alex Brown, Orly Elpeleg, Maurice J.B. van den Hoff, Barbara Mulder, Michael V. Airola, Stanislav Kmoch, Joey V. Barnett, Sally-Ann Clur, Michael A. Frohman, Connie R. Bezzina
Efferocytosis, the process through which apoptotic cells (ACs) are cleared through actin-mediated engulfment by macrophages, prevents secondary necrosis, suppresses inflammation, and promotes resolution. Impaired efferocytosis drives the formation of clinically dangerous necrotic atherosclerotic plaques, the underlying etiology of coronary artery disease (CAD). An intron of the gene encoding PHACTR1 contains a common variant rs9349379 (A > G) associated with CAD. As PHACTR1 is an actin-binding protein, we reasoned that if the rs9349379 risk allele G causes lower PHACTR1 expression in macrophages, it might link the risk-allele to CAD via impaired efferocytosis. We show here that rs9349379-G/G was associated with lower levels of PHACTR1 and impaired efferocytosis in human monocyte-derived macrophages and human atherosclerotic lesional macrophages compared with rs9349379-A/A. Silencing PHACTR1 in human and mouse macrophages compromised AC engulfment, and mice in which hematopoietic Phactr1 was genetically targeted in Western diet-fed Ldlr-/- mice showed impaired lesional efferocytosis, increased plaque necrosis, and thinner fibrous caps—all signs of vulnerable plaques in humans. Mechanistically, PHACTR1 prevented dephosphorylation of myosin light chain (MLC), which was necessary for AC engulfment. In summary, rs9349379-G lowers PHACTR1, which, by lowering phospho-MLC, compromised efferocytosis. Thus, rs9349379-G may contribute to CAD risk, at least in part, by impairing atherosclerotic lesional macrophage efferocytosis.
Canan Kasikara, Maaike Schilperoort, Brennan D. Gerlach, Chenyi Xue, Xiaobo Wang, Ze Zheng, George Kuriakose, Bernhard Dorweiler, Hanrui Zhang, Gabrielle Fredman, Danish Saleheen, Muredach P Reilly, Ira Tabas
Cx43, a major cardiac connexin, forms precursor hemichannels that accrue at the intercalated disc to assemble as gap junctions. While gap junctions are crucial for electrical conduction in the heart, little is known on potential roles of hemichannels. Recent evidence suggests that inhibiting Cx43 hemichannel opening with Gap19 has antiarrhythmic effects. Here, we used multiple electrophysiology, imaging and super-resolution techniques to understand and define the conditions underlying Cx43 hemichannel activation in ventricular cardiomyocytes, their contribution to diastolic Ca2+ release from the sarcoplasmic reticulum, and their impact on electrical stability. We showed that Cx43 hemichannels are activated during diastolic Ca2+ release in single ventricular cardiomyocytes and cardiomyocyte cell pairs from mouse and pig. This activation involved Cx43 hemichannel Ca2+ entry and coupling to Ca2+ release microdomains at the intercalated disc resulting in enhanced Ca2+ dynamics. Hemichannel opening furthermore contributed to delayed afterdepolarizations and triggered action potentials. In single cardiomyocytes, cardiomyocyte cell pairs and arterially perfused tissue wedges from failing human hearts, increased hemichannel activity contributed to electrical instability as compared to non-failing rejected donor hearts. We conclude that microdomain coupling between Cx43 hemichannels and Ca2+ release is a novel, targetable, mechanism of cardiac arrhythmogenesis in heart failure.
Maarten A.J. De Smet, Alessio Lissoni, Timur Nezlobinsky, Nan Wang, Eef Dries, Marta Pérez-Hernández, Xianming Lin, Matthew Amoni, Tim Vervliet, Katja Witschas, Eli Rothenberg, Geert Bultynck, Rainer Schulz, Alexander V. Panfilov, Mario Delmar, Karin R. Sipido, Luc Leybaert
The relationship between adiposity and metabolic health is well established. However, very little is known about the fat depot, known as paracardial fat (pCF), located superior to and surrounding the heart. Here, we show that pCF remodels with aging and a high-fat diet and that the size and function of this depot are controlled by alcohol dehydrogenase 1 (ADH1), an enzyme that oxidizes retinol into retinaldehyde. Elderly individuals and individuals with obesity have low ADH1 expression in pCF, and in mice, genetic ablation of Adh1 is sufficient to drive pCF accumulation, dysfunction, and global impairments in metabolic flexibility. Metabolomics analysis revealed that pCF controlled the levels of circulating metabolites affecting fatty acid biosynthesis. Also, surgical removal of the pCF depot was sufficient to rescue the impairments in cardiometabolic flexibility and fitness observed in Adh1-deficient mice. Furthermore, treatment with retinaldehyde prevented pCF remodeling in these animals. Mechanistically, we found that the ADH1/retinaldehyde pathway works by driving PGC-1α nuclear translocation and promoting mitochondrial fusion and biogenesis in the pCF depot. Together, these data demonstrate that pCF is a critical regulator of cardiometabolic fitness and that retinaldehyde and its generating enzyme ADH1 act as critical regulators of adipocyte remodeling in the pCF depot.
Jennifer M. Petrosino, Jacob Z. Longenecker, Srinivasagan Ramkumar, Xianyao Xu, Lisa E. Dorn, Anna Bratasz, Lianbo Yu, Santosh Maurya, Vladimir Tolstikov, Valerie Bussberg, Paul M.L. Janssen, Muthu Periasamy, Michael A. Kiebish, Gregg Duester, Johannes von Lintig, Ouliana Ziouzenkova, Federica Accornero
Tyro3, AXL, and MerTK (TAM) receptors are activated in macrophages in response to tissue injury and as such have been proposed as therapeutic targets to promote inflammation resolution during sterile wound healing, including myocardial infarction. While the role of MerTK in cardioprotection is well-characterized, the unique role of the other structurally similar TAMs, and particularly AXL, in clinically-relevant models of myocardial ischemia-reperfused infarction (IRI) is comparatively unknown. Utilizing complementary approaches, validated by flow cytometric analysis of human and murine macrophage subsets and conditional genetic loss and gain of function, we uncover a unique maladaptive role for myeloid AXL during IRI in the heart. Cross signaling between AXL and TLR4 in cardiac macrophages directed a switch to glycolytic metabolism and secretion of proinflammatory IL-1β, leading to increased intramyocardial inflammation, adverse ventricular remodeling, and impaired contractile function. AXL interestingly functioned independently of cardioprotective MerTK to reduce the efficacy of cardiac repair, but like MerTK, was proteolytically cleaved. Administration of a selective small molecule AXL inhibitor alone improved cardiac healing, which was further enhanced in combination with blockade of MerTK cleavage. These data support further exploration of macrophage TAM receptors as therapeutic targets for myocardial infarction.
Matthew DeBerge, Kristofor Glinton, Manikandan Subramanian, Lisa D. Wilsbacher, Carla V. Rothlin, Ira Tabas, Edward B. Thorp
LMNA mutations in patients are responsible for a dilated cardiomyopathy. Molecular mechanisms underlying the origin and development of the pathology are unknown. Herein, using mouse pluripotent embryonic stem cells (ESCs) and a mouse model both harboring the p.H222P Lmna mutation, we found early defects in cardiac differentiation of mutated ESCs and dilatation of mutated embryonic hearts at E13.5, pointing to a developmental origin of the disease. Using mouse ESCs, we demonstrated that cardiac differentiation of LmnaH222P/+ was impaired at the mesodermal stage. Expression of Mesp1, a mesodermal cardiogenic gene involved in epithelial-to-mesenchymal transition of epiblast cells, as well as Snai1 and Twist expression, was decreased in LmnaH222P/+ cells compared with WT cells in the course of differentiation. In turn, cardiomyocyte differentiation was impaired. ChIP assay of H3K4me1 in differentiating cells revealed a specific decrease of this histone mark on regulatory regions of Mesp1 and Twist in LmnaH222P/+ cells. Downregulation or inhibition of LSD1 that specifically demethylated H3K4me1 rescued the epigenetic landscape of mesodermal LmnaH222P/+ cells and in turn contraction of cardiomyocytes. Inhibition of LSD1 in pregnant mice or neonatal mice prevented cardiomyopathy in E13.5 LmnaH222P/H222P offspring and adults, respectively. Thus, LSD1 appeared to be a therapeutic target to prevent or cure dilated cardiomyopathy associated with a laminopathy.
Anne-Claire Guénantin, Imen Jebeniani, Julia Leschik, Erwan Watrin, Gisèle Bonne, Nicolas Vignier, Michel Pucéat