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LncRNA ZFAS1 as a SERCA2a Inhibitor to Cause Intracellular Ca2+ Overload and Contractile Dysfunction in a Mouse Model of Myocardial Infarction [Original Research]


Rationale: Ca2+ homeostasis, a critical determinant of cardiac contractile function, is critically regulated by sarcoplasmic reticulum Ca2+-ATPase 2a (SERCA2a). Our previous study has identified ZFAS1 as a new long non-coding RNA (lncRNA) biomarker of acute myocardial infarction (MI). Objective: To evaluate the effects of ZFAS1 on SERCA2a and the associated Ca2+ homeostasis and cardiac contractile function in the setting of MI. Methods and Results: ZFAS1 expression was robustly increased in cytoplasm and sarcoplasmic reticulum in a mouse model of MI and a cellular model of hypoxia. Knockdown of endogenous ZFAS1 by virus-mediated silencing shRNA partially abrogated the ischemia-induced contractile dysfunction. Overexpression of ZFAS1 in otherwise normal mice created similar impairment of cardiac function as that observed in MI mice. Moreover, at the cellular level ZFAS1 overexpression weakened the contractility of cardiac muscles. At the subcellular level, ZFAS1 deleteriously altered the Ca2+ transient leading to intracellular Ca2+ overload in cardiomyocytes. At the molecular level, ZFAS1 was found to directly bind SERCA2a protein and to limit its activity, as well as to repress its expression. The effects of ZFAS1 were readily reversible upon knockdown of this lncRNA. Notably, a sequence domain of ZFAS1 gene that is conserved across species mimicked the effects of the full-length ZFAS1. Mutation of this domain or application of an antisense fragment to this conserved region efficiently canceled out the deleterious actions of ZFAS1. ZFAS1 had no significant effects on other Ca2+-handling regulatory proteins. Conclusions: ZFAS1 is an endogenous SERCA2a inhibitor, acting by binding to SERCA2a protein to limit its intracellular level and inhibit its activity, and a contributor to the impairment of cardiac contractile function in MI. Therefore, anti-ZFAS1 might be considered a new therapeutic strategy for preserving SERCA2a activity thereby cardiac function under pathological conditions of the heart.

Desmin Phosphorylation Triggers Preamyloid Oligomers Formation and Myocyte Dysfunction in Acquired Heart Failure [Original Research]


Rationale: Disrupted proteostasis is one major pathological trait that heart failure (HF) shares with other organ proteinopathies, such as Alzheimer's and Parkinson's diseases. Yet, differently from the latter, whether and how cardiac preamyloid oligomers (PAOs) develop in acquired forms of HF is unclear. Objective: We previously reported a rise in mono-phosphorylated, aggregate-prone desmin in canine and human HF. We now tested if mono-phosphorylated desmin acts as the seed nucleating PAOs formation and determined if positron emission tomography (PET) is able to detect myocardial PAOs in non-genetic HF. Methods and Results: Here, we first show that toxic cardiac PAOs accumulate in the myocardium of mice subjected to transverse aortic constriction (TAC), and that PAOs co-migrate with the cytoskeletal protein desmin in this well-established model of acquired HF. We confirm this evidence in cardiac extracts from human ischemic and non-ischemic HF. We also demonstrate that Ser-31 phosphorylated (pSer31) desmin aggregates extensively in cultured cardiomyocytes. Lastly, we were able to detect the in vivo accumulation of cardiac PAOs using positron emission tomography (PET) for the first time in acquired HF. Conclusions: pSer31 is a likely candidate seed for the nucleation process leading to cardiac PAOs deposition. Desmin post-translational processing and misfolding constitute a new, attractive avenue for the diagnosis and treatment of the cardiac accumulation of toxic PAOs that can now be measured by PET in acquired HF.

Airn Regulates Igf2bp2 Translation in Cardiomyocytes [Short Communications]


Rationale: Increasing evidence indicates the presence of long noncoding RNAs (lncRNAs) in various cell types. Airn is an imprinting gene transcribed from the paternal chromosome. It is in antisense orientation to the imprinted, but maternally-derived, Igf2r gene, on which Airn exerts its regulation in cis. Although Airn is highly expressed in the heart, functions aside from imprinting remain unknown. Objective: Here, we studied the functions of Airn in the heart, especially cardiomyocytes. Methods and Results:: Silencing of Airn via siRNAs augmented cell death, vulnerability to cellular stress, and reduced cell migration. To find the cause of such phenotypes, the potential binding partners of Airn were identified via RNA pull-down followed by mass spectrometry, which indicated Igf2bp2 and Rpa1 as potential binding partners. Further experiments showed that Airn binds to Igf2bp2 to control the translation of a number of genes. Moreover, silencing of Airn caused less binding of Igf2bp2 to other mRNAs and reduced translation of Igf2bp2 protein. Conclusions: Our study uncovers a new function of Airn and demonstrates that Airn is important for the physiology of cardiomyocytes.

Progenitor Cells and Clinical Outcomes in Patients with Acute Coronary Syndromes [Original Research]


Rationale: Circulating progenitor cells (CPCs) mobilize in response to ischemic injury, but their predictive value remains unknown in acute coronary syndrome (ACS). Objective: We aimed to investigate the number of CPCs in ACS compared to those with stable coronary artery disease (CAD), relationship between bone marrow PCs and CPCs, and whether CPC counts predict mortality in patients with ACS. Methods and Results: In 2028 patients, 346 had unstable angina, 183 had an acute myocardial infarction (AMI) and the remaining 1499 patients had stable CAD. Patients with ACS were followed for the primary end point of all-cause death. CPCs were enumerated by flow cytometry as mononuclear cells expressing a combination of CD34+, CD133+, VEGFR2+, or CXCR4+. CPC counts were higher in subjects with AMI compared those with stable CAD even after adjustment for age, sex, race, body mass index, renal function, hypertension, diabetes, hyperlipidemia, and smoking; CD34+, CD34+/CD133+, CD34+/CXCR4+ and CD34+/VEGFR2+ CPC counts were 19%, 25%, 28%, and 142% higher in those with AMI, respectively, compared to stable CAD. There were strong correlations between the concentrations of CPCs and the PC counts in bone marrow aspirates in 20 patients with AMI. During a 2 (IQR 1.31-2.86)-year follow-up period of 529 ACS patients, 12.4% died. In Cox regression models adjusted for age, gender, body mass index, heart failure history, estimated GFR, and AMI, subjects with low CD34+ cell counts had a 2.46-fold (95% CI 1.18-5.13) increase in all-cause mortality, P=0.01. CD34+/CD133+ and CD34+/CXCR4+, but not CD34+/VEGFR2+ PC counts had similar associations with mortality. Results were validated in a separate cohort of 238 patients with ACS. Conclusions: CPC levels are significantly higher in patients after an AMI compared to those with stable CAD and reflect bone marrow PC content. Among patients with ACS, a lower number of hematopoietic-enriched CPCs are associated with a higher mortality.

Cardiac Kir2.1 and NaV1.5 Channels Traffic Together to the Sarcolemma to Control Excitability [Original Research]


Rationale: In cardiomyocytes, NaV1.5 and Kir2.1 channels interact dynamically as part of membrane bound macromolecular complexes. Objective: To test whether NaV1.5 and Kir2.1 preassemble during early forward trafficking and travel together to common membrane microdomains. Methods and Results: In patch-clamp experiments, co-expression of trafficking deficient mutants Kir2.1Δ314-315 or Kir2.1R44A/R46A with wildtype (WT) NaV1.5WT in heterologous cells reduced INa, compared to NaV1.5WT alone or co-expressed with Kir2.1WT. In cell surface biotinylation experiments, expression of Kir2.1Δ314-315 reduced NaV1.5 channel surface expression. Glycosylation analysis suggested that NaV1.5WT and Kir2.1WT channels associate early in their biosynthetic pathway, and fluorescence recovery after photobleaching experiments demonstrated that co-expression with Kir2.1 increased cytoplasmic mobility of NaV1.5WT, and vice versa, whereas co-expression with Kir2.1Δ314-315 reduced mobility of both channels. Viral gene transfer of Kir2.1∆314-315 in adult rat ventricular myocytes and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) reduced

Lysosomal Cholesterol Hydrolysis Couples Efferocytosis to Anti-Inflammatory Oxysterol Production [Original Research]


Rationale: Macrophages face a substantial amount of cholesterol following the ingestion of apoptotic cells and the lysosomal acid lipase (LIPA) has a major role in hydrolyzing cholesteryl esters in the endocytic compartment. Objective: Here, we directly investigated the role of LIPA-mediated clearance of apoptotic cells both in vitro and in vivo. Methods and Results: We show that LIPA inhibition causes a defective efferocytic response due to impaired generation of 25-OHC and 27-OHC. Reduced synthesis of 25-OHC after LIPA inhibition contributed to defective mitochondria associated membrane (MAM) leading to mitochondrial oxidative stress-induced NLRP3 inflammasome activation and caspase 1-dependent Rac1 degradation. A secondary event consisting of failure to appropriately activate liver X receptor-mediated pathways led to mitigation of cholesterol efflux and apoptotic cell clearance. In mice, LIPA inhibition caused defective clearance of apoptotic lymphocytes and stressed erythrocytes by hepatic and splenic macrophages, culminating in splenomegaly and splenic iron accumulation under hypercholesterolemia. Conclusions: Our findings position lysosomal cholesterol hydrolysis as a critical process that prevents metabolic inflammation by enabling efficient macrophage apoptotic cell clearance.

Glycosylation Profile of Immunoglobulin G Is Cross-Sectionally Associated with Cardiovascular Disease Risk Score and Subclinical Atherosclerosis in Two Independent Cohorts [Original Research]


Rationale: One measure of protein glycosylation (GlycA) has been reported to predict higher cardiovascular risk by reflecting inflammatory pathways Objective: To assess the role of a comprehensive panel of immunoglobulin (IgG) glycosylation traits on traditional risk factors for cardiovascular disease and on presence of subclinical atherosclerosis in addition to GlycA. Methods and Results: We measured 76 IgG glycosylation traits in 2970 women (age range 40-79 years) from the TwinsUK cohort and correlated it to their estimated 10-year atherosclerotic cardiovascular disease (ASCVD) risk score and their carotid and femoral plaque measured by ultrasound imaging. Eight IgG glycan traits are associated with the 10-year ASCVD risk score after adjusting for multiple tests and for individual risk factors - 5 with increased risk and 3 with decreased risk. These glycans replicated in 967 women from ORCADES cohort, six of them were also associated in 845 men. A linear combination of IgG glycans and GlycA is also associated with presence of carotid (OR[95%CI]=1.55 [1.25;1.93], P=7.5X10-5) and femoral (OR[95%CI]==1.32[1.06;1.64], P=0.01) plaque in a subset of women with atherosclerosis data after adjustment for traditional risk factors. One specific glycosylation trait, GP18 was negatively correlated with VLDL and triglyceride levels in serum and with presence of carotid plaque (OR[95%CI] = 0.60[0.50;0.71], P = 5x10-4). Conclusions: We find molecular pathways linking IgG to arterial lesion formation. Glycosylation traits are independently associated with subclinical atherosclerosis. One specific trait related to the sialylated N-glycan is negatively correlated with CVD risk, VLDL and triglyceride serum levels and presence of carotid plaque.

Single-Cell RNA-Seq Reveals the Transcriptional Landscape and Heterogeneity of Aortic Macrophages in Murine Atherosclerosis [Original Research]


Rationale: It is assumed that atherosclerotic arteries contain several macrophage subsets endowed with specific functions. The precise identity of these subsets is poorly characterized as they ha ve been defined by the expression of a restricted number of markers. Objective: We have applied single-cell RNA-seq as an unbiased profiling strategy to interrogate and classify aortic macrophage heterogeneity at the single-cell level in atherosclerosis. Methods and Results: We performed single-cell RNA sequencing of total aortic CD45+ cells extracted from the non-diseased (chow fed) and atherosclerotic (11 weeks of high fat diet) aorta of Ldlr-/- mice. Unsupervised clustering singled out 13 distinct aortic cell clusters. Among the myeloid cell populations, Resident-like macrophages with a gene expression profile similar to aortic resident macrophages were found in healthy and diseased aortae, whereas monocytes, monocyte-derived dendritic cells (MoDC), and two populations of macrophages were almost exclusively detectable in atherosclerotic aortae, comprising Inflammatory macrophages showing enrichment in Il1b, and previously undescribed TREM2hi macrophages. Differential gene expression and gene ontology enrichment analyses revealed specific gene expression patterns distinguishing these three macrophage subsets and MoDC, and uncovered putative functions of each cell type. Notably, TREM2hi macrophages appeared to be endowed with specialized functions in lipid metabolism and catabolism, and presented a gene expression signature reminiscent of osteoclasts, suggesting a role in lesion calcification. TREM2 expression was moreover detected in human lesional macrophages. Importantly, these macrophage populations were present also in advanced atherosclerosis and in Apoe-/- aortae, indicating relevance of our findings in different stages of atherosclerosis and mouse models. Conclusions: These data unprecedentedly uncovered the transcriptional landscape and phenotypic heterogeneity of aortic macrophages and MoDCs in atherosclerotic and identified previously unrecognized macrophage populations and their gene expression signature, suggesting specialized functions. Our findings will open up novel opportunities to explore distinct myeloid cell populations and their functions in atherosclerosis.

Atlas of the Immune Cell Repertoire in Mouse Atherosclerosis Defined by Single-Cell RNA-Sequencing and Mass Cytometry [Original Research]


Rationale: Atherosclerosis is a chronic inflammatory disease that is driven by the interplay of pro- and anti-inflammatory leukocytes in the aorta. Yet, the phenotypic and transcriptional diversity of aortic leukocytes is only poorly understood. Objective: We characterized leukocytes from healthy and atherosclerotic mouse aortas in-depth by single cell RNA-sequencing (scRNAseq) and mass cytometry (CyTOF) to define an atlas of the immune cell landscape in atherosclerosis. Methods and Results: Employing scRNAseq of aortic leukocytes from chow (CD) and western diet (WD) fed Apoe-/- and Ldlr-/- mice, we detected 11 principal leukocyte clusters with distinct phenotypical and spatial characteristics, while the cellular repertoire in healthy aortas was less diverse. Gene set enrichment analysis on a single cell level established that multiple pathways, such as for lipid metabolism, proliferation, and cytokine secretion, were confined to particular leukocyte clusters. Leukocyte populations were differentially regulated in atherosclerotic Apoe-/- and Ldlr-/- mice. We confirmed the phenotypic diversity of these clusters with a novel CyTOF 35-marker panel with metal-labelled antibodies and conventional flow cytometry. Cell populations retrieved by these protein-based approaches were highly correlated to transcriptionally defined clusters. In an integrated screening strategy of scRNAseq, CyTOF, and FACS, we detected three principal B-cell subsets with alterations in surface markers, functional pathways, and in vitro cytokine secretion. Finally, we used leukocyte cluster gene signatures to enumerate leukocyte frequencies in 126 human plaques by a genetic deconvolution strategy. This approach revealed that human carotid plaques and microdissected mouse plaques were mostly populated by macrophages, T-cells, and monocytes. In addition, the frequency of genetically defined leukocyte populations in carotid plaques predicted cardiovascular events in patients. Conclusions: The definition of leukocyte diversity by high-dimensional analyses enables a fine-grained analysis of aortic leukocyte subsets, reveals new immunological mechanisms and cell-type specific pathways, and establishes a functional relevance for lesional leukocytes in human atherosclerosis.

A Dominant Role for Regulatory T Cells in Protecting Females Against Pulmonary Hypertension [Original Research]


Rationale: Pulmonary arterial hypertension (PH) is a life-threatening condition associated with immune dysregulation and abnormal regulatory T cell (Treg) activity, but it is currently unknown whether and how abnormal Treg function differentially affects males and females. Objective: To evaluate whether and how Treg-deficiency differentially affects male and female rats in experimental PH. Methods and Results: Male and female athymic rnu/rnu rats, lacking Tregs, were treated with the vascular endothelial growth factor receptor-2 (VEGFR2) inhibitor SU5416 or chronic hypoxia and evaluated for PH; some animals underwent Treg immune reconstitution (IR) before SU5416 administration. Plasma prostacyclin (PGI2) levels were measured. Lung and right ventricles (RVs) were assessed for the expression of the vasoprotective proteins cyclooxygenase-2 (COX-2), prostacyclin synthase (PTGIS), programmed death ligand-1 (PDL-1), and heme oxygenase-1 (HO-1). Inhibitors of these pathways were administered to athymic rats undergoing Treg IR. Finally, human cardiac microvascular endothelial cells co-cultured with Tregs were evaluated for COX-2, PDL-1, HO-1, and estrogen receptor (ER) expression, and culture supernatants were assayed for PGI2 and IL-10. SU5416-treatment and chronic hypoxia produced more severe PH in female than male athymic rats. Females were distinguished by greater pulmonary inflammation, augmented RV fibrosis, lower plasma PGI2 levels, decreased lung COX-2, PTGIS, HO-1 and PDL-1 expression and reduced RV PDL-1 levels. In both sexes, Treg IR protected against PH development and raised levels of plasma PGI2 and cardiopulmonary COX-2, PTGIS, PDL-1, and HO-1. Inhibiting COX-2, HO-1, and programmed death-1 (PD1)/PDL1 pathways abrogated Treg protection. In vitro, human Tregs directly upregulated endothelial COX-2, PDL1, HO-1, ERs and increased supernatant levels of PGI2 and IL-10. Conclusions: In two animal models of PH based on Treg deficiency, females developed more severe PH than males. The data suggest that females are especially reliant on normal Treg function to counteract the effects of pulmonary vascular injury leading to PH.

COMMD Family Regulates Plasma LDL Levels and Attenuates Atherosclerosis Through Stabilizing the CCC Complex in Endosomal LDLR Trafficking [Original Research]


Rationale: COpper Metabolism MURR1 Domain-containing (COMMD) proteins are a part of the COMMD-CCDC22-CCDC93 (CCC) complexes facilitating endosomal trafficking of cell surface receptors. Hepatic COMMD1 inactivation decreases CCDC22 and CCDC93 protein levels, impairs the recycling of the low-density lipoprotein receptor (LDLR), and increases plasma LDL cholesterol levels in mice. However, whether any of the other COMMD members function similarly as COMMD1, and whether perturbation in the CCC complex promotes atherogenesis remain unclear. Objective: To unravel the contribution of evolutionarily conserved COMMD proteins to plasma lipoprotein levels and atherogenesis. Methods and Results: Using liver specific Commd1, Commd6 or Commd9 knockout mice we investigated the relation between the COMMD proteins in the regulation of plasma cholesterol levels. Combining biochemical and quantitative targeted proteomic approaches, we found that either hepatic COMMD1, COMMD6 or COMMD9 deficiency resulted in massive reduction in the protein levels of all ten COMMDs. This decrease in COMMD proteins levels coincided with destabilizing of the core (CCDC22, CCDC93, C16orf62) of the CCC complex, reduced cell surface levels of LDLR and LRP1, followed by increased plasma LDL cholesterol levels. To assess the direct contribution of the CCC core in the regulation of plasma cholesterol levels, Ccdc22 was deleted in mouse livers via CRISPR/Cas9-mediated somatic gene editing. CCDC22 deficiency also destabilized the complete CCC complex, and resulted in elevated plasma LDL cholesterol levels. Finally, we found that hepatic disruption of the CCC complex exacerbates dyslipidemia and atherosclerosis in ApoE3*Leiden mice. Conclusions: Collectively, these findings demonstrate a strong interrelationship between COMMD proteins and the core of the CCC complex in endosomal LDLR trafficking. Hepatic disruption of either of these CCC components causes hypercholesterolemia, and exacerbates atherosclerosis. Our results indicate that not only COMMD1, but all other COMMDs and CCC components may be potential targets for modulating plasma lipid levels in humans.

Protein S-Nitrosylation Controls Glycogen Synthase Kinase 3{beta} Function Independent of its Phosphorylation State [Original Research]


Rationale: Glycogen synthase kinase 3β (GSK3β) is a multifunctional and constitutively active kinase known to regulate a myriad of cellular processes. The primary mechanism to regulate its function is through phosphorylation-dependent inhibition at serine-9 residue. Emerging evidence indicates that there may be alternative mechanisms that control GSK3β for certain functions. Objective: Here we sought to understand the role of protein S-nitrosylation (SNO) on the function of GSK3β. SNO-dependent modulation of the localization of GSK3β and its ability to phosphorylate downstream targets was investigated in vitro and the network of proteins differentially impacted by phospho- or SNO-dependent GSK3β regulation and in vivo SNO modification of key signaling kinases during the development of heart failure was also studied. Methods and Results: We found that GSK3β undergoes site-specific SNO both in vitro, in HEK293 cells, H9C2 myoblasts, and primary neonatal rat ventricular myocytes (NRVM), as well as in vivo, in hearts from an animal model of heart failure and sudden cardiac death. S-nitrosylation of GSK3β significantly inhibits its kinase activity independent of the canonical phospho-inhibition pathway. S-nitrosylation of GSK3β promotes its nuclear translocation and access to novel downstream phospho-substrates which are enriched for a novel amino acid consensus sequence motif. Quantitative phospho-proteomics pathway analysis reveals that nuclear GSK3β plays a central role in cell cycle control, RNA splicing and DNA damage response. Conclusions: The results indicate that SNO has a differential effect on the location and activity of GSK3β in the cytoplasm versus the nucleus. SNO modification of GSK3β occurs in vivo and could contribute to the pathobiology of heart failure and sudden cardiac death.

Roles of PAD4 and NETosis in Experimental Atherosclerosis and Arterial Injury: Implications for Superficial Erosion [Original Research]


Rationale: Neutrophils likely contribute to the thrombotic complications of human atheromata. In particular, neutrophil extracellular traps (NETs) could exacerbate local inflammation and amplify and propagate arterial intimal injury and thrombosis. Peptidyl arginine deiminase 4 (PAD4) participates in NET formation, but understanding of this enzyme's role in atherothrombosis remains scant. Objective: This study tested the hypothesis that PAD4 and NETs influence experimental atherogenesis and in processes implicated in superficial erosion, a form of plaque complication we previously associated with NETs. Methods and Results: Bone marrow chimeric Ldlr deficient mice reconstituted with either wild type or Pad4 deficient cells underwent studies that assessed atheroma formation or procedures designed to probe mechanisms related to superficial erosion. PAD4 deficiency neither retarded fatty streak formation nor reduced plaque size or inflammation in bone marrow chimeric mice that consumed an atherogenic diet. In contrast, either PAD4 deficiency in bone marrow-derived cells, or administration of DNaseI to disrupt NETs, decreased the extent of arterial intimal injury in mice with arterial lesions tailored to recapitulate characteristics of human atheroma complicated by erosion. Conclusions: These results indicate that PAD4 from bone marrow-derived cells and NETs do not influence chronic experimental atherogenesis, but participate causally in acute thrombotic complications of intimal lesions that recapitulate features of superficial erosion.

Intra-Cardiac Release of Extracellular Vesicles Shapes Inflammation Following Myocardial Infarction [Short Communications]


Rationale: A rapid and massive influx of inflammatory cells occurs into ischemic area following myocardial infarction, resulting in local release of cytokines and growth factors. Yet, the mechanisms regulating their production are not fully explored. The release of extracellular vesicles (EV) in the interstitial space curbs important biological functions, including inflammation, and influences the development of cardiovascular diseases. So far, there is no evidence for in situ release of cardiac EVs following myocardial infarction. Objective: The present study tested the hypothesis that local EV generation in the infarcted heart coordinates cardiac inflammation following myocardial infarction. Methods and Results: Coronary artery ligation in mice transiently increases EV levels in the left ventricle when compared to sham animals. EVs from infarcted hearts were characterized as large vesicles (252±18nm) expressing cardiomyocyte and endothelial markers, and small EVs (118±4nm) harboring exosomal markers such as CD63 and CD9. Cardiac large EVs generated after myocardial infarction, but not small EVs or sham EVs, increased the release of interleukin-6, chemokines CCL2, CCL7 from FACS-sorted Ly6C+ cardiac monocytes. EVs of similar diameter were also isolated from fragments of interventricular septum obtained from patients undergoing aortic valve replacement, thus supporting the clinical relevance of our findings in mice. Conclusions: The present study demonstrates that acute myocardial infarction transiently increases the generation of cardiac EVs characterized as both exosomes and microvesicles, originating mainly from cardiomyocytes and endothelial cells. EVs accumulating in the ischemic myocardium are rapidly taken up by infiltrating monocytes and regulate local inflammatory responses.

Alternative Splicing of FOXP3 Controls Regulatory T Cell Effector Functions and Is Associated with Human Atherosclerotic Plaque Stability [Original Research]


Rationale: Regulatory T (Treg) cells suppress immune responses and have been shown to attenuate atherosclerosis. The Treg cell lineage specification factor FOXP3 is essential for Treg cells' ability to uphold immunological tolerance. In humans, FOXP3 exists in several different isoforms, however, their specific role is poorly understood. Objective: To define the regulation and functions of the two major FOXP3 isoforms, FOXP3fl and FOXP3Δ2, as well as to establish whether their expression is associated with ischemic atherosclerotic disease. Methods and Results: Human primary T-cells were transduced with lentiviruses encoding distinct FOXP3 isoforms. The phenotype and function of these cells were analyzed by flow cytometry, in vitro suppression assays and RNA-sequencing. We also assessed the effect of activation on Treg cells isolated from healthy volunteers. Treg cell activation resulted in increased FOXP3 expression that predominantly was made up of FOXP3Δ2. FOXP3Δ2 induced specific transcription of GARP, which functions by tethering the immunosuppressive cytokine TGF-β to the cell membrane of activated Treg cells. RT-PCR was used to determine the impact of alternative splicing of FOXP3 in relation with atherosclerotic plaque stability in a cohort of over 150 patients that underwent carotid endarterectomy. Plaque instability was associated with a lower FOXP3Δ2 transcript usage, when comparing plaques from patients without symptoms and patients with occurrence of recent (<1 month) vascular symptoms including minor stoke, transient ischemic attack or amaurosis fugax. No difference was detected in total levels of FOXP3 mRNA between these two groups. Conclusions: These results suggest that activated Treg cells suppress the atherosclerotic disease process and that FOXP3Δ2 controls a transcriptional program that acts protectively in human atherosclerotic plaques.

Regulation of Vascular Calcification by Growth Hormone-Releasing Hormone and its Agonists [Original Research]


Rationale: Vascular calcification (VC) is a marker of the severity of atherosclerotic disease. Hormones play important roles in regulating calcification; estrogen and parathyroid hormones exert opposing effects, the former alleviating VC and the latter exacerbating it. So far no treatment strategies have been developed to regulate clinical VC. Objective: To investigate the effect of growth hormone-releasing hormone (GHRH) and its agonist (GHRH-A) on the blocking of VC in a mouse model. Methods and Results: Young adult osteoprotegerin deficient (OPG-/-) mice were given daily subcutaneous injections of GHRH-A (MR409) for 4 weeks. Significant reductions in calcification of the aortas of MR409 treated mice were paralleled by markedly lower alkaline phosphatase (ALP) activity and a dramatic reduction in the expression of transcription factors including the osteogenic marker gene Runx2 and its downstream factors, osteonectin and osteocalcin. The mechanism of action of GHRH-A was dissected in smooth muscle cells (SMCs) isolated from human and mouse aortas. Calcification of SMCs induced by osteogenic medium (OM) was inhibited in the presence of GHRH or MR409, as evidenced by reduced ALP activity and Runx2 expression. Inhibition of calcification by MR409 was partially reversed by MIA602, a GHRH antagonist, or a GHRH receptor selective siRNA. Treatment with MR409 induced elevated cytosolic cAMP and its target, protein kinase A (PKA) which in turn blocked NADPH oxidase activity and reduced production of reactive oxygen species (ROS), thus blocking the phosphorylation of NFκB (p65), a key intermediate in the RANKL-Runx2/ALP osteogenesis program. A PKA-selective siRNA or the chemical inhibitor H89 abolished these beneficial effects of MR409. Conclusions: GHRH-A controls osteogenesis in SMCs by targeting cross talk between PKA and NFκB (p65) and through the suppression of ROS production that induces the Runx2 gene and ALP. Inflammation-mediated osteogenesis is thereby blocked. GHRH-A may represent a new pharmacological strategy to regulate VC.

Cardiac c-Kit Biology Revealed by Inducible Transgenesis [Original Research]


Rationale: Biological significance of c-Kit as a cardiac stem cell marker and role(s) of c-Kit+ cells in myocardial development or response to pathologic injury remain unresolved due to varied and discrepant findings. Alternative experimental models are required to contextualize and reconcile discordant published observations of cardiac c-Kit myocardial biology and provide meaningful insights regarding clinical relevance of c-Kit signaling for translational cell therapy. Objective: Demonstration of c-Kit myocardial biology through combined studies of both human and murine cardiac cells. Advancing understanding of c-Kit myocardial biology through creation and characterization of a novel, inducible transgenic c-Kit reporter mouse model that overcomes limitations inherent to knock-in reporter models, providing perspective to reconcile disparate viewpoints on c-Kit biology in the myocardium. Methods and Results: In vitro studies confirm a critical role for c-Kit signaling in both cardiomyocytes and cardiac stem cells. Activation of c-Kit receptor promotes cell survival and proliferation in stem cells and cardiomyocytes of either human or murine origin. For creation of the mouse model, the cloned mouse c-Kit promoter drives Histone2B-EGFP (H2BEGFP) expression in a doxycycline inducible transgenic reporter line. The combination of c-Kit transgenesis coupled to H2BEGFP readout provides sensitive, specific, inducible, and persistent tracking of c-Kit promoter activation. Tagging efficiency for EGFP+/c-Kit+ cells is similar between our transgenic versus a c-Kit knock-in mouse line, but frequency of c-Kit+ cells in cardiac tissue from the knock-in model is 55% lower than our transgenic line. The c-Kit transgenic reporter model reveals intimate association of c-Kit expression with adult myocardial biology. Both cardiac stem cells and a subpopulation of cardiomyocytes express c-Kit in uninjured adult heart, upregulating c-Kit expression in response to pathologic stress. Conclusions: c-Kit myocardial biology is more complex and varied than previously appreciated or documented, demonstrating validity in multiple points of coexisting yet heretofore seemingly irreconcilable published findings.

ER Chaperone GRP78 Protects Heart from Ischemia/Reperfusion Injury Through Akt Activation [Original Research]


Rationale: Restoration of coronary artery blood flow is the most effective means of ameliorating myocardial damage triggered by ischemic heart disease. However, coronary reperfusion elicits an increment of additional injury to the myocardium. Accumulating evidence indicates that the unfolded protein response (UPR) in cardiomyocytes is activated by ischemia/reperfusion (I/R) injury. Xbp1s, the most highly conserved branch of the UPR, is protective in response to cardiac I/R injury. GRP78, a master regulator of the UPR and an Xbp1s target, is up-regulated after I/R. However, its role in the protective response of Xbp1s during I/R remains largely undefined. Objective: To elucidate the role of GRP78 in the cardiomyocyte response to I/R using both in vitro and in vivo approaches. Methods and Results: Simulated I/R (sI/R) injury to cultured neonatal rat ventricular myocytes (NRVM) induced apoptotic cell death and strong induction of the UPR and GRP78. Over-expression of GRP78 in NRVM significantly protected myocytes from I/R-induced cell death. Furthermore, cardiomyocyte-specific over-expression of GRP78 ameliorated I/R damage to the heart in vivo. Exploration of underlying mechanisms revealed that GRP78 mitigates cellular damage by suppressing the accumulation of reactive oxygen species. We go on to show that the GR78-mediated cytoprotective response involves plasma membrane translocation of GRP78 and interaction with PI3 kinase, culminating in stimulation of Akt. This response is required as inhibition of the Akt pathway significantly blunted the anti-oxidant activity and cardioprotective effects of GRP78. Conclusions: I/R induction of GRP78 in cardiomyocytes stimulates Akt signaling and protects against oxidative stress, which together protect cells from I/R damage.

Heme Oxygenase-1 in Macrophages Drives Septic Cardiac Dysfunction via Suppressing Lysosomal Degradation of Inducible Nitric Oxide Synthase [Original Research]


Rationale: To date, our understanding of the role of heme oxygenase-1 (HO-1) in inflammatory diseases has mostly been limited to its catalytic function and the potential for its heme-related catabolic products to suppress inflammation and oxidative stress. Whether and how HO-1 in macrophages plays a role in the development of septic cardiac dysfunction has never been explored. Objective: Here, we investigated the role of macrophage-derived HO-1 in septic cardiac dysfunction. Methods and Results: Intraperitoneal injection of lipopolysaccharide (LPS) significantly activated HO-1 expression in cardiac infiltrated macrophages. Surprisingly, we found that myeloid conditional HO-1 deletion in mice evoked resistance to LPS-triggered septic cardiac dysfunction and lethality in vivo, which was accompanied by reduced cardiomyocyte apoptosis in the septic hearts and decreased peroxynitrite production and inducible nitric oxide synthase (iNOS) in the cardiac infiltrated macrophages, whereas proinflammatory cytokine production and macrophage infiltration were unaltered. We further demonstrated that HO-1 suppression abolished the LPS-induced iNOS protein rather than mRNA expression in macrophages. Moreover, we confirmed that the inhibition of HO-1 promoted iNOS degradation through a lysosomal rather than proteasomal pathway in macrophages. Suppression of the lysosomal degradation of iNOS by bafilomycin A1 drove septic cardiac dysfunction in myeloid HO-1-deficient mice. Mechanistically, we demonstrated that HO-1 interacted with iNOS at the flavin mononucleotide (FMN) domain, which further prevented iNOS conjugation with light chain 3 (LC3) and subsequent lysosomal degradation in macrophages. These effects were independent of HO-1's catabolic products: ferrous ion, carbon monoxide and bilirubin. Conclusions: Our results indicate that HO-1 in macrophages drives septic cardiac dysfunction. The mechanistic insights provide potential therapeutic targets to treat septic cardiac dysfunction.