In the normal heart, the atrioventricular node (AVN) is part of the sole pathway between the atria and ventricles and is responsible for transmitting and coordinating atrial and ventricular contractions. The AVN electrically connects the atria and ventricles of the heart and is part of the electrical conduction system. Conduction within this system is highly regulated by the autonomic nervous system. The complex neurochemical anatomy of this region has been studied extensively in mice and other small animals but not in humans. The goal of this study was to provide detailed neurochemical characterization of parasympathetic (cholinergic) and sympathetic (noradrenergic) innervation of the human AVN, which is the lead component of the conducting system. Using immunohistochemistry, we have investigated the innervation of the AVN region in samples collected from human hearts that were rejected for transplantation. Tissues were fixed in 4% paraformaldehyde, cryoprotected, and sectioned frozen at 30um thickness. Sections through the AVN were cut in the horizontal plane and collected in representative sets on charged slides. Each set of slides was stained for a different phenotypic marker using the VECTOR Elite ABC kits and ImmmPACT VIP Chromogen. To aid in locating the AVN, we used anti-Connexin-43(Cx-43). Unlike surrounding myocardium, nodal tissue lacks significant Cx-43. Cholinergic nerves were stained with anti-vesicular acetylcholine transporter (VAChT).Noradrenergic nerves were stained with anti-tyrosine hydroxylase (TH). We evaluated tissue from two patients and obtained similar results. The AVN received prominent input from cholinergic and noradrenergic nerves, with cholinergic being dominant. The AVN displayed a greater density of parasympathetic and sympathetic innervation compared to surrounding regions. Sympathetic innervation is what causes an increase in conduction speed in the AVN, while parasympathetic innervation is what causes a decrease in conduction speed. Based on previous pharmacological evidence and observation of innervation in other species, the AVN is a highly controlled area for cardiac regulation. Our research implies that parasympathetic innervation is more highly regulated in the AVN and that parasympathetic innervation could play a larger role in cardiac conduction than sympathetic innervation.

Long noncoding RNAs (lncRNAs) are emerging as novel regulators of macrophage biology and related inflammatory cardiovascular diseases. However, studies focused on lncRNAs in human macrophage subtypes, particularly human-specific lncRNAs that are not conserved in rodents, are limited. Through deep RNA-seq of human monocyte-derived macrophages, we identified SIMALR (suppressor of inflammatory macrophage apoptosis lincRNA), a human macrophage-specific long intergenic noncoding RNA (lincRNA), to be highly induced in the nucleus of LPS/IFNγ-stimulated macrophages. Treatment of LPS/IFNγ-stimulated THP1 human macrophages with SIMALR antisense oligonucleotides induced apoptosis of inflammatory macrophages, as shown by increased Annexin V+ macrophages, and increased protein expression of cleaved PARP, caspase 9, and caspase 3. Differential expression analysis of RNA-seq data from SIMALR knockdown versus control in human macrophages revealed Netrin 1 (NTN1), a known regulator of macrophage apoptosis, to be one of the top downregulated genes. NTN1 knockdown in LPS/IFNγ-stimulated THP1 macrophages induced apoptosis. This apoptotic phenotype was attenuated by treating LPS/IFNγ-stimulated macrophages with recombinant human NTN1 after SIMALR knockdown. Furthermore, NTN1 promoter-luciferase reporter activity was increased in HEK293T cells treated with lentiviral overexpression of SIMALR. NTN1 promoter activity is known to require HIF1α and RNA immunoprecipitation (RIP) showed that SIMALR binds HIF1α, suggesting that SIMALR may modulate HIF1α binding at the NTN1 promoter to regulate apoptosis of macrophages. In human translational studies, SIMALR was found to be upregulated in macrophages in unstable human atherosclerotic plaques, suggesting a possible mechanistic link between inflammation and cardiovascular diseases. In addition to SIMALR, through deep RNA-seq of human monocyte-derived macrophages, we identified RP11-184M15.1, a human macrophage-specific lincRNA, to be highly induced in the cytoplasm of IL-4-stimulated macrophage. Preliminary data showed that treatment of IL-4-stimulated THP1 human macrophages with RP11-184M15.1 small interfering RNA (siRNA) repressed apoptosis of resolving macrophages, as shown by decreased Annexin V+ macrophages, and reduced protein expression of cleaved PARP. Biotinylated RP11-184M15.1 pulldown coupled with mass spectrometry indicated an interaction between RP11-184M15.1 and zinc finger RNA-binding protein (ZFR). RIP corroborated the proposed interaction between RP11-184M15.1 and ZFR. RNAInter revealed mRNAs predicted to interact with ZFR, and some of those genes (e.g., ALYREF, CCNYL1) were also differentially expressed in RNA-seq data of control versus RP11-184M15.1 knockdown in IL-4-stimulated THP1 macrophages. qPCR validated that ALYREF and CCNYL1 expression are reduced with RP11-184M15.1 knockdown. In contrast, with ZFR siRNA, ALYREF and CCNYL1 mRNA expressions were elevated. Thus, a hypothesis to be further tested is that RP11-184M15.1 interacts with ZFR to regulate mRNA stability in IL-4-stimulated macrophages. Nuclear RNA export factor 1 (NXF1) was also validated by RIP to interact with RP11-184M15.1. NXF1 is a known interacting partner of ALYREF in the transcription-export (TREX) complex. With RP11-184M15.1 knockdown, the protein level of ALYREF decreased, and Ingenuity Pathway Analysis (IPA) of RNA-seq data of control versus RP11-184M15.1 knockdown revealed that THO complex subunit 5 homolog (THOC5), another component of the TREX complex, may be an upstream regulator. In addition, past studies have revealed that ALYREF and NXF1 are involved in nuclear export of inflammatory mRNAs and proinflammatory macrophage phenotype, respectively. With RP11-184M15.1 knockdown, there was decreased expression of inflammatory macrophage-associated genes. It may be possible that RP11-184M15.1 functions in mRNA export, along with NXF1 and ALYREF. In human translational studies, RP11-184M15.1 was found to be upregulated in macrophages in unstable human atherosclerotic plaques. Further work is needed to better understand the functions and molecular mechanism of RP11-184M15.1. In summary, we found that SIMALR may interact with HIF1α to regulate macrophage apoptosis via NTN1. Our preliminary work also revealed that RP11-184M15.1 may regulate apoptosis, mRNA stability and mRNA export in anti-inflammatory macrophages. Both lincRNAs may be upregulated in unstable human atherosclerotic plaques. By studying SIMALR and RP11-184M15.1, we were able to illustrate the importance of interrogating the functions of human-specific lincRNAs despite the lack of rodent models, and demonstrated roles in macrophage inflammation that may be relevant to human cardiovascular disease.

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Introduction: Takotsubo cardiomyopathy or stress cardiomyopathy is a syndrome of transient left ventricular (LV) dysfunction mimicking myocardial infarction, but lacking obstruction of coronary artery disease (CAD) or acute plaque rupture. A characteristic differentiation from CAD is that regional motional abnormality extends beyond a territory perfused with a single epicardial coronary artery. Clinically, it is characterized by apical ballooning of the LV due to due to depression of mid and apical segments, with hyperkinesis of cardiac basal walls. Women are affected more than men, predominantly in the postmenopausal age. Case Report: A 54-year-old Caucasian female with a history of COPD, hypertension, uncontrolled diabetes mellitus, hyperlipidemia, depression and ongoing tobacco use presented with complaints of worsening shortness of breath two days prior to admission. She denied chest pain, worsened cough, palpitations, nausea or vomiting. On examination, she was in distress and anxious, with labored breathing. Upon examining the chest, decreased air entry was present in both lung fields with bibasilar wheezing. Initial lab tests showed mild respiratory acidosis, with pH of 7.24, pCO2 of 47.4 and pO2 of 65. Troponins on the day of admission was Soon after admission, she started complaining of severe right neck pain. Repeat EKG revealed localized lateral J point, anteroseptal q waves and 4mm ST-segment elevation in leads V3 and V4 reciprocal changes and without chest pain. Repeat troponins were slightly elevated to 0.42 ng/ml and CK-MB was elevated to 20.2 ng/ml. A transthoracic echocardiogram showed regional abnormalities in left ventricle with the apex, mid to distal septum and the anterior part of septum was akinetic. Discussion: Takotsubo cardiomyopathy presents in 1 to 2 percent of troponin-positive acute coronary syndrome (ACS) with various clinical manifestations and various outcomes. Some patients have favorable outcomes based on their clinical performance and extent of cardiac muscle involvement. As in the case we presented, this syndrome can be entirely idiopathic, without a definitive underlying cause. Supportive management while hospitalized and early identification of complications improve the prognosis.

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