Effect of α7 nicotinic acetylcholine receptor activation on cardiac fibroblasts: A mechanism underlying RV fibrosis associated with cigarette smoke exposure

INTRODUCTION: Right ventricular dysfunction is associated with numerous smoking-related illnesses including chronic obstructive pulmonary disease (COPD) where it is present even in absence of pulmonary hypertension. It is unknown if exposure to cigarette smoke has direct effects on RV function and cardiac fibroblast proliferation or collagen synthesis. In this study, we evaluated cardiac function and fibrosis in mice exposed to cigarette smoke (CS) and determined mechanisms of smoke-induced changes in cardiac fibroblast signaling and fibrosis. METHODS: AKR mice were exposed to cigarette smoke for six weeks followed by echocardiography and evaluation of cardiac hypertrophy, collagen content, and pulmonary muscularization. Proliferation and collagen content were evaluated in primary isolated rat cardiac fibroblasts (CF) exposed to cigarette smoke extract (CSE) or nicotine. Markers of cell proliferation, fibrosis, and proliferative signaling were determined by immunoblot or Sircol collagen assay. RESULTS: Mice exposed to CS had significantly decreased RV function as determined by TAPSE. There were no changes in LV parameters. RV collagen content was significantly elevated but there was no change in RV hypertrophy or pulmonary vascular muscularization. CSE directly increased cardiac fibroblast proliferation and collagen content in CF. Nicotine alone reproduced these effects. CSE and nicotine-induced fibroblast proliferation and collagen content were mediated through α7 nicotinic acetylcholine receptors and were dependent on PKC-α, PKC-δ, and reduced p38-MAPK phosphorylation. CONCLUSION: CS and nicotine have direct effects on cardiac fibroblasts to induce proliferation and fibrosis which may negatively affect right heart function

CloVR: A virtual machine for automated and portable sequence analysis from the desktop using cloud computing

Next-generation sequencing technologies have decentralized sequence acquisition, increasing the demand for new bioinformatics tools that are easy to use, portable across multiple platforms, and scalable for high-throughput applications. Cloud computing platforms provide on-demand access to computing infrastructure over the Internet and can be used in combination with custom built virtual machines to distribute pre-packaged with pre-configured software. We describe the Cloud Virtual Resource, CloVR, a new desktop application for push-button automated sequence analysis that can utilize cloud computing resources. CloVR is implemented as a single portable virtual machine (VM) that provides several automated analysis pipelines for microbial genomics, including 16S, whole genome and metagenome sequence analysis. The CloVR VM runs on a personal computer, utilizes local computer resources and requires minimal installation, addressing key challenges in deploying bioinformatics workflows. In addition CloVR supports use of remote cloud computing resources to improve performance for large-scale sequence processing. In a case study, we demonstrate the use of CloVR to automatically process next-generation sequencing data on multiple cloud computing platforms. The CloVR VM and associated architecture lowers the barrier of entry for utilizing complex analysis protocols on both local single- and multi-core computers and cloud systems for high throughput data processing.https://doi.org/10.1186/1471-2105-12-35

Consensus guidelines for the use and interpretation of angiogenesis assays

The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference

RBM25/LUC7L3 function in Cardiac Sodium Channel Splicing Regulation of Human Heart Failure

Alternative splicing is a post-transcriptional mechanism that can substantially change the pattern of gene expression. Up to 95% of human genes have multi-exon alternative spliced forms, suggesting that alternative splicing is one of the most significant components of the functional complexity of the human genome. Nevertheless, alternative splicing regulation has received comparatively little attention in the study of cardiac diseases. When investigating SCN5A splicing abnormalities in heart failure, we found 47 of 181 known splicing regulators were upregulated in HF when compared to controls, which indicate that splicing regulation may play a key role in heart failure. Our results shows that AngII and hypoxia, signals common to HF, result in increased LUC7L3 and RBM25 splicing regulators, increased binding of RBM25 to SCN5A mRNA, increased SCN5A splice variant abundances, decreased full-length SCN5A mRNA and protein, and decreased Na+ current. These observations could shed light on a mechanism whereby cardiac function and arrhythmic risk are associated and allow for refined predictions of which patients may be at highest arrhythmic risk or suffer from Na+ channel blocking anti-arrhythmic drug complications

MIR448 antagomir reduces arrhythmic risk after myocardial infarction by upregulating the cardiac sodium channel

Cardiac ischemia is associated with arrhythmias; however, effective therapies are currently limited. The cardiac voltage-gated sodium channel α subunit (SCN5A), encoding the Nav1.5 current, plays a key role in the cardiac electrical conduction and arrhythmic risk. Here, we show that hypoxia reduces Nav1.5 through effects on a miR, miR-448. miR-448 expression is increased in ischemic cardiomyopathy. miR-448 has a conserved binding site in 3′-UTR of SCN5A. miR-448 binding to this site suppressed SCN5A expression and sodium currents. Hypoxia-induced HIF-1α and NF-κB were major transcriptional regulators for MIR448. Moreover, hypoxia relieved MIR448 transcriptional suppression by RE1 silencing transcription factor. Therefore, miR-448 inhibition reduced arrhythmic risk after myocardial infarction. Here, we show that ischemia drove miR-448 expression, reduced Nav1.5 current, and increased arrhythmic risk. Arrhythmic risk was improved by preventing Nav1.5 downregulation, suggesting a new approach to antiarrhythmic therapy

The characteristics of APs recorded in Wt and RYR2^-/- ventricular-like CM.

<p>The characteristics of APs recorded in Wt and RYR2^-/- ventricular-like CM.</p

The effect of mitochondrial Ca²⁺ flux on spontaneously beating in hiPSC-derived ventricular-like CMs.

<p><b>A</b>) Typical cytoplasmic Ca²⁺ transients in a hiPSC-derived CM. <b>B</b>) Inhibition effects of mitochondrial Ca²⁺ influx blocker, 1 μmol/L Ru360 (in pipette solution), on the beating rate of cytoplasmic Ca²⁺ transients. The spontaneous beating rate is reduced by 21.2 ± 4.3% (n = 10). <b>C</b>) The mitochondrial NCX (NCLX) blocker, 3 μmol/L CGP-37157, abolishes the automaticity of CMs (n = 11). <b>D</b>) Cytoplasmic Ca²⁺ transients are abolished by mitochondrial depolarization by 300 nmol/L FCCP and 1 μmol/L oligomycin (n = 9).</p

Cytoplasmic Ca²⁺ transients in RYR2^-/- ventricular-like ESC-derived CMs.

<p><b>A</b>) Typical cytoplasmic Ca²⁺ transients in a RYR2^-/- CM. <b>B</b>) Cytoplasmic Ca²⁺ transients are eliminated by the NCX blocker, 5 μmol/L KB-R7943 and the Na⁺ channel blocker, 30 μmol/L TTX (n = 4 for each group). <b>C</b>) Blocking IP₃Rs with 2 μmol/L 2-APB reduces but does not eliminate the cytoplasmic Ca²⁺ transients (n = 8). Data were normalized with those before blocker was used. *<i>p</i><0.05, compared with those before antagonist applied. <b>D</b>) Cytoplasmic Ca²⁺ transients are abolished by mitochondrial depolarization with 300 nmol/L FCCP and 1 μmol/L oligomycin (n = 5). Inhibiting mitochondrial Ca²⁺ release with 3 μmol/L CGP-37157 also terminates cytoplasmic Ca²⁺ transients (n = 6).</p

Regulation of abnormal automaticity by mitochondrial Ca²⁺ flux in ischemic mouse ventricular CMs.

<p>MCU blocker, 1 μmol/L Ru360 (in the pipette solution), reduces the ischemia-induced spontaneous beating rate of cytoplasmic Ca²⁺ transients in mouse ventricular CMs (*<i>p</i><0.05, compared with control groups. n = 11).</p