75caa1d61c55fc0fa9c7795e728e36f0 302804.pdf 3d98e05e3139cfaaf7cc7ebeb54d13344356c889 302804.pdf 3413b646488cd7f6ff4ba40e64d9f3eff47933a7a8e80124bfa5b7a20f1b7f06 302804.pdf Title: Expression and Impact of Adenosine A3 Receptors on Calcium Homeostasis in Human Right Atrium Subject: Increased adenosine A2A receptor (A2AR) expression and activation underlies a higher incidence of spontaneous calcium release in atrial fibrillation (AF). Adenosine A3 receptors (A3R) could counteract excessive A2AR activation, but their functional role in the atrium remains elusive, and we therefore aimed to address the impact of A3Rs on intracellular calcium homeostasis. For this purpose, we analyzed right atrial samples or myocytes from 53 patients without AF, using quantitative PCR, patch-clamp technique, immunofluorescent labeling or confocal calcium imaging. A3R mRNA accounted for 9% and A2AR mRNA for 32%. At baseline, A3R inhibition increased the transient inward current (ITI) frequency from 0.28 to 0.81 events/min (p < 0.05). Simultaneous stimulation of A2ARs and A3Rs increased the calcium spark frequency seven-fold (p < 0.001) and the ITI frequency from 0.14 to 0.64 events/min (p < 0.05). Subsequent A3R inhibition caused a strong additional increase in the ITI frequency (to 2.04 events/min; p < 0.01) and increased phosphorylation at s2808 1.7-fold (p < 0.001). These pharmacological treatments had no significant effects on L-type calcium current density or sarcoplasmic reticulum calcium load. In conclusion, A3Rs are expressed and blunt spontaneous calcium release at baseline and upon A2AR-stimulation in human atrial myocytes, pointing to A3R activation as a means to attenuate physiological and pathological elevations of spontaneous calcium release events. Keywords: human atrial myocyte; adenosine A3 receptor; adenosine A2A receptor; sarcoplasmic reticulum; calcium spark; transient inward current; L-type calcium current; electrophysiology Author: Carmen Tarifa, Verónica Jiménez-Sábado, Rafael Franco, José Montiel, José Guerra, Francisco Ciruela and Leif Hove-Madsen Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Thu Feb 23 09:05:37 2023 CET ModDate: Thu Feb 23 09:17:28 2023 CET Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 12 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1746358 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- ZZTQUO+VnURWPalladioL-Bold Type 1 Custom yes yes no 10 0 XAWNGI+VnURWPalladioL Type 1 Custom yes yes no 15 0 QJQUTX+URWPalladioL-Bold Type 1 Custom yes yes no 19 0 LGKLEZ+URWPalladioL-Roma Type 1 Custom yes yes no 24 0 KZVFSF+URWPalladioL-Ital Type 1 Custom yes yes no 28 0 IRWHZX+TimesNewRomanPSMT Type 1C WinAnsi yes yes no 46 0 CKUPDE+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 49 0 CAMXTK+EURM10 Type 1 Builtin yes yes no 71 0 JYUNWZ+CMSY10 Type 1 Builtin yes yes no 75 0 IMFCDL+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 85 0 IMFCDM+PalatinoLinotype CID TrueType Identity-H yes yes yes 88 0 IMFCDN+PalatinoLinotype TrueType WinAnsi yes yes no 94 0 IMFCDP+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 97 0 MPUDFL+PazoMath Type 1 Builtin yes yes no 104 0 IMFCDL+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 111 0 IMFCDN+PalatinoLinotype TrueType WinAnsi yes yes no 114 0 IMFCDP+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 117 0 IMFCDL+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 127 0 IMFCDN+PalatinoLinotype TrueType WinAnsi yes yes no 130 0 IMFCDP+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 133 0 IMFCDL+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 144 0 IMFCDN+PalatinoLinotype TrueType WinAnsi yes yes no 147 0 IMFCDP+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 150 0 IMFCDL+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 157 0 IMFCDM+PalatinoLinotype CID TrueType Identity-H yes yes yes 160 0 IMFCDN+PalatinoLinotype TrueType WinAnsi yes yes no 166 0 IMFCDP+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 169 0 IMFFKL+Cambria CID TrueType Identity-H yes yes yes 172 0 FERHKG+CMR10 Type 1 Builtin yes yes no 194 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-11-13 02:58:15 CET RepresentationInformation: 302804.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2024-11-12 10:14:46 CET Size: 1746358 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 356 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Results Destination: section.2 Children: Item: Title: Adenosine A3R Expression Destination: subsection.2.1 Item: Title: Impact of Adenosine A3Rs on Calcium Homeostasis at Baseline Destination: subsection.2.2 Item: Title: Impact of Crosstalk between A3R and A2AR on Spontaneous Calcium Release Destination: subsection.2.3 Item: Title: Impact of Crosstalk between A3R and A2AR on L-type Calcium Current Destination: subsection.2.4 Item: Title: Discussion Destination: section.3 Children: Item: Title: Main Findings Destination: subsection.3.1 Item: Title: Impact of the A3R on Calcium Homeostasis at Baseline Destination: subsection.3.2 Item: Title: Impact of Crosstalk between A3Rs and A2ARs on Calcium Homeostasis Destination: subsection.3.3 Item: Title: Study Limitations Destination: subsection.3.4 Item: Title: Clinical Implications and Conclusion Destination: subsection.3.5 Item: Title: Materials and Methods Destination: section.4 Children: Item: Title: Myocyte Isolation Destination: subsection.4.1 Item: Title: Quantitative Real-Time PCR Destination: subsection.4.2 Item: Title: Patch-Clamp Technique Destination: subsection.4.3 Item: Title: Immunofluorescent Labelling Destination: subsection.4.4 Item: Title: Confocal Imaging Destination: subsection.4.5 Item: Title: Data Analysis Destination: subsection.4.6 Item: Title: References Destination: subsection.4.6 Info: Title: Expression and Impact of Adenosine A3 Receptors on Calcium Homeostasis in Human Right Atrium Author: Carmen Tarifa, Verónica Jiménez-Sábado, Rafael Franco, José Montiel, José Guerra, Francisco Ciruela and Leif Hove-Madsen Subject: Increased adenosine A2A receptor (A2AR) expression and activation underlies a higher incidence of spontaneous calcium release in atrial fibrillation (AF). Adenosine A3 receptors (A3R) could counteract excessive A2AR activation, but their functional role in the atrium remains elusive, and we therefore aimed to address the impact of A3Rs on intracellular calcium homeostasis. For this purpose, we analyzed right atrial samples or myocytes from 53 patients without AF, using quantitative PCR, patch-clamp technique, immunofluorescent labeling or confocal calcium imaging. A3R mRNA accounted for 9% and A2AR mRNA for 32%. At baseline, A3R inhibition increased the transient inward current (ITI) frequency from 0.28 to 0.81 events/min (p < 0.05). Simultaneous stimulation of A2ARs and A3Rs increased the calcium spark frequency seven-fold (p < 0.001) and the ITI frequency from 0.14 to 0.64 events/min (p < 0.05). Subsequent A3R inhibition caused a strong additional increase in the ITI frequency (to 2.04 events/min; p < 0.01) and increased phosphorylation at s2808 1.7-fold (p < 0.001). These pharmacological treatments had no significant effects on L-type calcium current density or sarcoplasmic reticulum calcium load. In conclusion, A3Rs are expressed and blunt spontaneous calcium release at baseline and upon A2AR-stimulation in human atrial myocytes, pointing to A3R activation as a means to attenuate physiological and pathological elevations of spontaneous calcium release events. 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