2007). The aortic pressure, atrial pressure, and electrocardiogram (ECG) were recorded using the PowerLab 8/SP data acquisition system and Chart software version 5 (AD Instruments Ltd, Oxford, U.K.). Atrial effective period (AERP) and conduction velocity (CV) were measured by the recording of bipolar electrograms from the epicardial surface of the left atrium using a 5 × 5 electrode array as described previously (Kim et al. 2011, 2012). The inducibility of atrial tachyarrhythmias was examined by 5 sec of burst pacing at cycle lengths of <10 msec (Kim et al. 2011, 2012). At the end of experiments, hearts Temozolomide supplier were removed from the perfusion apparatus, dissected, and wet heart weight, left atrial weight, and left ventricular weight recorded. Left atria were embedded in Tissue Tek OCT® (Sakura Finetek Europe B.V., Alphen aan den Rijn, Netherlands) and snap frozen in preparation for histological analysis. Histology and immunocytochemistry Cryo‐sections (10 μm) of left atrial tissue were prepared as described previously (Jones et al. 2004). Frozen sections were fixed using 4% paraformaldehyde for 2 min and washed with PBS. Sections were dehydrated using an increasing ethanol series (70%, 85%, and 95% at −20°C). For the histological analysis of fibrosis, fixed sections were stained with Masson’s trichrome, digitized images obtained, and the blue pixel content measured relative to the total tissue area using Adobe Photoshop CS2. A total connexin‐43 antibody (MAB 3068, Chemicon International Inc., Temecula, CA) and a phospho‐connexin‐43 (connexin‐43P) antibody (3511S, Cell Signaling Technology, Beverly, MA) specific to phosphorylation at serine 368 (Solan et al. 2003) were used. AlexaFluor®488‐conjugated anti‐mouse IgG1 secondary (“”type””:””entrez-nucleotide””,””attrs””:””text””:””A21121″”,””term_id””:””512319″”,””term_text””:””A21121″”A21121, Invitrogen, Paisley, U.K.) were applied at 1 μg/mL. Using methods that we have reported previously (Kim et al. 2011), confocal images were collected using a LSM‐510 laser scanning microscope using the same settings (i.e., objective lens, laser power, photomultiplier gain, and pixel size) for each image (Carl Zeiss Ltd, Cambridge, U.K.). Images of equal area (95.5 × 88.9 μm) were selected at random from sections (a single image per section) from control WKY (WKY, n = 8 sections from four rats), control SHR (SHR control, n = 8–9 sections from four rats), candesartan‐treated SHR (SHR can, n = 8–9 sections from three rats), and hydralazine‐treated SHR (SHR hydra, n = 8 sections from three rats) tissue sections and connexin‐43‐specific fluorescent staining quantified using ImageJ (v1.48, NIH). Statistics Data are presented as mean ± SEM. Student’s t‐test and one‐way ANOVA with Bonferroni multiple comparisons post hoc tests were performed, as appropriate, using Prism 5.03 (GraphPad Software Inc, San Diego, CA). P < 0.