Analysis of ryanodine receptor (RyR) and phosphorylated ryanodine receptor (p-RyR) levels in SR and AF pigs.

<p><b>a</b> Total RyR protein did not differ between animal groups. <b>b, c</b> PKA-phosphorylated RyR at Ser2808 was reduced, while CaMKII-phosphorylated RyR at Ser2814 was not affected by AF. <b>d</b> Relative p-RyR_Ser2808 but not p-RyR_Ser2814 content was diminished in atrial tissue obtained from n = 5 animals per group. Original Western blots and mean normalized optical density data are provided (*<i>P</i> < 0.05).</p

Expression of L-type Ca²⁺ channels (LTCC) and Na+-Ca²⁺ exchanger (NCX) 1 in AF animals and in SR controls.

<p><b>a, b</b> Regulatory LTCC α₂ subunit was significantly downregulated in AF animals, whereas the pore forming α_1c subunit was not affected. <b>c</b> AF lead to significant upregulation of the NCX1 transporter. Representative Western blots and mean (± SEM) optical density data normalized to GAPDH are displayed (n = 5 animals per group). *<i>P</i> < 0.05; ***<i>P</i> < 0.001.</p

AF-induced remodeling of Ca²⁺-calmodulin-dependent protein kinase II (CaMKII) δ and protein kinase A (PKA).

<p>Representative Western blots and mean optical density values normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are displayed relative to sinus rhythm (SR) controls. <b>a,</b> Absolute expression levels of the reference protein, GAPDH, were note affected by AF. <b>b, c</b> Normalized protein expression of total CaMKIIδ and phosphorylated CaMKIIδ (Thr286) in AF animals compared to SR. <b>d</b> Relative CaMKIIδ autophosphorylation at Thr286 was not different between animal groups. <b>e, f</b> Protein levels of catalytic PKA Cα/β subunits (PKA_C) and of phosphorylated regulatory RIIα subunits (PKA_RII) in AF and SR animals. Mean values obtained from five animals per group are provided ± SEM; *<i>P</i> < 0.05.</p

Remodeling of Ca²⁺ handling proteins during atrial fibrillation and heart failure.

<p>Changes in protein expression are indicated by arrows (orange color). Solid black arrows indicate transport directions of Ca²⁺ or Na⁺ ions, respectively. Decreased levels of Ca²⁺-calmodulin-dependent protein kinase (CaMK) IIδ and protein kinase A (PKA) causes hypophosphorylation of phospholamban (PLN) and ryanodine receptor (RyR) 2, leading to reduced Ca²⁺ uptake into the sarcoplasmic reticulum through sarcoplasmic reticulum Ca²⁺-ATPase (Serca) 2a. Increased intracellular Ca²⁺ levels and upregulation of Na⁺-Ca²⁺ exchanger (NCX) 1 expression enhance electrogenic Na⁺-Ca²⁺ exchange. This mechanism is attenuated by reduced L-type calcium channel (LTCC) expression that limits systolic Ca²⁺ influx.</p

Alterations of sarcoplasmic reticulum Ca²⁺-ATPase (Serca) 2a and its regulator phospholamban (PLN) during AF.

<p>Total PLN (<b>a</b>) and PLN phosphorylated by CaMKII (Thr17; <b>b</b>) or PKA (Ser16; <b>c</b>) was downregulated in AF animals. <b>d</b> Phosphorylation levels relative to total PLN. <b>e</b> Serca2a protein analysis revealed a trend towards reduced protein expression associated with AF. Mean ± SEM optical density data normalized to GAPDH obtained from n = 5 Western blots per group are displayed. *<i>P</i> < 0.05; **<i>P</i> < 0.01.</p

Clinical findings in AF pigs subjected to atrial tachypacing.

<p><b>a</b> Representative ECG recordings at baseline and after atrial fibrillation (AF) induction by atrial burst pacing show sinus rhythm on day 0 and AF on day 7. <b>b</b> Mean heart rates assessed by daily ECG measurements from AF animals (n = 5). <b>c-f</b> Atrial effective refractory periods (AERP; <b>c</b>, <b>d</b>) and corrected sinus node recovery time (SNRT; <b>e</b>, <b>f</b>) at baseline and prior to euthanization at day 7 in AF pigs (n = 5). <b>g, h</b> Echocardiographic analysis of left ventricular ejection fraction (EF) and left atrial (LA) diameter. Data are given as mean ± SEM; *<i>P</i> < 0.05; **<i>P</i> < 0.01.</p

The small molecule hERG inhibitor terazosin protects GB cells from doxazosin-induced apoptosis.

<p>(A, B) Terazosin treatment did not impair LNT-229 cell viability (A) and caused less suppression of hERG protein compared to doxazosin (B). Combined drug exposure attenuated doxazosin-associated hERG protein suppression (B). Cell death quantified using the XTT assay was inhibited in concentration-dependent fashion (C). Mean (± SEM) data are presented (n = 4 to 6 assays; *p<0.05; **p<0.01).</p

Pro-apoptotic effects of doxazosin in human glioblastoma cells.

<p>(A–D) Fluorescence microphotographs corresponding to terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling (TUNEL) assays. Increased green nuclear fluorescence reflects DNA degradation and apoptosis of LNT-229 glioblastoma cells treated with doxazosin for 24 h, compared to solvent controls. (E, F) Microscopic findings after treatment of LNT-229 cells with vehicle (E) or 30 µM doxazosin (F) further illustrate doxazosin-associated cell death. Scale bars, 100 µm. (G) Concentration-response relationship obtained from 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide salt (XTT) cell viability assays reveals an EC₅₀ value of 35.3 µM in LNT-229 cells. (H) Time course of doxazosin-associated apoptosis. Cell death was determined using the XTT-based assay (n = 3 to 6 assays). Data are represented as mean ± SEM.</p

Quantification of apoptosis and cell cycle arrest in LNT-229 glioblastoma cells.

<p>(A–C) Representative flow cytometric analyses indicate pro-apoptotic effects of doxazosin. Cells were treated with doxazosin for 24 h and analyzed by fluorescent annexin V labeling to detect phosphatidylserine (PS) externalization as early and specific apoptotic feature. Early apoptotic cells are located in the lower right (LR) quadrant. Propidium iodide co-staining served to indicate late apoptotic cells (upper right quadrant). (D–F) Cell cycle arrest associated with doxazosin treatment (24 h), demonstrated by flow cytometry. Propidium iodide fluorescence intensity correlates with the amount of cellular DNA content. Decreased diploid DNA content reflects a reduced number of cells in G2 and M phases after doxazosin application (E, F) compared to solvent controls (D), whereas the fraction of haploid cells in the G0/G1 phase was elevated. Results and data from representative experiments are shown.</p

Caspase 9 activation <i>en route</i> to apoptosis.

<p>(A, B) Increased levels of cleaved caspase 9 and unaffected total caspase 9 protein expression were detected in LNT-229 cells treated with doxazosin. Representative Western blots and mean (± SEM) optical densities normalized to doxazosin-free conditions are presented for cells exposed to increasing concentrations of doxazosin (n = 3 independent assays; **p<0.01; GAPDH, glyceraldehyde-3-phosphate dehydrogenase).</p