Modulation of cardiac mechanosensitive ion channels involves superoxide, nitric oxide and peroxynitrite

In murine ventricular myocytes, activation of mechanosensitive ion channels (MSCs) includes activation of non-selective cation currents and deactivation of inwardly rectifying potassium currents. Using pharmacological inhibitors and knockout models, we analyzed signaling steps that are critical to transduce the mechanical signal (stretch) into electrophysiological events (MSC). We provide evidence for an activation of NAD(P)H oxidase and NOS3 in response to stretch putatively via the angiotensin II receptor type 1. The involvement of superoxide and nitric oxide was verified by the block of MSC using specific scavengers (tiron and PTIO, respectively). Superoxide and nitric oxide are known to combine very rapidly to form peroxynitrite. Accordingly, MSC were blocked by the peroxynitrite scavenger uric acid and could be mimicked by application of exogenous peroxynitrite. Peroxynitrite formation may activate phospholipases generating amphipaths that modulates channel function via changing the curvature of the surrounding lipid bilayer. This conclusion is supported by our findings that MSC were suppressed by inhibitors of phospholipases but could be mimicked by exogenous phospholipases or by amphipaths (oleic acid, Triton X-100).     

Cyclical mechanical stretch-induced apoptosis in myocytes from young rats but necrosis in myocytes from old rats

Mechanical load as stimulus for apoptosis and necrosis could be responsible for the loss of cardiomyocytes. Ventricular myocytes from young (3 mo) and old (14-24 mo) rats underwent cyclical mechanical stretch (CMS; 5% elongation, 1 Hz) for 24 h. Spontaneous apoptosis was in myocytes from young rats 0.33 +/- 0.12% and from old rats 1.05 +/- 0.35% [Tdt-mediated dUTP nick-end labeling (TUNEL) assay]; associated with a decrease of Bcl-2. CMS increased the apoptosis to 0.58 +/- 0.18% in myocytes from young rats. Western blot analysis showed that CMS reduced Bcl-2 and increased p53 (young rats). Bax was not changed by CMS. These were confirmed by cytochrome c release (31 +/- 13%) and by the enrichment of cytosolic nucleosomes (11 +/- 8%). CMS did not influence the apoptosis in myocytes from old rats (TUNEL assay, Bcl-2, Bax, or p53). CMS did not cause necrosis in myocytes from young rats. CMS increased the number of necrotic cells by showing the cell membrane rupture in myocytes from old rats (50 +/- 13% 5-hexadecanoylaminofluorescein-positive and 38 +/- 6% propidium iodide-positive cells) as well as by measuring the lactate dehydrogenase release. The results suggest that CMS-induced apoptosis in myocytes of young rats but necrosis in myocytes from old rats, which could be attributed to more stress sensitivity of cells from old rats.     

Comprehensive characterization of heat shock protein 27 phosphorylation in human endothelial cells stimulated by the microbial dithiole thiolutin

Thiolutin is a sulfur-based microbial compound with known activity as an angiogenesis inhibitor. Relative to previously studied angiogenesis inhibitors, thiolutin is a remarkably potent inducer of heat shock protein 27 (Hsp27) phosphorylation. This phosphorylation requires p38 kinase but is independent of increased p38 phosphorylation. To elucidate how thiolutin regulates Hsp27 phosphorylation and ultimately angiogenesis, Hsp27 was immunoprecipitated using nonphosphorylated and phospho-Ser78 specific antibodies from lysates of thiolutin treated and untreated human umbilical vein endothelial cells and analyzed by LC-MS. Separate LC-MS analyses of Lys-C, Lys-C plus trypsin, and Lys-C plus Glu-C digests provided 100% sequence coverage, including the identification of a very large 13 kDa Lys-C fragment using a special sample handling procedure (4 M guanidine HCl) prior to the LC-MS analysis to improve the large peptide recovery. The analysis revealed a novel post-translational modification of Hsp27 involving truncation of the N-terminal Met and acetylation of the penultimate Thr. Analysis of a Glu-C fragment containing two phosphorylation sites, Ser78 and Ser82, and a tryptic fragment containing the other phosphorylation site, Ser15, enabled quantitative stoichiometry of Hsp27 phosphorylation by LC-MS. The strategy revealed details of Hsp27 phosphorylation, including significant di-phosphorylation at both Ser78 and Ser82, that would be difficult to obtain by traditional approaches because oligomerization of the hydrophobic N-terminal region of the molecule prevents efficient enzymatic cleavage. The combination of Western blotting, immunoprecipation, and LC-MS provides a quantitative analysis of thiolutin-stimulated Hsp27 phosphorylation and further defines the role of Hsp27 in the antiangiogenic activities of thiolutin and related dithiolethiones.