208 The PKC Epsilon/AMPK ALPHA/ENOS Pathway is Implicated as a Mechanism by which Remote Ischaemic Conditioning Attenuates Endothelin-1 Mediated Cardiomyocyte Hypertrophy. (31st May 2014)

• Record Type: Journal Article
• Authors: Vanezis, Andrew; Thaitirarot, Chokanan; Butt, Madiha; Squire, Iain; Samani, Nilesh; Rodrigo, Glenn
• Is Part Of: Heart. Volume 100:(2014)Supplement 3
• Page Start: A114
• Page End: A114
• Publication Date: 2014-05-31
• Subjects: remote ischaemic conditioning -- hypertrophy -- nitric oxide; Heart -- Diseases -- Treatment -- Periodicals; Cardiology -- Periodicals; 616.12
• DOI: 10.1136/heartjnl-2014-306118.208
• Languages: English
• ISSNs: 1355-6037

Abstract

Abstract : Introduction: Remote ischaemic conditioning (rIC) has mainly been implicated in protection from ischemia/reperfusion injury. Using a model of endothelin-1 (ET-1) driven cardiomyocyte hypertrophy, we have previously shown that rIC attenuates ET-1 induced hypertrophy via PKCε translocation and AMPKα phosphorylation, suggesting a broader cardioprotective role for rIC. Here we investigate downstream mechanisms in this process. Methods: Blood was taken from healthy volunteers after 3 cycles of 5 min of upper arm cuff inflation/deflation and then centrifuged. The resulting rIC-serum was applied to H9c2 cardiomyoblasts in culture for 30 min. Cells were treated with ET-1 to stimulate hypertrophy. Cell area was determined using immunofluorescence after 48 h and protein levels were detected using Western blotting after 30 min and 48 h. Compound-C was used to inhibit AMP-activated protein kinase alpha (AMPKα), L-NAME to inhibit nitric oxide synthase (NOS) and 1H-[1, 2, 4]Oxadiazolo[4, 3-a]quinoxalin-1-one (ODQ) to inhibit NO-activation of soluble guanylate cyclase (sGC). Results: ET-1 increased cell surface area by ~44% from 1.41 × 10 4 ± 0.08 μm 2 in control cells to 2.03 × 10 4 ± 0.07 μm 2 in ET-1 treated cells (n = 4–8, 500–1000 cells per treatment, p < 0.001). The ET-1 induced hypertrophy was blocked by pre-treatment with rIC-serum (1.45 × 10 4 ± 0.06 μm 2, p < 0.001). L-NAME partially attenuated the blocking of hypertrophy by rIC-serum (1.79 × 10 4 ± 0.08 μm 2, p < 0.001) as did ODQ (1.69 × 10 4 ± 0.08 μm 2, p < 0.001). Furthermore, rIC-serum caused a significant increase in phosphorylated eNOS levels compared to ET-1 only treated cells after 30 min (1.76 ± 0.28 fold, p < 0.001, n = 4). This acute increase in phosphorylated eNOS was blocked by compound-C (1.21 ± 0.28 fold, p = 0.193) present during rIC and was lost 48 h after rIC-serum treatment (1.25 ± 0.45 fold, p = 0.514). Conclusions and Implications: The PKCε/AMPKα/eNOS pathway is important in rIC attenuation of ET-1 induced cardiomyoblast hypertrophy. Downstream cGMP/PKG is likely to play a prominent role in this anti-hypertrophic effect and further experiments are planned to test this hypothesis.