208 The PKC Epsilon/AMPK ALPHA/ENOS Pathway is Implicated as a Mechanism by which Remote Ischaemic Conditioning Attenuates Endothelin-1 Mediated Cardiomyocyte Hypertrophy

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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 × 104 ± 0.08 μm2 in control cells to 2.03 × 104 ± 0.07 μm2 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 × 104 ± 0.06 μm2, p < 0.001). L-NAME partially attenuated the blocking of hypertrophy by rIC-serum (1.79 × 104 ± 0.08 μm2, p < 0.001) as did ODQ (1.69 × 104 ± 0.08 μm2, 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.

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