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208 The PKC Epsilon/AMPK ALPHA/ENOS Pathway is Implicated as a Mechanism by which Remote Ischaemic Conditioning Attenuates Endothelin-1 Mediated Cardiomyocyte Hypertrophy
  1. Andrew Vanezis,
  2. Chokanan Thaitirarot,
  3. Madiha Butt,
  4. Iain Squire,
  5. Nilesh Samani,
  6. Glenn Rodrigo
  1. University of Leicester

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.

  • remote ischaemic conditioning
  • hypertrophy
  • nitric oxide

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