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).
- remote ischaemic conditioning
- nitric oxide
Statistics from Altmetric.com
If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.