Effect of remote ischemic preconditioning on phosphorylated protein signaling in children undergoing tetralogy of Fallot repair: a randomized controlled trial

Abstract

Background: Our previous randomized controlled trial demonstrated cardiorespiratory protection by remote ischemic preconditioning (RIPC) in children before cardiac surgery. However, the impact of RIPC on myocardial prosurvival intracellular signaling remains unknown in cyanosis. RIPC may augment phosphorylated protein signaling in myocardium and circulating leukocytes during tetralogy of Fallot (ToF) repair.

Methods and results: Children (n=40) undergoing ToF repair were double-blind randomized to RIPC (n=11 boys, 9 girls) or control (sham RIPC: n=9 boys, 11 girls). Blood samples were taken before, immediately after, and 24 hours after cardiopulmonary bypass. Resected right ventricular outflow tract muscle and leukocytes were processed for protein expression and mitochondrial respiration. There was no difference in age (7.1 ± 3.4 versus 7.1 ± 3.4 months), weight (7.7 ± 1.8 versus 7.5 ± 1.9 kg), or bypass or aortic cross-clamp times between the groups (control versus RIPC, mean±SD). No differences were seen between the groups for an increase in the ratio of phosphorylated to total protein for protein kinase B, p38 mitogen activated protein kinase, signal transducer and activator of transcription 3, glycogen synthase kinase 3β, heat shock protein 27, Connexin43, or markers associated with promotion of necrosis (serum cardiac troponin I), apoptosis (Bax, Bcl-2), and autophagy (Parkin, Beclin-1, LC3B). A high proportion of total proteins were in phosphorylated form in control and RIPC myocardium. In leukocytes, mitochondrial respiration and assessed protein levels did not differ between groups.

Conclusions: In patients with cyanotic heart disease, a high proportion of proteins are in phosphorylated form. RIPC does not further enhance phosphorylated protein signaling in myocardium or circulating leukocytes in children undergoing ToF repair.

Clinical trial registration: URL: (http://www.anzctr.org.au/trial_view.aspx?id=335613. Unique identifier: Australian New Zealand Clinical Trials Registry number ACTRN12610000496011.

Keywords: cyanosis; heart; mitochondria; pediatric surgery; remote ischemic preconditioning; tetralogy of Fallot.

Figure 1.

RVOT tissue protein expression of prosurvival signaling pathway kinases obtained during CPB from patients subjected to Control (Sham) or RIPC treatments. Western blots of protein bands were densitometrically detected using antibodies targeting phosphorylated and total Akt (A), p38MAPK (B), GSK3β (C), and HSP27 (D). Data (relative densitometric units) were normalized to loading controls detected by antibodies to either GAPDH or β‐tubulin and are presented as the ratio of phosphorylated to total protein expression (mean±SD). RVOT indicates right ventricular outflow tract; CPB, cardiopulmonary bypass; RIPC, remote ischemic preconditioning; p38MAPK, p38 mitogen activated protein kinase; GSK3β, glycogen synthase kinase 3β; HSP27, heat shock protein 27.

Figure 2.

RVOT tissue protein expression of STAT3 (A) and Connexin43 (B), as described in the Methods section. Data (relative densitometric units) are normalized to loading controls and presented as the ratio of phosphorylated to total protein expression (mean±SD). RVOT indicates right ventricular outflow tract; STAT3, signal transducer and activator of transcription 3; RIPC, remote ischemic preconditioning.

Figure 3.

RVOT tissue protein expression of the autophagy proteins Parkin (A), light chain 3B (LC3B) (B), Beclin 1 (C), and the proapoptotic protein Bax and antiapoptotic Bcl‐2, expressed as the ratio Bax:Bcl‐2, (D). Data are presented as relative (densitometric) units or as a ratio (mean±SD). RVOT indicates right ventricular outflow tract; RIPC, remote ischemic preconditioning.