No evidence for activated autophagy in left ventricular myocardium at early reperfusion with protection by remote ischemic preconditioning in patients undergoing coronary artery bypass grafting

Abstract

Objective: Remote ischemic preconditioning (RIPC) by repeated brief limb ischemia/reperfusion reduces myocardial injury in patients undergoing coronary artery bypass grafting (CABG). Activation of signal transducer and activator of transcription 5 (STAT5) in left ventricular (LV) myocardium at early reperfusion is associated with such protection. Autophagy, i.e., removal of dysfunctional cellular components through lysosomes, has been proposed as one mechanism of cardioprotection. Therefore, we analyzed whether or not the protection by RIPC is associated with activated autophagy.

Methods: CABG patients were randomized to undergo RIPC (3×5 min blood pressure cuff inflation/5 min deflation) or placebo (cuff deflated) before skin incision (n = 10/10). Transmural myocardial biopsies were taken from the LV before cardioplegia (baseline) and at early (5-10 min) reperfusion. RIPC-induced protection was reflected by decreased serum troponin I concentration area under the curve (194±17 versus 709±129 ng/ml × 72 h, p = 0.002). Western blotting for beclin-1-phosphorylation and protein expression of autophagy-related gene 5-12 (ATG5-12) complex, light chain 3 (LC3), parkin, and p62 was performed. STAT3-, STAT5- and extracellular signal-regulated protein kinase 1/2 (ERK1/2)-phosphorylation was used as positive control to confirm signal activation by ischemia/reperfusion.

Results: Signals of all analyzed autophagy proteins did not differ between baseline and early reperfusion and not between RIPC and placebo. STAT5-phosphorylation was greater at early reperfusion only with RIPC (2.2-fold, p = 0.02). STAT3- and ERK1/2-phosphorylation were greater at early reperfusion with placebo and RIPC (≥2.7-fold versus baseline, p≤0.05).

Conclusion: Protection through RIPC in patients undergoing CABG surgery does not appear to be associated with enhanced autophagy in LV myocardium at early reperfusion.

Figure 1. Serum concentration of troponin I.

Serum concentration of troponin I before (preop) and over 72 hours after coronary artery bypass grafting (CABG) in patients undergoing remote ischemic preconditioning (RIPC) or not (placebo). Decreased troponin I concentrations confirmed protection by RIPC. Insert: area under the curve (AUC) for serum troponin I concentrations over 72 h.

Figure 2. Expression of autophagy-related gene 5–12 complex (ATG5-12).

Original Western blots of protein content of ATG5-12 in myocardial biopsies obtained at baseline (BL) before initiation of cardiopulmonary bypass and at 5–10 min reperfusion (R10) from patients undergoing remote ischemic preconditioning (RIPC) or not (placebo). Immunoreactivity of ATG5-12 was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Ratios are presented in bar graphs.

Figure 3. Phosphorylation and expression of beclin-1.

Original Western blots of protein content of phosphorylated (p) and total beclin-1 in myocardial biopsies obtained at baseline (BL) before initiation of cardiopulmonary bypass and at 5–10 min reperfusion (R10) from patients undergoing remote ischemic preconditioning (RIPC) or not (placebo). Immunoreactivity of pbeclin-1 was normalized to the respective total protein. Immunoreactivity of total protein was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Ratios are presented in bar graphs.

Figure 4. Expression of light chain 3 (LC3).

Original Western blots of protein content of LC3 in myocardial biopsies obtained at baseline (BL) before initiation of cardiopulmonary bypass and at 5–10 min reperfusion (R10) from patients undergoing remote ischemic preconditioning (RIPC) or not (placebo). LC3-II (lower band) was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and LC3-I (upper band), respectively. Ratios are presented in bar graphs.

Figure 5. Expression of parkin.

Original Western blots of protein content of parkin in myocardial biopsies obtained at baseline (BL) before initiation of cardiopulmonary bypass and at 5–10 min reperfusion (R10) from patients undergoing remote ischemic preconditioning (RIPC) or not (placebo). Immunoreactivity of parkin was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Ratios are presented in bar graphs.

Figure 6. Expression of p62.

Original Western blots of protein content of p62 in myocardial biopsies obtained at baseline (BL) before initiation of cardiopulmonary bypass and at 5–10 min reperfusion (R10) from patients undergoing remote ischemic preconditioning (RIPC) or not (placebo). Immunoreactivity of p62 was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Ratios are presented in bar graphs.

Figure 7. Activation of signal transducer and activator of transcription 5 (STAT5) by ischemia/reperfusion.

Original Western blots of phosphorylated (p) and total STAT5 content in myocardial biopsies obtained at baseline (BL) before initiation of cardiopulmonary bypass and at 5–10 min reperfusion (R10) from patients undergoing remote ischemic preconditioning (RIPC) or not (placebo). Immunoreactivitiy of pSTAT5 was normalized to the respective total protein. Ratios are presented in bar graphs.

Figure 8. Activation of signal transducer and activator of transcription 3 (STAT3) by ischemia/reperfusion.

Original Western blots of phosphorylated (p) and total STAT3 content in myocardial biopsies obtained at baseline (BL) before initiation of cardiopulmonary bypass and at 5–10 min reperfusion (R10) from patients undergoing remote ischemic preconditioning (RIPC) or not (placebo). Immunoreactivitiy of pSTAT3 was normalized to the respective total protein. Ratios are presented in bar graphs.

Figure 9. Activation of signal transducer and activator of extracellular signal-regulated protein kinase 1/2 (ERK1/2) by ischemia/reperfusion.

Original Western blots of phosphorylated (p) and total ERK1/2 content in myocardial biopsies obtained at baseline (BL) before initiation of cardiopulmonary bypass and at 5–10 min reperfusion (R10) from patients undergoing remote ischemic preconditioning (RIPC) or not (placebo). Immunoreactivitiy of pERK1/2 was normalized to the respective total protein. Ratios are presented in bar graphs.