AdipoRon Attenuates Inflammation and Impairment of Cardiac Function Associated With Cardiopulmonary Bypass–Induced Systemic Inflammatory Response Syndrome

Figure 1. AdipoRon improves cardiocirculatory recovery after deep hypothermic circulatory arrest and resistance to weaning from the heart‐lung machine. A, Heart rate (HR) and mean arterial pressure (MAP) values were recorded after the start of the operation (OP) throughout the cardiopulmonary bypass (CPB) procedure at the indicated predefined time points. B, HR and MAP values, as well as (C) existence of cardiac rhythm were recorded throughout the weaning and postweaning phases at the indicated predefined time points. HR measurements were restricted by an upper detection limit of 300 beats per minute (bpm). Results are presented as mean±SEM (n=7 animals per group). Differences of HR and MAP values were analyzed statistically by comparing the experimental groups CPB‐Vehicle and CPB‐AdipoRon at the indicated time points. No statistically significant intergroup differences were observed. Values of P≥0.10 are not shown.

Figure 2. AdipoRon modulates cardiopulmonary bypass (CPB)–induced changes in blood concentrations of K+, lactate, and cytokines. Blood levels of (A) lactate and (B) K+ as well as (C) the cytokines TNF‐α (tumor necrosis factor α), IL (interleukin) 1β, IL‐6, and IL‐10 were measured in AdipoRon‐ and vehicle‐treated rats before CPB (T1), at 25°C in the cooling phase (T2), at 20°C (T3) and 35°C (T4) in the rewarming phase, and after 60 minutes of reperfusion (T5). Results for T1 to T5 are presented as mean±SEM (n=7 animals per group). Differences of blood parameter values were analyzed statistically by comparing the experimental groups CPB‐Vehicle and CPB‐AdipoRon at the indicated time points. No statistically significant intergroup differences were observed. Values of P≥0.10 are not shown.

Figure 3. AdipoRon attenuates cardiopulmonary bypass (CPB)–induced myocardial inflammation. Myocardial mRNA expression of (A) the cytokines/chemokines TNF‐α (tumor necrosis factor α), IL (interleukin) 1β, CCL2 (C‐C chemokine ligand 2), and IL‐10, as well as (B) the enzymes nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox2 subunit) and inducible nitric oxide synthase (iNOS) was measured relative to HPRT1 (hypoxanthine phosphoribosyltransferase 1) by quantitative real‐time polymerase chain reaction in AdipoRon‐ and vehicle‐treated animals after completion of the experimental CPB procedure. C, Expression of nuclear factor κB inhibitor α (IκBα) in myocardial tissue of AdipoRon‐ and vehicle‐treated rats was analyzed by immunoblot. Left panel: representative pictures of the IκBα (molecular weight: 39 kDa) and GAPDH, molecular weight: 37 kDa) band patterns. Right panel: column bars indicate quantified levels of IκBα/GAPDH expression ratios. Results are presented as mean+SEM (n=7 animals per group). Differences of marker expression levels between experimental groups were analyzed statistically by performing the indicated pairwise comparisons.

Figure 4. AdipoRon does not affect cardiopulmonary bypass (CPB)–induced myocardial injury. A, Myocardial activation of apoptosis marker caspase‐3 (CASP‐3) in AdipoRon‐ and vehicle‐treated animals was analyzed by immunoblot after completion of the experimental CPB procedure. Upper panel: representative pictures of the uncleaved (molecular weight: 35 kDa) and cleaved CASP‐3 (molecular weight: 17 kDa) band patterns. Lower panel: column bars indicate quantified levels of cleaved CASP‐3/CASP‐3 expression ratios. B, Serum levels of troponin T were measured at the end of the reperfusion phase (T5) in AdipoRon‐ and vehicle‐treated animals. Results are presented as mean+SEM (n=7 animals per group). Differences of marker expression levels between experimental groups were analyzed statistically by performing the indicated pairwise comparisons.

Figure 5. AdipoRon upregulates cardiopulmonary bypass (CPB)–induced myocardial activation of AMP‐activated protein kinase (AMPK). Phosphorylation of AMPK in myocardial tissue of AdipoRon‐ and vehicle‐treated rats was analyzed by immunoblot after completion of the experimental CPB procedure. Upper panel: representative pictures of the resulting phosphorylated AMPK (P‐AMPK; molecular weight: 62 kDa) and AMPK (molecular weight: 62 kDa) band patterns. Lower panel: column bars indicate quantified P‐AMPK/AMPK expression ratios. Results are presented as mean+SEM (n=7 animals per group). Differences of marker expression levels between experimental groups were analyzed statistically by performing the indicated pairwise comparisons.

Figure 6. AdipoRon inhibits the emergence of a systemic inflammatory response syndrome–associated inflammatory phenotype in cardiac myocytes. Cardiac myocytes were preincubated with AdipoRon (concentration range 40–100 µmol/L) or vehicle (dimethyl sulfoxide) for 2 hours before stimulation with lipopolysaccharide (1 mg/mL) for 1 hour. A, Concentration of TNF‐α (tumor necrosis factor α) in culture supernatants, (B) phosphorylation status of AMP‐activated protein kinase (AMPK; molecular weight: 62 kDa) in cell lysates, and (C) content of lactate dehydrogenase (LDH) in culture supernatants was quantified by ELISA (n=5), immunoblot (n=2), and activity assay (n=5), respectively. Cardiac myocytes were preincubated with AdipoRon (80 µmol/L) or vehicle for 2 hours before stimulation with lipopolysaccharide (1 mg/mL) for 1 hour. mRNA expression of (D) the cytokine IL (interleukin) 1β and the chemokine CCL2 (C‐C chemokine ligand 2), as well as (E) the enzymes nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox2 subunit) and inducible nitric synthase (iNOS) were measured relative to 18S ribosomal RNA (rRNA) by quantitative real‐time polymerase chain reaction (n=6–7). Results are presented…

Figure 7. AdipoRon inhibits the emergence of a systemic inflammatory response syndrome–associated inflammatory phenotype in cardiac fibroblasts. Cardiac fibroblasts were preincubated with AdipoRon (concentration range 40–100 µmol/L) or vehicle (dimethyl sulfoxide) for 2 hours before stimulation with lipopolysaccharide (1 mg/mL) for 1 hour. A, Concentration of TNF‐α (tumor necrosis factor α) in culture supernatants, (B) phosphorylation status of AMP‐activated protein kinase (AMPK; molecular weight: 62 kDa) in cell lysates and (C) content of lactate dehydrogenase (LDH) in culture supernatants was quantified by ELISA (n=5), immunoblot (n=2), and activity assay (n=5), respectively. Cardiac fibroblasts were preincubated with AdipoRon (80 µmol/L) or vehicle for 2 hours before stimulation with TNF‐α (10 ng/mL) for 1 hour. mRNA expression of (D) the chemokine CCL2 (C‐C chemokine ligand 2), (E) the enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox2 subunit), and (F) the cytokine IL (interleukin) 10 was measured relative to 18S ribosomal RNA (rRNA) by quantitative real‐time polymerase chain reaction (n=6). G, Phosphorylation of AMPK in cell lysates was analyzed by immunoblot…

Figure 8. AdipoRon inhibits the emergence of a systemic inflammatory response syndrome–associated inflammatory phenotype in vascular endothelial cells. Vascular endothelial cells were preincubated with AdipoRon (concentration range 40–100 µmol/L) or vehicle (dimethyl sulfoxide) for 2 hours before stimulation with lipopolysaccharide (1 mg/mL) for 1 hour. A, Phosphorylation status of AMP‐activated protein kinase (AMPK; molecular weight: 62 kDa) in cell lysates was quantified by immunoblot (n=2). Vascular endothelial cells were preincubated with AdipoRon (80 µmol/L) or vehicle for 2 hours before stimulation with lipopolysaccharide (1 mg/mL) for 1 hour. mRNA expression of (B) the cytokines TNF‐α (tumor necrosis factor α) and IL (interleukin) 1β and (C) the enzyme inducible nitric oxide synthase (iNOS) was measured by quantitative real‐time polymerase chain reaction (n=6). D, The content of lactate dehydrogenase (LDH) in culture supernatants was quantified by activity assay (n=6). Vascular endothelial cells were preincubated with AdipoRon (80 µmol/L) or vehicle for 2 hours before stimulation with TNF‐α (10 ng/mL) for 1 hour. mRNA expression of (E) the chemokine CCL2 (C‐C chemokine ligand 2) and…

Figure 9. AdipoRon inhibits systemic inflammatory response syndrome–associated proinflammatory TLR4 (Toll‐like receptor 4) and TNF‐α (tumor necrosis factor α) signaling in cardiac myocytes by mechanisms involving upregulation of IL (interleukin) 10 and activation of AMP‐activated protein kinase (AMPK). A, Cardiac myocytes were preincubated with AdipoRon (80 µmol/L) or vehicle (dimethyl sulfoxide) for 2 hours before stimulation with lipopolysaccharide (1 mg/mL) for 1 hour (left panel) or TNF‐α (10 ng/mL) for 15 minutes (right panel). Activation of nuclear factor κB (NF‐κB) p65 in cell lysates was measured by DNA‐binding ELISA (n=5). Cardiac myocytes were preincubated with AdipoRon (80 µmol/L) or vehicle ) for 2 hours before stimulation with lipopolysaccharide (1 mg/mL) for 1 hour. B, mRNA expression of the cytokine IL (interleukin) 10 was quantified relative to 18S ribosomal RNA (rRNA) by quantitative real‐time polymerase chain reaction (n=6–7). C, Phosphorylation of AMPK in cell lysates was analyzed by immunoblot (n=5). Left panel: representative pictures of the resulting phosphorylated AMPK (P‐AMPK; molecular weight: 62 kDa) and AMPK (molecular weight: 62 kDa) band patterns. Right panel:…

Figure 10. 5‐Aminoimidazole‐4‐carboxamide 1‐β‐D‐ribofuranoside (AICAR) inhibits the emergence of a systemic inflammatory response syndrome–associated inflammatory phenotype in cardiac myocytes. Cardiac myocytes were preincubated with AICAR (1 mmol/L) or vehicle (H2O) for 2 hours before stimulation with lipopolysaccharide (1 mg/mL) for 1 hour. A, Concentration of TNF‐α (tumor necrosis factor α) in culture supernatants and (B) content of lactate dehydrogenase (LDH) in culture supernatants was quantified by ELISA (n=7) and activity assay (n=7), respectively. Cardiac myocytes were preincubated with AICAR (1 mmol/L) or vehicle (H2O) for 2 hours before stimulation with lipopolysaccharide (1 mg/mL) for 1 hour. mRNA expression of (C) the chemokine CCL2 (C‐C chemokine ligand 2), (D) the enzyme inducible nitric oxide synthase (iNOS), and (E) the cytokine IL (interleukin) 10 was measured relative to 18S ribosomal RNA (rRNA) by quantitative real‐time polymerase chain reaction (n=6). F, Phosphorylation of AMP‐activated protein kinase (AMPK) in cell lysates was analyzed by immunoblot (n=5). Upper panel: representative pictures of the resulting phosphorylated AMPK (P‐AMPK; molecular weight: 62 kDa) and…