Adverse Effects on β‐Adrenergic Receptor Coupling: Ischemic Postconditioning Failed to Preserve Long‐Term Cardiac Function

Figures

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  Figure 1. Study designs and treatment protocols. A, Stimulation of β‐ARs at the end of a 2‐hour reperfusion in the Langendorff model. B, Stimulation of β‐ARs 7 days after infarction in the working heart model. C, Cardiomyocytes were isolated from IPC or IPoC hearts and cultured for 24 hours. Cell shortening was determined before and after stimulation with ISO using a cell‐edge detection system. D, Perfusion of IPoC hearts 7 days after an infarction. After 10 minutes’ infusion with paroxetine, the CO was measured continuously for 5 minutes with additional stimulation using ISO. β‐AR indicates β‐adrenergic receptor; CO, cardiac output; IPC, ischemic preconditioning; IPoC, ischemic postconditioning; ISO, isoproterenol.

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  Figure 2. Proof of cardiac protection for IPC and IPoC algorithms in a Langendorff model of myocardial ischemia and reperfusion. A, Five minutes after the onset of reperfusion, the LDH level was determined in the perfusate as a surrogate parameter for tissue damage. B, After 15 minutes of reperfusion reactive oxygen species (ROS) were determined in the left ventricular myocardium using DHE staining. C, Activation of caspase‐3 was determined by immunohistochemistry after 120 minutes of reperfusion. D, The structural preservation of the myocardium was qualitatively assessed using connexin 43 staining. E, Functional recovery was expressed as a percentage of the preischemic LVDP. Data are means±SD of n=6 to 8 hearts. *P≤0.05 vs Nx, # P≤0.05 vs IR. DHE indicates dihydroethidium; IPC, ischemic preconditioning; IPoC, ischemic postconditioning; IR, ischemia/reperfusion; LDH, lactate dehydrogenase; LVDP, left ventricular developed pressure; Nx, normoxia.

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  Figure 3. Influence of IR, IPC, and IPoC on β‐adrenergic signaling 2 hours after starting reperfusion. A, Representative immunoblots and quantification by densitometric analysis of (B) β1‐AR dimers (D, MW 95 kDa) and (C) β1‐AR monomers (M, MW 50 kDa). Quantification of the (D) phosphorylated (p‐GRK2) and (E) total GRK2 protein. F, The p‐GRK2 to β1‐AR (D) ratio was calculated for each animal, and group means were expressed as fold change of Nx. Data are means±SD of n=6 hearts. *P≤0.05 vs Nx, # P≤0.05 vs IPC. The significance of PKC for GRK2 phosphorylation. G, ISO‐induced inotropic response of IPoC hearts perfused with the PKC inhibitor CEC. H, The effect of PKC inhibition on GRK2 phosphorylation in IPoC hearts. Immunoblot bands were normalized to GAPDH and expressed as a percentage of Nx. Data are means±SD of n=6 hearts. *P≤0.05 vs IPoC. CEC indicates chelerythrine chloride; D, dimer; GAPDH, Glyceraldehyde 3‐phosphate dehydrogenase; GRK2, G protein‐coupled receptor kinase 2; IPC, ischemic preconditioning; IPoC, ischemic postconditioning; IR, ischemia/reperfusion; ISO, isoproterenol; M, monomer; Nx, normoxic; p‐GRK2, phosphorylated GRK2; PKC, protein kinase C; β1‐AR, β1‐adrenergic receptor.

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  Figure 4. Influence of IR, IPC, and IPoC on infarct size and cardiac function in vivo. A, Plasma levels of cardiac troponin I (TnI) were determined 1 hour after the onset of reperfusion as an early index of myocardial infarct size. B, Left ventricular systolic function, represented as fractional shortening (%), was determined 7 days following the reperfusion. Data are means±SD of n=6 to 10 animals. *P≤0.05 vs Sham, # P≤0.05 vs IR. IPC indicates ischemic preconditioning; IPoC, ischemic postconditioning; IR, ischemia/reperfusion.

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  Figure 5. Expression pattern of β‐ARs. A, Representative immunoblots and densitometric analysis of (B) the β1‐AR, (C) the β2‐AR and (D) the β3‐AR. Remodeling and intracellular coupling of cardiac β‐ARs. E, Representative blots and densitometric data of the (F) phosphorylated and (G) total GRK2 protein. H, The pGRK2 to β1‐AR ratio was calculated for each animal, and group means were expressed as fold change of Sham. I, The mRNA expression of GRK2 was analyzed in left ventricular tissue and normalized to HPRT. J, β‐Arrestin1/2, also critically involved in β‐AR coupling, was exclusively upregulated in IPoC hearts. K, Na+/K+ ATPase was determined as a surrogate marker for the membrane density. Immunoblot bands were normalized to GAPDH and expressed as a percentage of Sham. Data are means±SD of n=6 hearts. *P≤0.05 vs Sham, # P≤0.05 vs IR. AR indicates adrenergic receptor; arr, arrestin; GAPDH, Glyceraldehyde 3‐phosphate dehydrogenase; GRK2, G protein‐coupled receptor kinase 2; HPRT, hypoxanthine‐guanine phosphoribosyltransferase; IPC, ischemic preconditioning; IPoC, ischemic postconditioning; IR, ischemia/reperfusion; p‐GRK2, phosphorylated GRK2.

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  Figure 6. Remodeling of β‐ARs as the main cause for the loss of the inotropic reserve in IPoC hearts. A, Responsivity of cardiomyocytes isolated from IPC or IPoC hearts to ISO was analyzed using a cell‐edge detection system. B, Induction of GRK2 varied depending on whether IPC or IPoC was applied. Data are means±SD of n=4 hearts/216 cells. *P≤0.05 vs IPC. IPoC‐induced loss of the β‐adrenergic functional reserve. C, IPoC hearts 7 days after an infarction were perfused using the working heart technique. After 10 minutes’ infusion with paroxetine, the CO was measured continuously for 5 minutes with additional stimulation using ISO. Data are means±SD of n=4 hearts. *P≤0.05 vs ISO. AR indicates adrenergic receptor; CO, cardiac output; GAPDH, Glyceraldehyde 3‐phosphate dehydrogenase; GRK2, G protein‐coupled receptor kinase 2; IPC, ischemic preconditioning; IPoC, ischemic postconditioning; ISO, isoprenaline; Parox, paroxetine; p‐GRK2, phosphorylated GRK2.

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  Figure 7. Regulatory mechanisms for GRK2. A, Representative immunoblots and densitometric data of the (B) phosphorylated and (C) total MDM2 protein. D, Western blot image and (E) analysis of PI3 kinase expression. Immunoblot bands were normalized to GAPDH and expressed as a percentage of Sham. Data are means±SD of n=6 hearts. *P≤0.05 vs Sham, # P≤0.05 vs IR. GAPDH indicates Glyceraldehyde 3‐phosphate dehydrogenase; GRK2, G protein–coupled receptor kinase 2; IPC, ischemic preconditioning; IPoC, ischemic postconditioning; IR, ischemia/reperfusion; MDM2, mouse double minute 2 homolog/E3 ubiquitin‐protein ligase Mdm2; p‐GRK2, phosphorylated GRK2; PI3K, phosphoinositide 3‐kinase; p‐MDM2, phosphorylated MDM2.

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  Figure 8. IPoC‐induced desensitization of β‐ARs. In the short term, rapid and transient activation of the GRK2 could reduce the reperfusion injury and protect the myocardium against hypercontractions. In the long term, however, stabilization of GRK2 and its phosphorylated fraction leads to a desensitization or decoupling of β‐ARs with a resultant loss of the positive inotropic functional reserve. AR indicates adrenergic receptor; GRK2, G protein‐coupled receptor kinase 2; MDM2, mouse double minute 2 homolog/E3 ubiquitin‐protein ligase Mdm2; PI3, phosphoinositide 3‐kinase; PKC, protein kinase C.