Calcium/calmodulin-dependent protein kinase IV limits organ damage in hepatic ischemia-reperfusion injury through induction of autophagy

Abstract

Sterile inflammatory insults, such as ischemia-reperfusion (I/R) injury, result from pathogenic factors, including damage-associated molecular pattern signaling, activation of innate immunity, and upregulation of proinflammatory cytokines. At the same time, a number of protective, or prosurvival, pathways are also activated, and the extent of end-organ damage is ultimately determined by the balance between these two systems. In liver I/R, members of the calcium/calmodulin-dependent protein kinase (CaMK) family are known to be activated, but their individual roles are largely unknown. In this study, we show that one CaMK member, CaMKIV, is protective in hepatic I/R by activating the prosurvival pathway of autophagy in hepatocytes. CaMKIV knockout mice experience significantly worse organ damage after I/R and are deficient in hepatocyte autophagic signaling. Restoration of autophagic signaling with rapamycin reduces organ damage in CaMKIV knockout mice to wild-type levels. In vitro, we show that CaMKIV activation induces autophagy in mouse hepatocytes, and that CaMKIV activation protects hepatocytes from oxidative stress-induced cell death. In conclusion, the protective autophagic signaling pathway serves to reduce organ damage following I/R and is regulated by activation of CaMKIV signaling in hepatocytes.

Fig. 1.

Calcium/calmodulin-dependent protein kinase (CaMK) IV is activated and protective during liver ischemia-reperfusion (I/R). A: Western blot of phopsho (p)-CaMKIV with liver tissues from time course I/R. B: serum alanine aminotransferase (sALT) of wild-type (WT) and CaMKIV knockout (KO) mice at 6 h of reperfusion; n = 6 mice/groups. *P < 0.05. C: hematoxylin-eosin staining of liver sections of WT and CaMKIV KO mice after 6 h of reperfusion. D: quantification of necrotic area in hematoxylin-eosin-stained liver sections. Graph represents average of 3 slides/liver and n = 6/group. E: WT and CaMKIV KO mice were subjected to I/R for various time points, and ischemic liver tissue was analyzed by Western blot for cleaved caspase-3. Figure is a representative image of similar experiments repeated three times. F: sALT levels in WT and CaMK kinase (CaMKK) KO mice after 6 h I/R. Values are means ± SE; n = 6 mice/group. *P < 0.01.

Fig. 2.

Autophagy is deficient in CaMKIV KO mice. A: Western blot for LC3B and beclin 1 on liver tissues of WT and CaMKIV KO mice after I/R. The top bar graph represents the conversion rate of LC3B I to II, and bottom graph indicates relative density of beclin 1 to β-actin. Blots were representative of three similar experiments. *P < 0.05, WT vs. KO at the same time point. B: LC3B mRNA levels after 6 h of reperfusion in WT and CaMKIV KO mice. *P < 0.05 WT vs. KO. C: transmission electron microscopy images of liver sections from WT and CaMKIV KO mice after 3 h of reperfusion. Inset shows representative images. D: autophagic vacuole (AV) volume in liver sections of WT and CaMKIV KO mice after 3 h of reperfusion. For details refer to materials and methods. Values are means ± SE. *P < 0.01.

Fig. 3.

Restoring autophagy with rapamycin reduces damage in CaMKIV KO mice. Mice were treated with 3 mg/kg rapamycin intraperitoneally 90 min before induction of I/R. A: sALT levels in WT mice treated with rapamycin after 6 h I/R; n = 4 mice/group. *P < 0.05. B: Western blot analysis was performed on liver tissue homogenate of control and rapamycin-treated mice for p-P70S6K, an indicator of mammalian target of rapamycin activity, and LC3B. C: immunofluorescent staining for LC3B in cultured hepatocytes: blue, nuclei; green, LC3B. D: sALT in WT, CaMKIV KO, and rapamycin-treated CaMKIV KO mice after 6 h of reperfusion. Values are means ± SE; n = 4 mice/group. *P < 0.05 compared with KO control. E: Western blot for LC3B in WT, CaMKIV KO, and rapamycin-treated CaMKIV KO mice after 6 h of reperfusion.

Fig. 4.

CaMKIV activates autophagy in vitro in hepatocytes. A: cultured hepatocytes were subjected to normoxia (N; 21% O₂) for 6 h (control), or hypoxia (H; 1% O₂) for various time points. Whole cell lysate protein was examined by Western blot for autophagy by LC3B. Figure is a representative image of similar experiments repeated three times. B: hepatocytes were transfected with small interfering RNA (siRNA) against CaMKIV and subjected to hypoxia for analysis of autophagy by LC3B Western blot. C: hepatocytes were subjected to hypoxia, with or without the CaMKK inhibitor STO609. D: hepatocytes were transiently transfected with control vector (pDNA 3.1+) or dominant-active mutant CaMKIV-dCT, and Western blot was performed for LC3B. E, top: hepatocytes were transfected with control vector or CaMKIV-dCT and exposed to either normoxia, 1% hypoxia for 12 h, or H₂O₂ (500 μM) for 8 h before lactate dehydrogenase assay was performed on the cell culture supernatants. Values are means ± SE. OD, outer diameter. Bottom: the FLAG blot indicates expression of plasmids in hepatocytes.

Fig. 5.

CaMKIV-cAMP response element binding protein (CREB) signaling pathway mediates autophagy. A: Western blot analysis of p-CREB in WT and CaMKIV KO mice after I/R. Values are means ± SE. *P < 0.05, WT vs. KO at the same time point. B: p-CREB in hypoxic hepatocytes transfected with CaMKIV siRNA. C: hepatocytes transfected with control vector, or CaMKIV-dCT. Images were representative of three experiments with similar results. D: Western blot for LC3B in hypoxia-stimulated hepatocytes after transfection with CaMKIV-dCT. When exposed to 1% hypoxia, cells were concomitantly treated with or without a CREB inhibitor (2.5 μM) for 1 h.