In this study we analyzed the AMPK isoform expression and activity in the heart during the development and in heart failure. We also compared the mouse heart to the human heart to determine whether the mouse heart is a good model for the human AMPK in the failing and non-failing hearts. There are several major findings of the study 1) AMPK expression is higher in the fetal heart than the adult, and part of the fetal expression profile of the AMPK isoforms reappears in the failing heart. 2) The protein expression and the activity of AMPK both increased significantly in the failing heart. 3) Changes in the specific isoforms are different in human failing heart compared to the mouse.
The AMPK cascade functions to maintain cellular energy homeostasis and regulate cell growth and survival [2
]. Deletion of both isoforms of the catalytic subunit of AMPK is embryonic lethal suggesting a critical role of this pathway during the development. Here we found that the expression of AMPK was greater in the fetal heart, and in particular, the expressions of the α1 and γ2 isoforms were 3-fold higher in the E15 mouse hearts and the expression fell significantly in the neonatal hearts. It is interesting that the trough of expression for most of the isoforms is around the time of birth. However, the tissue collection process in the fetal and neonatal mice affected the Thr172
phosphorylation hence the AMPK activity we were unable to assess whether this pattern of expression was paralleled by the changes in the AMPK activity. Previous study by Makinde et al. observed an increase of AMPK activity in newborn rabbit hearts from day 1 to day 7 [21
]. This may reflect an increase in AMPK phosphorylation during that period.
Since the α1 and γ2 were minor isoforms for these subunits in the normal adult hearts, our results show a significant isoform switch for these subunits during the development. We also observed an upregulation of the γ2 isoform expression in the failing heart suggesting a reversal of the γ subunit isoforms toward the fetal pattern. The isoform-specific function of the γ subunit in the heart is poorly understood. However, it has been shown that human mutations of γ2-AMPK cause aberrant activation of AMPK activity resulting in glycogen storage, cardiac hypertrophy and arrhythmia. Thus, it is worth investigating whether the upregulation of the γ2 isoform in the failing heart, observed both in mice and humans, is mechanistically involved in the development of pathological hypertrophy and heart failure.
Changes in the isoform-specific AMPK activity have been shown in rodent hearts during ischemia and pressure overload [7
]. Here we find that the isoform distribution of AMPK in the human hearts is not identical to the mouse hearts and the changes in their expression in the human failing hearts only partially overlap with that of mouse failing hearts. Although the upregulation of γ2 isoform were observed in both mouse and human failing hearts, the isoform expression for the α and β subunits were changed in distinct directions. The mouse failing heart switched towards α2 and β2 subunits while the human failing hearts expressed more α1 and β1 subunits. We expect that these changes will not only increase the total amount of AMPK heterotrimers but also alter the subunit isoform composition of the kinase, i.e. more AMPK complexes in the failing mouse heart contain α2 and β2 isoforms compared to the controls while in the failing human heart, AMPK heterotrimers containing α1 and β1 isoforms are increased. By measuring the isoform-specific AMPK activity in samples after full phosphorylation of α-AMPK, we found that in mouse heart α2 complexes accounted for the majority (65%) of the AMPK activity with α1 complexes accounted for the remaining 35%. In contrast, the human heart has higher α1-AMPK activity and it further increases in the failing heart where α1- and α2-AMPK have equal contribution to the total activity. The functional significance of the altered composition of the α and β subunit isoforms of the AMPK in the heart is unknown. However, it has been shown in non-cardiac cell types that AMPK α2-containing complexes are found in both the cytoplasm and the nucleus while AMPK α1-containing complexes are predominantly localized in the cytoplasm and have been also observed at the plasma membrane in some cell types such as carotid body cells [23
]. Leptin treatment of C2C12 cells caused the translocation of AMPK containing α2 and β2 isoform into the nucleus after Thr172 phosphorylation of α2 whereas AMPK containing α2 and β1 isoform was anchored in the cytoplasm through the myristoylation [26
]. Therefore, future work delineating isoform specific functions of AMPK in the heart will be essential for better understanding of the signaling cascade in heart failure.
AMPK is activated in animal models of pathological hypertrophy and heart failure [7
] although one study showed inactivation of AMPK by oxidative stress in the heart of spontaneous hypertensive rats [28
]. In the present study we found a similar increase of AMPK activity in human failing hearts as that in mice and rats. Furthermore, our study suggests that increased phosphorylation of α-AMPK is primarily responsible for the greater AMPK activity in the failing heart, which may be linked to changes in AMPK subunit composition rather than increased expression of AMPK. In mouse hearts, although the expression level of AMPK has changed in the failing heart, we detected only a modest increase of activity in the failing hearts when the tissue homogenates was treated with LKB1 to match the α-AMPK phosphorylation in failing and non-failing samples. In the human failing hearts, however, both enhanced phosphorylation and increased AMPK protein expression contribute significantly to the upregulation of AMPK activity. Taken together, these observations raise the possibility that some of the regulatory mechanisms of the AMPK cascade in the heart are species specific. It is also worth noting that compared to the mouse study in which the tissue harvest process is well controlled the AMPK activities in human heart samples could be affected by the procedures applied to each individual patients. Nevertheless, the mouse and human results are directionally consistent. Since human heart samples are quite limited for research mouse heart will continue to be used for studies of AMPK in heart failure. Our study suggests that the results of rodent studies should be careful interpreted taking into consideration of possibly different regulatory mechanisms of AMPK cascade including isoform-specific function of AMPK across the species.
There are some other limitations of this study. Since we used cardiac tissue for all the experiments, we were unable to distinguish the changes of AMPK expression and activity originated from cardiac myocytes versus that from the non-myocyte, i.e. endothelial, fibroblasts or smooth muscle cells. By blotting for α-sarcomeric actin, a protein expressed in cardiac myocyte but not in smooth muscle or non-muscle cells, we did confirm that the cardiac myocyte components in the protein extracts of failing and non-failing heart sample were comparable. Thus, our results are not due to the over representation of non-myocytes in the sample preparation. There are other limitations that should be taken into consideration, for example, we did not separate the LV from the RV in the mouse study, and the human study results might be influenced by the pharmacological treatments, the etiology of heart failure, and the age and gender of the patients. Due to the small number of patients included here we were unable to stratify for these factors, which warranted the future clinical study with a greater number of patients.
In summary, the protein level and the isoform distribution of AMPK in the heart change significantly during normal development as well as in heart failure. Our study has identified similarities and differences in the AMPK expression profile in response to chronic stresses during the development of heart failure in mice and humans. These results provide important basis for targeting AMPK for therapeutics, and furthermore, warrant investigation of isoform-specific function of AMPK in the heart.