In a series of experiments, we demonstrate that HNG offers cardioprotection in a mouse model of MI-R injury, including a decrease in infarct size by 50% and significant improvement in LV function. We demonstrate that this protective effect of HNG is dose dependent, with a maximum effect at 2 mg/kg, is independent of the route of administration (intra-peritoneally or intracardiac) and is elicitable independent of the timing of delivery (1 hr pre-treatment or at the time of reperfusion).
HN protein is endogenously expressed in the heart with the greatest levels found in cardiomyocytes. Furthermore, we show that the expression of the protein is regulated, with levels increasing in the left ventricle in response to a severe stress such as MI-R. This increase is consistent with its role in conferring robust cytoprotection in animal models of Alzheimer’s disease, serum deprivation and stroke(5
). In studies described here, we demonstrate for the first time, a potential role for HNG in offering protection against MI-R injury in vivo
in a clinically relevant model system. Considering that HNG is a potent analog of HN (1,000 times more potent than native HN), we propose that augmenting the physiological response (an increase in endogenous HN in response to MI-R) by administration of exogenous HNG leads to enhanced cardioprotection.
It is especially interesting that the beneficial effects are elicitable when given 1 hr prior to MI-R or during reperfusion; this suggests that the treatment with HNG initiates protective signaling cascades that aid survival when exposed to a severe insult (i.e., cardiac ischemia). Moreover, the demonstration that HNG affords cardioprotection even when given during reperfusion highlights clinical therapeutic potential in acute coronary ischemia and myocardial infarction. Treatment with HNG resulted in preservation of left ventricular dimensions post MI-R and resulted in significant improvement in cardiac function as evidenced by better ejection fraction, stroke volume and cardiac output. The improvements in cardiac function seen in HNG-treated animals could be directly related to the smaller size of infarct.
HN has been shown to act thru CNTFR-α/gp130/WSX-1 receptor complex in offering protection against apoptosis (11
); many downstream signaling pathways such as MAPK, PI3K and Jak-STAT could be activated in response to signaling through this complex (28
). In fact, HN has been shown to induce its cytoprotective effects through activation of Jak-Stat, Akt, JNK and p38 MAPK signaling pathways (15
). All components of this receptor and signaling complex are present in the heart and skeletal muscle and signaling through gp 130 receptors has been shown to activate MAPK and Jak-STAT in the heart (28
) and induce AMPK activation in skeletal muscle (30
In our study, significant activation of AMPK was noticed in the heart within 15 min of treatment with HNG and persisted up to 24 hr. This could be potentially important as activation of AMPK can work through multiple mechanisms which include: 1) offering a metabolic advantage through switch to an energy conservation mode by stimulating energy-yielding processes such as promoting glucose transport and accelerating glycolysis while inhibiting anabolic processes such as triglyceride and protein synthesis, and 2) activating additional cardioprotective pathways such as endothelial nitric oxide synthase (eNOS) which promotes vasodilation, decreases oxidative stress and increases peroxisome proliferator-activated receptor-γ coactivator (PGC)α, an important regulator of mitochondrial biogenesis and function (21
), and 3) decreasing apoptosis as has been shown in cardiomyocytes, endothelial cells, thymocytes, astrocytes via improved glucose utilization and inhibition of cytochrome C release from the mitochondria (31
). Indeed, we demonstrate that HNG treatment is associated with phosphorylation of eNOS at serine residue 1177 (eNOSSer1177
). Phosphorylation of this residue is a critical requirement for eNOS activation and has been reported to be mediated by AMPK during myocardial ischemia and AMPK-eNOS signaling has been shown to mediate the cardioprotective effects of metformin (22
). The increased generation of nitric oxide by eNOS could offer cardioprotection through its effects on vasodilation, inhibition of oxidative stress, platelet aggregation, leukocyte chemotaxis and apoptosis (22
). Concerns have been raised regarding the increased fatty acid oxidation with activation of this pathway, which could exacerbate acidosis during the reperfusion phase. Nevertheless, the role of AMPK in this process still seems likely beneficial as exemplified in AMPK-DN mice subjected to MI-R, which demonstrate a larger infarct size, more apoptosis, and worse cardiac function than wild type (34
Activation of STAT-3 has been shown to be a crucial step in the neuroprotective effects (15
) as well as the insulin-sensitizing effects of HN (3
). A protective role of STAT-3 activation following ischemia/reperfusion injury has been shown earlier (35
). Indeed, activation of STAT-3 promotes cardiomyocyte survival and hypertrophy as well as cardiac angiogenesis in response to various pathophysiological stimuli. Mice with cardiomyocyte-restricted deletion of STAT-3 (cardiac specific STAT3-KO) show enhanced susceptibility to injury caused by myocardial ischemia (36
), further highlighting the role for this pathway in cardiac survival post-MI-R. Activation of Akt has been shown to be important in the protective role of HN in stroke (20
). However, we demonstrate that there is no significant change in pAkt or pSTAT-3 in the heart suggesting that the cardioprotective effects of HNG in this model are not mediated through these pathways.
Cardiac myocyte death during MI-R occurs through necrosis, as well as apoptosis (37
). The activation of AMPK as well as the ability of HN to inactivate pro-apoptotic peptides such as Bax, Bid, Bim suggested that modulation of apoptosis pathway could be a mechanism through which HNG offers cardio-protection(13
). Indeed, Bax was significantly down regulated in the myocardium of HNG treated animals. The sustained rise of Bax seen in controls following MI-R was significantly attenuated in animals treated with HNG suggesting Bax down-regulation as another potential mechanism through which HNG offers better survival. This is similar to the mechanism implicated in cardio-protection via ischemic pre-conditioning and intermittent hypoxia where down regulation of Bax decreases myocardial apoptosis and therefore infarct size (39
). A role for Bax in regulating cardiac apoptosis following M-IR is illustrated in Bax−/−
mice which demonstrate significantly smaller infarct size compared to wild type, while Bax+/−
demonstrate an intermediate infarct size following MI-R(40
). Along with up-regulation of Bax, there is also up-regulation of the anti-apoptotic Bcl-2 protein in the vehicle-treated animals that underwent MI-R. In the HNG treated animals, the rise in Bcl-2 levels is also attenuated resulting in a similar Bax/Bcl-2 ratio as controls. This is not consistent with studies that showed favorable change in Bax/Bcl2 ratio as one of the mechanisms that mediates cardiac survival (41
). In addition, we observed no differences in cleaved caspase-3 expression between vehicle and HNG-treated groups. A role of HNG in attenuating apoptosis is also demonstrated in vitro
where cardiomyocytes exposed to daunorubicin, an anthracycline chemotherapeutic drug known to induce apoptosis, show significantly decreased apoptosis and improved survival in the presence of HNG.
The lack of a significant difference in AMPK, eNOS between controls and HNG treated animals post MI-R is intriguing especially considering the significant activation of AMPK and eNOS in the heart in acute signaling experiments. It is possible that the activation of these signaling pathways in response to a severe stressor such as MI-R overwhelms the difference attributable to HNG. It is also plausible that we may have missed a response because of the time points we chose or potential differences in these and other members of the apoptosis pathway may become apparent if this data could be interpreted in the context of the size of the infarct. It also raises the point that in addition to the proposed intermediate pathways, other mechanisms may be involved in the cardio-protection offered by humanin. Future studies in Bax−/− and AMPK DN mice should shed light on the relative role of each pathway in offering cardio-protection in response to HNG.
In summary, these studies demonstrate a novel role for HNG in offering cardioprotection in a mouse model of MI-R. Our data suggest that this protection may be mediated through activation of AMPK-eNOS signaling as well as alteration in pro-apoptotic factors. The significant decrease in infarct size accompanied by improvement in cardiac function following a single treatment with HNG demonstrates clinical utility in the treatment for acute myocardial ischemia. Considering that diabetes mellitus increases the risk for cardiovascular disease and exacerbates the severity of acute myocardial infarction and our recent work showing the salubrious role of HN and analogs on insulin sensitivity and diabetes, future studies may reveal a role for HNG in the treatment of ischemic heart disease in the setting of diabetes.