Our study demonstrates that the receptor for Tpo (c-mpl) is present in the heart and localized to the myocytes. We found that a single treatment with Tpo before ischaemia exerts an immediate protective effect against injury from myocardial ischaemia/reperfusion both in vitro
and in vivo
as manifested by a reduction in necrosis and apoptosis and an increase in the recovery of ventricular function following ischaemia. This immediate cardioprotective effect of Tpo is concentration- and dose-dependent with optimal protection occurring at 1.0 ng/mL in vitro
and 0.05 μg/kg iv in vivo
. Tpo also reduced infarct size when given after the onset of ischaemia or at reperfusion. Increased resistance to injury from myocardial ischaemia/reperfusion conferred by Tpo is mediated by JAK-2, p42/44 MAPK, and KATP
channels. Furthermore, increased resistance to injury is observed immediately after treatment with Tpo, indicating that the induction of new genes is not necessary for its cardioprotective effect to be manifested. Tpo confers its protective effect against injury from myocardial ischaemia/reperfusion without increasing platelet levels or haematocrit. Our finding that Tpo is cardioprotective in the isolated buffer-perfused heart suggests that it has direct cardioprotective properties, independent of its ability to promote platelet production. These original observations may cause a paradigm shift in how we view the mechanisms and biological effects of Tpo. In support of this idea, Tpo has been shown to act as a protective agent against doxorubicin-induced cardiac injury.18
In platelets, activation of c-mpl by Tpo induces secondary tyrosine kinase activity in cytoplasmic proteins and recruits cytoplasmic JAK-2 resulting in phosphorylation of specific tyrosine residues on c-mpl.13
We show Tpo acts via activation of JAK-2 in hearts, suggesting signalling through c-mpl is necessary to confer cardioprotection. Tpo also activates STAT3 within minutes following administration, suggesting the signalling pathway stimulated by Tpo is capable of conducting a signal to the nucleus. Further studies are needed to determine the events following Tpo-induced STAT activation and translocation to the nucleus and their role in cardioprotection.
The signalling pathway by which Tpo protects against injury to the heart caused by ischaemia is mediated in part by p42/44 MAPK and KATP
channels. Tpo increased phosphorylation of p44 but not p42 MAPK during reperfusion with cardioprotection produced by Tpo abrogated by the p42/44 MAPK inhibitor PD98059. p42/44 MAPK activation is known to reduce ischaemia/reperfusion injury in the heart and to upregulate the bcl family of anti-apoptotic genes. In support of this, we demonstrated that Tpo reduced the extent of apoptosis induced by ischaemia and reperfusion by the TUNEL assay. Activation of KATP
channels is also associated with increased resistance to injury from myocardial ischaemia/reperfusion conferred by ischaemic and pharmacological pre-conditioning. Currently there are two KATP
channels thought to be present in the cardiac myocyte, one in the sarcolemma (sarc KATP
channel) and one in the inner mitochondrial membrane (mito KATP
channel). Our data show that the sarcolemmal KATP
channel appears to act as trigger and the mitochondrial KATP
channel as an effector of Tpo-induced cardioprotection. There is evidence to suggest that opening either channel may be important in producing cardioprotection, although the bulk of evidence suggests that it is the mito KATP
channel that is responsible for mediating the protective effect of ischaemic pre-conditioning.19
Inhibition of the sarc KATP
channel prevented activation of JAK-2 and p44 MAPK, supporting the notion that the sarc KATP
channel acts as a trigger for Tpo-induced cardioprotection. The role of additional components in the signalling pathway by which the heart is protected by Tpo against injury remains unknown.
Tpo also confers protection against injury when given after the onset of ischaemia, i.e. after the onset of symptoms. In patients experiencing symptoms of a myocardial infarction, or those who are about to undergo cardiac surgery, Tpo could be administered acutely to decrease injury to the heart from ischaemia/reperfusion. Thus, Tpo may represent an important and potent agent for immediately increasing cardioprotection. Cardioprotection by ischaemic pre-conditioning is a powerful endogenous phenomenon in which brief episodes of a subtoxic ischaemic insult induce robust protection against more prolonged, lethal ischaemia. The molecular mechanisms underlying ischaemic pre-conditioning are still being elucidated and clinical application remains elusive, as it has not yet gained widespread acceptance as a treatment strategy. Pharmacological pre-conditioning against ischaemia could offer a more practical way of harnessing the molecular mechanisms responsible for increased cardioprotection. Our study shows that pharmacological pre-conditioning through Tpo is effective and represents a novel cardioprotective strategy in the setting of elective myocardial ischaemia and reperfusion as encountered during cardiac surgery and during acute myocardial infarction. Tpo has been evaluated in patients with cancer who require platelet transfusion support. Furthermore, SB-497115, an oral Tpo receptor agonist, is currently undergoing phase II and III clinical trials in adults receiving chemotherapy for advanced solid tumours and in immune thrombocytopenic purpura. AMG531, an analogue of Tpo, is currently being evaluated in a phase III study for the treatment of thrombocytopenia.
The dose of Tpo used in vivo
of 0.05 μg/kg to confer immediate cardioprotection is approximately 10 times lower than that used in humans to stimulate platelet production in cancer patients20
and to mobilize peripheral blood progenitor cells.21,22
In initial human trials of Tpo, doses of 1–5 μg/kg increased platelet production five to 10 times in healthy individuals and in those about to receive chemotherapy for malignancy. Similarly in rats, the dose of PEGylated Tpo used to reduce thrombocytopenia (100 μg/kg)23
and to ameliorate thrombocytopenia in carboplatin-treated rats (1–30 μg/kg)24
is also considerably higher than the dose used in our study. The single dose of Tpo (0.05 or 1.0 μg/kg) we used did not result in an increased platelet count or haematocrit over the 16-day follow-up period.
In summary, a single treatment with Tpo confers immediate protection against injury caused by ischaemia–reperfusion in the heart, with protection mediated by JAK-2, STAT3, p42/44 MAPK, and KATP channels. The level of protection conferred by Tpo is comparable with that achieved by ischaemic pre-conditioning and is manifest at a dose that does not result in increased platelet count or haematocrit. Further studies are warranted to define the pathways by which binding of Tpo to c-mpl confers cardioprotection. Our results suggest that Tpo directly protects the heart and may represent a novel approach for the treatment of acute myocardial infarction.