Role of Nitric Oxide Synthases in the Infarct Size-Reducing Effect Conferred by Heat Stress in Isolated Rat Hearts

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This study provides the first demonstration of the implication of NO in the heat stress-induced delayed cardioprotection. We observed that prior heat stress significantly reduced infarct size in the isolated rat heart subjected to an ischaemia-reperfusion sequence, in accordance with previous studies (Donnelly et al, 1992; Marber et al, 1993; Joyeux et al, 1997). This myocardial ischaemic tolerance was abolished by the administration of both _L-NAME and _L-NIL prior to heat stress. The use of both inhibitors allowed the investigation of the role of the different NOS isoforms. While _L-NAME is unselective, _L-NIL is a selective inhibitor of iNOS (Moore et al., 1994). In our study, the treatment applied had to satisfy two points. First, NOS isoforms had to be inhibited at the moment of heat stress. This point has been assessed by Schwartz and co-workers (1997). They have demonstrated that following similar _L-NAME and _L-NIL treatment, corresponding NOS isoforms were inhibited since LPS-induced hypotension was corrected. By the same manner, LPS-induced increase in both urinary excretion of nitrates-nitrites and cGMP level was abolished (Schwartz et al., 1997; Lortie et al., 2000). Secondly, treatments had to be reversible since in this study we have investigated the role of NO as a trigger of the heat stress-induced cardioprotection and not as a mediator during the ischaemia-reperfusion sequence. We have previously verified this point in vivo in the rat (Lagneux et al., 2000). This study showed that 24 h after the end of treatment, mean arterial blood pressure of anaesthetised rats was within the normal range, and that the hypotension induced by bradykinin, which is triggered by NO production (Davisson et al., 1996), was comparable to that of control animals.

It seems that HS induces NO production, since Malyshev and co-workers (1995) have observed a sharp transient increase in NO generation 1 h after HS in different organs of the rat and notably the myocardium. Our study shows that the non-selective NOS inhibitor, _L-NAME, completely abolished the HS-induced protection against myocardial infarction in rat heart. Thus, NO formation seems to play an essential role in this cardioprotective phenomenon. We also demonstrate that the selective iNOS inhibitor, _L-NIL, is as effective as _L-NAME, providing the first evidence that NO produced by iNOS is involved in HS-induced cardioprotection.

 NO has also been shown to be involved in other cardioprotective phenomena. Indeed, Bolli’s group has recently presented convincing evidence that NO is a trigger of the delayed protection conferred by ischaemic preconditioning in the conscious rabbit (Bolli et al., 1998). Pretreatment with a NOS inhibitor during the initial ischaemic stimulus blocked protection (Qiu et al., 1997), and conversely a NO donor in lieu of ischaemia induced delayed cardioprotection (Takano et al., 1998; Banerjee et al., 1999). Moreover, iNOS seems to be the principal NOS isoform involved in this cardioprotective phenomenon, since a selective iNOS inhibitor completely abolished the delayed protection induced by the ischaemic preconditioning in vivo in the rabbit (Imagawa et al., 1999). This is in accordance with a study from Guo and co-workers (1999) which demonstrates in vivo in the mouse that the late phase of ischaemic preconditioning is associated with a selective up-regulation of myocardial iNOS. NO seems also trigger the delayed protective effect of monophosphoryl lipid A (MLA) in the isolated rat heart, since co-administration of NOS inhibitors and MLA abolished the preservation of ventricular function induced by MLA alone (Tosaki et al., 1998; György et al., 1999).

Our immunohistochemical analysis showed an increase in myocardial HSP 27 and 72 synthesis induced 24 hours after heat stress, which was not modified by the blockade of all NOS isoforms. It seems thus that the heat stress-induced cardioprotection does not appear to be related to induction of HSP 27 and 72 synthesis, since pretreatment with _L-NAME abolished myocardial ischemic tolerance while it had no effect on the increase in myocardial HSP levels. Several studies point to a relation between HSP 27 and 72 induction and cardioprotection. Hence, Marber and co-workers (1993) have observed that prior hyperthermia induces a high level of myocardial HSP 72 expression along with the enhanced myocardial tolerance to ischaemic injury. Moreover, the level of HSP 72 has been directly correlated to the degree of heat stress-induced cardioprotection in the rat (Hutter et al., 1994) and in the rabbit (Marber et al., 1994). Furthermore, improved functional recovery or reduced infarct size has been observed in transgenic mouse and rat hearts overexpressing HSP 72 and subjected to an ischaemia-reperfusion sequence (Marber et al., 1995; Plumier et al., 1995; Hutter et al., 1996; Suzuki et al., 1997). By the same manner, it has been shown that the overexpression of HSP 27 or 72 protects rat cardiomyocytes against ischaemic insult (Martin et al., 1997; Mestril et al., 1996).

Our study shows that protection of myocardium can be blocked independently of the level of HSP 27 and 72 induction. This finding is in agreement with previous studies showing that protein kinase C (PKC) inhibition or a₁-adrenoceptor blockade abolished the cardioprotection conferred by heat stress with no effect on myocardial HSP 72 synthesis (Joyeux et al., 1997 and 1998a). One possible explanation is that NOS inhibition, as PKC inhibition or a₁-adrenoceptor blockade, could alter the phosphorylation and/or the functional state of HSPs thus rendering them ineffective in protecting the myocardium. Further experiments are required to explore this hypothesis.

Although HSPs are widely studied as primary effectors of heat stress-induced protection, other mediators can be evoked (Joyeux et al., 1999). Hence, ATP-sensitive potassium (K_ATP) channel opening appears to mediate the heat stress-induced delayed cardioprotection in the rat (Joyeux et al., 1998b) and in the rabbit (Hoag et al., 1997; Pell et al., 1997). Moreover, some physiological effects of NO seem to be due to the K_ATP channel activation. For example, peripheral vascular vasodilatory response have been found to involve specific K_ATP channels (Champion & Kadowitz, 1997) and it seems that NO could potentiate the K_ATP channel current in isolated guinea-pig ventricular cells (Shinbo & Iijima, 1997). A recent study on rabbit ventricular myocytes suggests that NO could act directly as a mitochondrial K_ATP channel opener (Sasaki et al., 2000). Furthermore, it has been observed that K_ATP channel blockers abolish the ability of the NO donor to protect cultured myocytes against ischaemic injury (Stambaugh et al., 1999). Thus, it could be hypothesised that the NO-dependant opening of K_ATP channels mediates HS-induced cardioprotection.

In summary, this study provides the first demonstration of the implication of NO as trigger of resistance to myocardial infarction induced by heat stress in the isolated rat heart, since both _L-NAME and _L-NIL pretreatments abolished the heat stress-induced cardioprotection. Although iNOS appears to play a role in this cardioprotective phenomenon, the role of the other isoforms remains to be determined. Finally, NO appears to mediate this cardioprotection by a mechanism independent of HSP 27 and 72 induction. Further investigations are required to clarify the signal transduction pathways which co-ordinate the heat stress response and the potential role of HSP 27 and 72, and of other stress-inducible proteins, in mediating adaptative cytoprotection.