There is a growing awareness that co-morbid conditions, particularly those that are also major risk factors for the development of ischaemic heart disease such as diabetes mellitus, hypertension and hyperlipidaemia, may modify cytoprotective responses in acute ischaemia–reperfusion (Ferdinandy et al., 2007). For example, in hearts from obese Zucker diabetic fatty and lean Goto–Kakizaki type 2 diabetic rats, ischaemic preconditioning did not afford protection against reperfusion injury (Kristiansen et al., 2004). In hyperlipidaemia, cardioprotection induced by ischaemic preconditioning and post-conditioning is lost (Giricz et al., 2006; Iliodromitis et al., 2006), and it has also been reported that the infarct size-limiting effect of ischaemic preconditioning is lost in hyperlipidaemic rabbits (Juhasz et al., 2004). Consistent with these observations, in the present study we have observed that neither exogenous NO, cGMP nor BNP protects the myocardium of hyperlipidaemic rats against ischaemia–reperfusion injury, as illustrated by the lack of decrease in infarct size and myocardial LDH efflux. As the majority of the effects of cGMP are exerted through PKG (see Burley et al., 2007; Ferdinandy et al., 2007), we investigated its myocardial expression and oxidative status. Here, we found that hyperlipidaemia did not modulate the basal expression of PKG, but the ratio of its oxidized dimeric form was more abundant in hearts of hyperlipidaemic animals. Although the underlying reason for the increased oxidative dimerization in hyperlipidaemia is still unknown, we have reported previously an increased superoxide production in hearts of hyperlipidaemic rats (Onody et al., 2003) that may be responsible for this phenomenon. In addition, increased vascular superoxide and decreased phosphorylation of a PKG target were found in hyperlipidaemic Watanabe rabbits (Oelze et al., 2000). Moreover, in hyperlipidaemic Watanabe rabbits treated with a direct NO donor (glyceryl trinitrate), there was increased vascular production of peroxynitrite in parallel with a substantially decreased PKG activity (Warnholtz et al., 2002). These results suggest that in hyperlipidaemia, the increased oxidative stress may contribute to the decreased PKG activity, and that the use of NO donors such as glyceryl trinitrate or SNAP in hyperlipidaemic animals can further increase oxidative stress, resulting in a decreased PKG activity. In accordance with this suggestion, Burgoyne et al. (2007) reported that hydrogen peroxide increases the formation of PKG dimers in vitro and in isolated hearts. The somewhat controversial findings that dimerization activated PKG (Burgoyne et al., 2007), and that hyperlipidaemia increased PKG dimerization, but compromised the activity of PKG suggest that in hyperlipidaemia, the oxidative state of the PKG is not a major determinant of its downstream effects. This hypothesis is further supported by the finding that treatments elevating intracellular cGMP concentration do not induce PKG-dependent cardioprotection in hyperlipidaemia. It has been previously shown that targeted disruption of PKG signalling leads to the loss of cGMP-dependent cardioprotection (Das et al., 2008), but this phenomenon has not been studied in connection with hyperlipidaemia. In this study, we assessed the activity of PKG indirectly by investigating the phosphorylation of troponin I, which was decreased in hyperlipidaemia. The functional relevance of decreased phosphorylation of troponin I in the attenuation of PKG-induced cardioprotection observed in hyperlipidaemia is not clear. However, it has been shown that PKG-mediated phosphorylation of troponin I could result in a reduction of responsiveness of myofilament Ca2+ in rat ventricular myocytes (Layland et al., 2002) that might be responsible for some of the cardioprotection in ischaemia/reperfusion injury (Stamm and del Nido, 2004).
The major limitation of the present study is the lack of direct evidence for decreased activity of PKG and of the effect of the treatment protocols on myocardial cGMP concentration. Unfortunately, specific substrates of PKG that permit unambiguous estimation of the kinase's activity in intact tissues are not known. As we have indicated previously (Burley et al., 2007), global estimates of cGMP concentration in tissue extracts may not reflect localization and biological activity of the cyclic nucleotide. Therefore, it is not possible to conclude firmly at this stage that hyperlipidaemia-induced attenuation of PKG activation accounts for the loss of the cardioprotective effects of the applied treatments. Clearly, further studies investigating alterations in PKG activity, guanylyl cyclase and phosphodiesterase activity and cGMP content and localization are required to provide comprehensive insights into the detrimental effects of chronic hyperlipidaemia on the cGMP–PKG system.
In conclusion, these data reveal that hyperlipidaemia is associated with loss of cardioprotective responses through interruption of a major cardioprotective signalling mechanism in the myocardium. In contrast to the normal myocardium, the hyperlipidaemic myocardium is clearly insensitive to the protective effects of NO and natriuretic peptide. Although our present study has not identified the nature of the cardioprotective signalling defect in hyperlipidaemia, we speculate that this could lie downstream of PKG activation, as we found evidence of down-regulation of basal PKG activity and troponin I phosphorylation. However, detailed exploration of the effects of hyperlipidaemia on PKG activation and its putative downstream substrates associated with cardioprotection is warranted, especially in view of the increasing recognition of this pathway as a pharmacological target in ischaemia–reperfusion injury and the prevalence of hyperlipidaemia in human patients at risk of ischaemia/reperfusion injury.