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Hence, the aim of our study was to focus on a role for Gai3 in cardiac ischemia. Here, we show for the first time that the absence of Gai2 or Gai3 have opposite effects on the severity of myocardial IR injury in knockout mice. In particular, Gai2-deficiency led to enhanced myocardial infarct size whereas the absence of Gai3 was highly protective. Whereas the first observation confirms and extends previous studies, the latter finding was unexpected. The increased infarct size visible in Gai2-deficient mice underlines a protective role of Gai2 signaling which was reported in previous studies making use of different experimental approaches. For instance, in vivo administration of PTX being considered a functional pan-Gi-inhibitor in combination with an infarct model demonstrated a cardio-protective effect of these G-proteins in rat hearts. We performed similar experiments using our acute mouse model of 60 min. of regional myocardial ischemia followed by 120 min. reperfusion in vivo. Interestingly, infarct sizes of the PTX-treated animals were even more pronounced as compared to those seen in Gai2-deficient mice, i.e. 67.064.8% vs. 56.663.7%, respectively, whereas the values for the controls in either group were almost the same. The latter data argue for a reliable procedure as indicated by similar values in both control groups. PTX modifies Gai-proteins by ADP-ribosylation of a cystein residue in the extreme C-terminus of sensitive Gai-proteins. In the afore-mentioned study in rats the degree of PTX-induced in vivo ADP-ribosylation of cardiac Gai-proteins was assessed by employing a radioactive in vitro approach. Interestingly, this analysis revealed that only a small subpopulation of Gi-proteins in the myocardial membrane was PTX-modified. This is a phenomenon we also see in our studies. Since PTX modifies Gai-proteins with different efficiency, it cannot be excluded that PTX acted in a rather isoform selective way. Moreover, different cells and tissues may exhibit variable sensitivity and kinetics towards PTX. Therefore it remains unclear which Gai-isoforms in which tissues and organs have contributed to the observed cardio-protective effect. Another study also targeted the interaction of GPCRs with cardiac Gi-proteins in a more specific approach. Mice were created with a transgene expressing an inhibitory carboxyl-terminal 63 amino acid peptide of Gai2 in cardiac tissue acting in a dominant negative fashion. These mice, when subjected to ischemia/reperfusion induced heart injury, demonstrated an exacerbated ischemic injury as compared to controls. Although the effects of the inhibitory Gai2- minigene on Gi-dependent signaling pathways were significant, the contribution of the Gai2- and Gai3-specific pathways to the observed cardio-protective effect was not investigated. In a recent paper a complementary genetic approach to study the effect of Gai2-signaling on cardiac ischemia in vitro was described. Knock-in mice were examined in which the endogenous Gai2 gene was replaced with an RGS-insensitive G184S Gai2 mutant that was SAR131675 1433953-83-3 unable to interact with RGS proteins. This resulted in an enhancement of Gai2 signaling by reversal of its negative regulation by RGS proteins thereby protecting the heart from ischemic injury. Although this study was in accordance with the concept of Gai2-dependent protection of the heart, it ignored a possible role of Gai3. Moreover, these mice showed a dramatic and complex phenotype affecting the heart and several other organs which may produce secondary effects on heart function and resulting in premature death. Similar concerns have been raised about the Gai2 knockout model that we have used in our current study. Initially, these mice have been reported to display a histopathological phenotype resembling ulcerative colitis and adenocarcinoma of the colon. However, when these mice were housed under pathogen-free conditions no obvious signs of intestinal inflammation were visible during the course of the study and they did not show the previously reported lethality phenotype. This allowed us to specifically study the roles of the two Gai-isoforms in cardiac ischemia injury in vivo. Surprisingly, mice lacking Gai3 showed a significantly reduced infarct size following IR injury. It was intriguing that the deletion of one Gai isoform results in the up regulation of the remaining ones. In fact, we detected an up regulation in heart tissue; a phenomenon we have observed previously in all tissues and cells we analyzed so far. As a consequence, the particular knock-out model exhibits two important features, i.e. the deletion of the target Gai-isoform and the enhanced expression of the remaining ones. Therefore the deleterious or protective effect might not only be the result of the loss of one isoform but also the enhanced signaling of the remaining ones. For example, the increased infarct size seen in Gai2-deficient mice could either be due to missing Gai2 or over-expressed Gai3. Conversely, the reduced infarct size in Gai3-deficient mice could either be due to the over-expressed Gai2 or absent Gai3. In that respect, it will be interesting to re-evaluate the previous studies discussed above. The main conclusions from these studies were to attach a predominant role of Gai2 in ischemia reperfusion injury. However, these studies ignored that an altered Gai2 signaling could affect Gai3 expression - as observed here and in previous studies - and signaling. This is of special importance since Gai3 has been shown to play crucial roles in both, its GDP-bound and GTP-bound form. The current view is that Gai2 and Gai3 have largely overlapping roles.