PTX modifies TC-MCH 7c 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 TCS 46b 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 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.