Generation of a conditional mouse mutant model of gene therapy for heart failure

There is evidence that the beta adrenergic receptor may play an important role in the emergency and the progression of heart failure. Continued stimulation of the adrenergic receptor, which starts as a consequence of a compensatory response of the heart, may turn detrimental. Chronic receptor desensitization and the aberrant signaling that it can trigger is believed to be an important stimulus for the irreversible changes and the widespread apoptosis that occur in the failing heart. The aim of this project was to develop a new experimental platform suitable both for testing the "adrenergic hypothesis" of heart failure and evaluating possible therapeutic strategies based on myocardial expression of genetically engineered adrenergic receptors. To this end, we created a transgenic mouse which expresses in a tetracycline dependent way G protein-fused beta2 adrenergic receptors that are resistant to agonist-induced desensitization. We also prepared a number of permanently expressing cell lines in which the biological properties of the fused receptor can be easily studied. The in vitro characterisation of the signalling properties of the fusion protein was carried on in stably transfected HEK293cell line.The ability of the chimeric receptor in trasducing adrenergic signals and its increased sensitivity to agonists compared to wild type receptor has already been assessed as well as the desensitization properties of the modified receptor. We further investigated the recovery of chimeric receptor after internalisation. Quantitative fluorometry analysis revealed that while as much as 50% of wild type receptor can be recoverd on to the plasma membrane within 15-30 min after exposure to antagonist, only a negligible amount of chimeric receptor can be recycled in the same time frame. Blocking the neo-synthesis of proteins by cycloeximide, does not affect wild type receptor, but abolishes the modest recovery on the cell surface of chimeric receptor that is observable after 60 minutes. In the last two years four independent lineages of transgenic mice(TG) were generated in which the beta2AR -Gs is under the control of a Tet Responder Element. These "responder" TG animals have been cross-bred with the "activator" MHC-tTA transgenic strain (Jackson Labs) expressing the tetracycline transactivator under the control of the cardiac specific promoter Myosin Heavy Chain (MHC). We obtained in this way a binary inducible transgenic mouse in which the gene of the modified beta2 receptor can be turned off by the inclusion of doxycycline in drinking water. We selected by Northern blot analysis the TG strain with the higher expression of the transgene in the hearth to study the basal heart performance.We investigated the effects of the expression of the beta 2AR -Gs receptors on the cardiac performance both under basal and adrenergic stimulated conditions. Structural and functional evaluation were performed by combining standard echocardiographic imaging and hemodynamic assessment of ventricular pressure-volume relationships. Basal heart rate and dP/dtmax were, respectively, 12 and 30% higher in hearts from transgenic mice than in controls. Hearts from transgenic mice did not respond to isoproterenol. Thus cardiac expression of beta2 AR-Gs receptors modifies the intrisic beta-adrenergic activity, but also the responses to exogenous adrenergic stimulation.