Preconditioning protocols that protect the heart from ischemic injury may aid in the development of new therapies. However, the temporal window of cardioprotection is limited to a few days after the preconditioning stimulus. Here we report a sustained cardioprotected phenotype in mice expressing a tetracycline transactivator (tTA) transcription factor under the control of the alpha-myosin heavy chain (alphaMHC) promoter. alphaMHC-tTA mice were originally designed for tetracycline-regulated gene expression in the heart (Tet system). However, we found that after 45 min of global ischemia at 37 degrees C, left ventricular developed pressure (LVDP) of Langendorff-perfused alphaMHC-tTA mouse hearts rapidly recovered in 5 min to 60% of initial levels, whereas LVDP of wild-type (WT) littermates recovered to only 10% of the initial level. Improved postischemic recovery of function for alphaMHC-tTA hearts was associated with a 50% decrease of infarct size and a significantly smaller release of lactate dehydrogenase to the coronary effluent. Improved postischemic recovery was not attributable to differences in coronary flow that was similar for WT- and alphaMHC-tTA hearts during recovery. Moreover, improved postischemic recovery of alphaMHC-tTA hearts was not abolished by inhibitors of classical cardioprotective effectors (mitochondrial ATP-sensitive K+ channels, PKC, or adenosine receptors), suggesting a novel mechanism. Finally, the tetracycline analog doxycycline, which inhibits binding of tTA to DNA, did not abolish improved recovery for alphaMHC-tTA hearts. The sustained cardioprotected phenotype of alphaMHC-tTA hearts may have implications for developing new therapies to minimize cardiac ischemic injury. Furthermore, investigations of cardioprotection using the Tet system may be aberrantly influenced by sustained preconditioning induced by cardiac transgenesis with tTA.