BACKGROUND: Linkage of the 11-amino-acid transduction domain of HIV TAT to a heterologous protein allows the protein to be transduced readily into cells. METHODS AND RESULTS: In this study, we inserted the apoptosis repressor with caspase recruitment domain (ARC) or beta-galactosidase (beta-gal) cDNA into the pTAT-hemagglutinin bacterial expression vector to produce genetic in-frame TAT-ARC or TAT-beta-gal fusion proteins for use in cell culture and in Langendorff perfusion of adult rat hearts. TAT-beta-gal and TAT-ARC were conjugated with Texas Red and could be detected in >95% of cells. TAT-ARC was able to protect H9c2 cells against cell death mediated by hydrogen peroxide, as measured by protection against the loss of mitochondrial membrane potential and preservation of nuclear morphology. Isolated adult hearts were perfused with recombinant TAT-beta-gal or TAT-ARC (20 nmol/L) for 15 minutes and then subjected to 30 minutes of global no-flow ischemia, followed by 2 hours of reperfusion. Protein transduction was assessed by Western blotting of cell lysates and cytosolic and mitochondrial fractions and by fluorescence microscopy of Texas Red-conjugated TAT proteins. TAT-beta-gal and TAT-ARC readily transduced into perfused hearts and were homogeneously distributed. Infarct size was determined by 2,3,5-triphenyltetrazolium chloride staining, and creatine kinase release was measured. Transduction of TAT-ARC was cardioprotective when administered before global ischemia and reperfusion. CONCLUSIONS: Our results demonstrate that TAT-linked fusion protein transduction into the myocardium is feasible and that transduction of TAT-ARC is protective in cell culture and in the perfused heart.
BACKGROUND: Linkage of the 11-amino-acid transduction domain of HIV TAT to a heterologous protein allows the protein to be transduced readily into cells. METHODS AND RESULTS: In this study, we inserted the apoptosis repressor with caspase recruitment domain (ARC) or beta-galactosidase (beta-gal) cDNA into the pTAT-hemagglutinin bacterial expression vector to produce genetic in-frame TAT-ARC or TAT-beta-gal fusion proteins for use in cell culture and in Langendorff perfusion of adult rat hearts. TAT-beta-gal and TAT-ARC were conjugated with Texas Red and could be detected in >95% of cells. TAT-ARC was able to protect H9c2 cells against cell death mediated by hydrogen peroxide, as measured by protection against the loss of mitochondrial membrane potential and preservation of nuclear morphology. Isolated adult hearts were perfused with recombinant TAT-beta-gal or TAT-ARC (20 nmol/L) for 15 minutes and then subjected to 30 minutes of global no-flow ischemia, followed by 2 hours of reperfusion. Protein transduction was assessed by Western blotting of cell lysates and cytosolic and mitochondrial fractions and by fluorescence microscopy of Texas Red-conjugated TAT proteins. TAT-beta-gal and TAT-ARC readily transduced into perfused hearts and were homogeneously distributed. Infarct size was determined by 2,3,5-triphenyltetrazolium chloride staining, and creatine kinase release was measured. Transduction of TAT-ARC was cardioprotective when administered before global ischemia and reperfusion. CONCLUSIONS: Our results demonstrate that TAT-linked fusion protein transduction into the myocardium is feasible and that transduction of TAT-ARC is protective in cell culture and in the perfused heart.
Authors: Chengqun Huang; Smadar Yitzhaki; Cynthia N Perry; Wayne Liu; Zoltan Giricz; Robert M Mentzer; Roberta A Gottlieb Journal: J Cardiovasc Transl Res Date: 2010-05-11 Impact factor: 4.132
Authors: Dmitry Kondrikov; Fabio V Fonseca; Shawn Elms; David Fulton; Steven M Black; Edward R Block; Yunchao Su Journal: J Biol Chem Date: 2009-11-28 Impact factor: 5.157