Abstract 349: Signal Transducer and Activator of Transcription 3 (Stat3) in the Matrix of Cardiomyocyte Mitochondria

STAT3 (signal transducer and activator of transcription 3) transduces signals from the plasma membrane to the nucleus and is central for the cardioprotection by ischemic pre- and postconditioning. However, preliminary data suggest that STAT3 is also located in mitochondria. The aim of the present study was to confirm the presence of STAT3 in cardiomyocyte mitochondria, to elucidate its submitochondrial localization and to identify interacting proteins and the import mechanism of STAT3. STAT3 was detected by Western blot analysis in mitochondrial preparations from rat left ventricle (LV), which were negative for marker proteins of other subcellular compartments (sarcolemma, sarcoplasmic reticulum, nucleus, and cytosol). This finding was confirmed by confocal laser scan microscopy on mouse LV mitochondria (n=4). In contrast, no STAT3 was detected in mitochondria isolated from the LV of mice with a cardiomyocyte-specific deletion of STAT3 (n=3). Immunoprecipitation and confocal laser scan microscopy showed that, apart from total STAT3, also Ser727- and Tyr705-phosphorylated STAT3 was present in rat mitochondria. The analysis of subfractionated mitochondria by Western blot displayed STAT3 predominantly in the fraction negative for marker proteins of the outer membrane (voltage dependent anion channel), inner membrane (ATP synthase alpha), and the intermembrane space (cytochrome c), but positive for marker proteins of the matrix such as cyclophilin D (n=4). Immunoprecipitation of STAT3 from right ventricular and mitochondrial proteins showed a co-precipitation of connexin 43 (Cx43), which is present both at the sarcolemma and at the inner mitochondrial membrane and is also involved in cardioprotection, but not with GSK3β, MnSOD or cytochrome c. Furthermore, STAT3 co-precipitated with Tom20 (translocase of the outer membrane 20), which regulates the import of proteins into the mitochondria. Taken together, total and phosphorylated STAT3 are present in the matrix of cardiomyocyte mitochondria, STAT3 is possibly imported via a Tom20-dependent pathway, and STAT3 interacts with mitochondrial Cx43, and is thus possibly involved in Cx43-mediated cardioprotection.

Nuclear halo from Bradysia hygida (Diptera:Sciaridae) salivary gland polytene cells

A protocol for recovered nuclear halos from insect polytene nuclei after the extraction of the nuclear proteins using LIS detergent is reported in this work. Analysis was carried out using fluorescence and confocal laser scan microscopy. The extraction of nuclear halos was possible only with nuclei-fraction isolation in hypotonic buffer without spermine and spermidine. The recovered nuclear halos from Bradysia hygida salivary gland polytene nuclei, contributed greatly to the study of the structure and function of these special organelles.