a number of peptides and antibodies binding to the ATPase and substrate binding domains of GRP78/BiP have been reported. Some of these affect the growth promoting and angiogenic action of GRP78/BiP positively or negatively but their modes of action have not been extensively investigated

Mitochondria prepared with the protease meth- Experiments on isolated cardiomyocytes Cardiomyocyte isolation. Ventricular cardiomyocytes were isolated using collagenase and protease digestion as described previously. The only modification was that the perfusion speed was 2.3 mLmin21 and there was no CaCl2 in the enzyme solution. Once digested, the ventricular tissue was separated and mechanically dispersed by shaking at 37uC. Cardiomyocytes were filtered and then the CaCl2 concentration in the buffer was gradually increased to a final concentration of 1 mM. Superfusion and stimulation of cardiomyocytes. Cardiomyocytes were superfused at a rate of 1.3 mLmin21 with HEPES buffer NVP-BHG712 solution consisting of, in mM, 137 NaCl, 5 KCl, 1.2 MgSO47H2O, 1.2 NaH2PO42H2O, 20 HEPES, 15 D-glucose anhydrous and 2 CaCl2 . The microscope stage was heated by a temperature controller so that the solution bathing the cells was 3233uC. The stimulation voltage was set at just above the threshold required for the cell to beat. Changes in cardiomyocyte morphology during metabolic inhibition. Cardiomyocytes were initially superfused with HEPES buffer solution and field stimulated at 0.2 Hz throughout the protocol. The solution superfusing the cells was switched from HEPES buffer to HEPES buffer containing 2 mM NaCN and no glucose which has been shown to mimic the effects of hypoxia. After a period of metabolic inhibition the cardiomyocyte goes into rigor. After 10 min in rigor, the perfusion was switched back to HEPES buffer for 10 min. Parameters such as time to stop beating, time to rigor and contractile recovery after reperfusion were recorded. Intracellular Ca2+ measurements. Isolated cardiomyocytes were loaded with the fluorescent dye Fura-2 AM ester at a concentration of 2 mM and gently shaken at 37uC for 15 min. Loaded cardiomyocytes were superfused with HEPES buffer, stimulated at different frequencies and the intracellular Ca2+ concentrations were measured. The ratio of 340:380 was used as an indication of i. The excitation wavelengths were set so that 20 ratio measurements were taken per second. The excitation 16041400 dichroic mirror was 415 nm and the emission passed through a 510620 nm bandpass filter. The photomultiplier was connected to Felix 32 Analysis version 1.2 software. ROS turnover in a cardiomyocyte suspension. The fluorescent dye 5–chloromethyl-29, 79-dichlorodihydrofluorescein diacetate was added to a cardiomyocyte suspension for 15 min at room temperature. After loading, the solution was centrifuged at 1006g for 1 min at room temperature and the cell pellet was resuspended in HEPES buffer or HEPES buffer without glucose but supplemented with 0.5 mM palmitate bound to 1% fatty acid free BSA, as described previously. 200 mL aliquots of the cardiomyocyte suspension were added to a 96-well plate, excited at 485 nm and fluorescence was detected at 520 nm using a fluorescent plate reader . od were used to determine the O2 consumption rates at 37uC using a High-Resolution Respirometry Oxygraph-2K, as described previously. Mitochondria were added to 2 mL of KCl buffer in the chamber and the O2 consumption was recorded. The KCl buffer contained, in mM, 125 KCl, 20 MOPS, 10 Tris, 0.01 EGTA, 2.5 KH2PO4, 2.5 MgCl2 and 2% fatty acid free BSA. The buffer was 17628524 set to pH 7.1 at 37uC with KOH. The O2 consumption was recorded in state 2 to assess basal respiration, state 3.5 thought to mimic ATP turnover in vivo and state 3 respiration to assess maximal respiration ra

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