sq-23377 and Myocardial-Ischemia

To determine the role of calcineurin and Src tyrosine kinase in the regulation of inducible nitric oxide synthase (iNOS) expression and protection in cardiomyocytes.. iNOS expression was studied in isolated neonatal rat ventricular myocyte cultures in response to bacterial lipopolysaccharide (LPS) or following transfection with constitutively active calcineurin or Src and in hearts isolated from wild-type or calcineruin Abeta knockout mice. Cell injury in response to simulated ischemia-reperfusion was studied following overexpression of active calcineurin. Regulation of the iNOS gene promoter by calcineurin was studied using promoter-luciferase reporter and chromatin immunoprecipitation assays.. Overexpression of constitutively active Src co-operated with [Ca2+]c elevation to induce iNOS expression, and LPS-induced iNOS expression was abrogated by pharmacological inhibition of calcineurin or tyrosine kinase. LPS also induced tyrosine kinase-dependent but calcineurin-independent phosphorylation of Src Tyr418. LPS induced myocardial iNOS expression in wild-type but not calcineurin Abeta knockout mice. Overexpression of constitutively active calcinuerin in isolated cardiomyocytes caused dephosphorylation and nuclear accumulation of the c1 isoform of nuclear factor of activated T-cells (NFATc1), induced strong iNOS expression, and induced NOS-dependent protection against simulated ischemia-reperfusion prior to cardiomyocyte hypertrophy. Co-transfection of a mouse iNOS promoter-luciferase reporter in combination with active calcineurin and wild-type or dominant negative Src confirmed that constitutive activation of calcineurin was sufficient for transactivation. Chromatin immunoprecipitation confirmed calcineurin-dependent in vivo binding of NFATc1 to consensus sites within the iNOS promoter.. These results support a cardioprotective role for calcineurin mediated by NFAT-dependent induction of iNOS expression and co-operativity between calcineurin and Src.

Topics: Animals; Calcineurin; Calcium; Cells, Cultured; Immunoprecipitation; Ionomycin; Ionophores; Male; Mice; Mice, Knockout; Microscopy, Fluorescence; Myocardial Ischemia; Myocytes, Cardiac; NFATC Transcription Factors; Nitric Oxide Synthase Type II; Rats; Signal Transduction; src-Family Kinases; Transfection; Up-Regulation

Ischemia produces striking electrophysiological abnormalities in blood-perfused hearts that may be caused, in part, by effects of ischemia on intracellular calcium. To test this hypothesis, intracellular Ca2+ concentration ([Ca2+]i) transients were recorded from the epicardial surface of blood- and saline-perfused rabbit hearts using the long-wavelength indicator Fura Red. Calcium transients were much larger than the movement artifact, representing up to 29% of the total signal. Switching the perfusate from saline to blood did not affect the time course of the transients or the apparent level of [Ca2+]i. Compartmentation of Fura Red fluorescence was estimated by exposure to Mn2+. The results were cytosol 60 +/- 3%, organelles 12 +/- 2%, and autofluorescence plus partly deesterified Fura Red 29 +/- 4%. [Ca2+]i transients were calibrated in situ by perfusion of the extracellular space with high-Ca2+ and Ca(2+)-free EGTA solutions. Peak systolic [Ca2+]i was 663 +/- 74 nM, and end-diastolic [Ca2+]i was 279 +/- 59 nm. Ischemia was produced by interruption of aortic perfusion for 2.5 min during pacing (150 beats/min). Ischemia produced broadening of the [Ca2+]i transient, along with beat-to-beat alternations in the peak systolic and end-diastolic level of [Ca2+]i (calcium transient alternans). [Ca2+]i transient alternans occurred in 82% of blood-perfused hearts vs. 43% of saline-perfused hearts. The discrepancy between large and small transients (mean alternans ratio) was larger in the blood-perfused hearts (0.23 +/- 0.04 vs. 0.07 +/- 0.03, P = 0.005). These observations are important because of the apparent relationship of [Ca2+]i transient alternans to electrical alternans and arrhythmias during ischemia.

Topics: Animals; Benzofurans; Calcium; Calibration; Female; Fluorescent Dyes; Heart; Imidazoles; Ionomycin; Kinetics; Male; Manganese; Myocardial Ischemia; Myocardium; Rabbits; Spectrometry, Fluorescence; Subcellular Fractions; Time Factors