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Quantifying subcellularly resolved Ca2+ signals in cardiac myocytes is essential for understanding Ca2+ fluxes in excitation-contraction and excitation-transcription coupling. Translation of changes in Ca2+-dependent fluorescence into changes in [Ca2+] relies on the indicator's behavior in situ, but properties of fluorescent indicators in different intracellular compartments may differ. Thus, we determined the in situ calibration of a frequently used Ca2+ indicator, Fluo-4, and evaluated its use in reporting cytoplasmic and nucleoplasmic Ca2+ signals in isolated cardiac myocytes.Calibration solutions were made by mixing known quantities of EGTA and CaEGTA solutions and the free [Ca2+] was confirmed with a Ca2+-sensitive electrode. Solutions contained metabolic inhibitors and cyclopiazonic-acid (5μM) to block active Ca2+ transport and the Ca2+ ionophore A-23187 (10μM) to allow equilibration of [Ca2+] between bath solution and cell interior. Ventricular rat and mouse myocytes were loaded with Fluo-4/AM (8μM, 20min). Fluo-4 fluorescence (excitation/emission: 488/>505nm) was recorded using a Nipkow dual disc-based confocal microscope.Concentration-response curves were obtained and a significant difference in the apparent Ca2+ binding affinities (Kd) of Fluo-4 between cytoplasmic (993±56nM; 1026±65nM) and nucleoplasmic (1211±73nM; 1251±71nM) compartments was observed for both mouse and rat cells, respectively (both n=15, P

AIM: Urocortin II (UcnII) exerts beneficial effects in heart failure. In cardiac myocytes, UcnII exerts positive inotropic and lusitropic effects through a PKA-dependent pathway. We tested the hypothesis that, in addition, UcnII stimulates endothelial NO synthase (eNOS) and evaluated the underlying signaling pathways and mechanisms.METHODS: UcnII-induced phosphorylation of Akt and eNOS was measured using phospho-specific antibodies. Isolated cardiac myocytes were loaded with 5x10-6M DAF-FM and UcnII-induced changes in NO production were assessed by changes in DAF-FM fluorescence in electrically paced myocytes (0.5 Hz, room temperature) by means of confocal microscopy.RESULTS: In rabbit ventricular myocytes, UcnII caused increases in phosphorylation of Akt at Ser473 (+89.4±21.4%) and Thr308 (+60.4±39.7%) and phosphorylation of eNOS at Ser1177 (+49.6±25.9%; n=6-11, all P

Alterations in intracellular Ca2+ handling may play a critical role in the pathophysiology of myocardial failure. Contractile dysfunction in end-stage human heart failure may be attributed to reduced [Ca] transients (CaTs). We, therefore, quantified electrically stimulated [Ca] transients in cardiac myocytes (CMs) isolated from non-failing donor and explanted end-stage failing hearts.Myocardial tissue samples were obtained from explanted failing hearts at the time of transplantation (n = 4) and unused donors (n = 6). Ejection fraction was significantly reduced in end-stage failing group as evaluated by transthoracic echocardiography (60.8±2.0% vs 13.75±1.3%, P