N-n-Butyl Haloperidol Iodide Preserves Cardiomyocyte Calcium Homeostasis During Hypoxia

N-n-Butyl haloperidol iodide （F₂） is a novel compound derived from haloperidol. In our previous work, F₂ was found to be an L-type calcium channel blocker which played a protective role in rat heart ischemic–reperfusion injury in a dose-dependent manner. Study has shown that the disbalance of calcium homeostasis in ventricular myocytes is connected with ischemia-reperfusion injury. Calcium transients is the sum of calcium sparks in ventricular myocytes, so any change in calcium transients reflects the function change of ventricular myocytes during hypoxia/ischemia. In the current study, we aimed to investigate the effects and some possible mechanisms of F₂ on calcium transients in hypoxic/ischemic rat cardiac myocytes.METHODS1. Single ventricular cardiac myocytes were isolated from the hearts of adult male Sprague-Dawley rats by a standard enzymatic technique. For confocal Ca²⁺ imaging, freshly isolated ventricular cardiac myocytes were loaded with 5μM fluo-4 AM （Molecular Probes, USA） for 15 min at room temperature and then washed with a Tyrode solution that contained 2.5 mM Ca²⁺ to remove excess dye. Confocal imaging involved use of an Olympus FluoView FV1000 confocal microscope. The experiments were conducted at room temperature. Contractions and Ca²⁺ transients were elicited by field-stimulating myocytes through a pair of platinum electrodes with a 0.5-ms supra-threshold voltage square pulse.2. After 20 min of superfusion with normal Tyrode solution, isolated ventricular cardiac myocytes were superfused 30min hypoxia solution. Superfusion with buffer was controlled by gravity to maintain a flow rate of 6 ml/min. Calcium transients images were reocorded at 10min, 20min. 30min with confocal microscope.3. After 20 min of superfusion with normal Tyrode solution, isolated ventricular cardiac myocytes were superfused in a randomized, blinded fashion to 1 of 3 simulated hypoxia solutions by the addition of stock solution containing 0.1, 1, 10μM F₂. Calcium transients images were recorded at 10min, 20min. 30min with confocal microscope.4. To estimate the Ca²⁺ content of SR, rapidly apply 15μM caffeine, which releases all SR Ca²⁺ after perfusing in a randomized, blinded fashion to 1 of 4 simulated hypoxia solutions by the addition of stock solution containing0, 0.1, 1, 10μM F₂. The images were recorded with confocal microscope.5. To estimate the rate of Ca²⁺ transport by SERCA2a, the contribution of NCX and PMCA was eliminated. Ventricular myocytes were incubated with 5μM thapsigargin （TG） blocking SERCA2a uptake of calcium for 6 min after 30-min hypoxia at room temperature. Under these conditions, the rate constant of decline reflects the rate of Ca²⁺ transport by NCX and PMCA （VNCX+PMCA）. The rate of Ca²⁺ transport by SERCA2a was calculated as VSERCA=VTOTAL - VNCX+PMCA.6. To estimate the rate of Ca²⁺ transport by NCX, the myocytes were incubated with 5μM Thapsigargin, 5μM carboxyeosin （CE）, a specific PMCA blocker, and 10μM Ru360 to block uptake into mitochondria for 6 min after 30-min hypoxia at room temperature. Therefore, under these conditions, the rate constant of decline reflects the rate of Ca²⁺ transport by NCX （VNCX）.RESULTS1. A series changes happened after 30-min hypoxia, including the resting [Ca²⁺]i increased, the amplitude of calcium transients reduced and RT25-75, T50, DT75-25 prolonged.2. F₂ could inhibited the resting [Ca²⁺]i increased, the amplitude of calcium transients reduced and RT25-75, T50, DT75-25 prolonged which caused by 30-min hypoxia in a dose-dependent in some degree.3. The SR Ca²⁺ content reduced after 30-min hypoxia and F₂ could inhibit this reduction in a dose-dependent in some degree.4. The rate of Ca²⁺ transport by SERCA2a reduced after 30-min hypoxia and F₂ could inhibited the reduction of SERCA uptake Ca²⁺ rate. 5. F₂ did not affect the rate of NCX remove Ca²⁺ after 30-min hypoxia.CONCLUSIONS1. The balance of calcium homeostasis in ventricular myocytes was disturbed by 30 min hypoxia. The changes included the resting [Ca²⁺]i increased, the amplitude reduce of calcium transients and the prolonger of RT25-75, DT75-25 and T50.2. F₂ could reduced the changes of calcium signals in ventricular myocytes which caused by hypoxia.3. The protective role of F₂ on SERCA2a activity is the major mechanism of F₂ improves the calcium removement during calcium transients and the SR Ca²⁺ content after 30-min hypoxia.4. F₂ could affect the activity of RyRs indirectly by influencing the SR Ca²⁺ content after 30 min hypoxia.5. The target point of F₂ inhibiting Ca²⁺ influx during hypoxia is L-type calcium channels not NCX.6.The prevention of calcium overload and protection on SR Ca²⁺ content, the activity of SERCA2a and RyRs is the reason of F₂ affected T50 and the amplitude of calcium transients.