| A lot of researches have demonstrated that chronic intermittent hypobaric hypoxia (CIHH) confers significant cardioprotetive effects on adult heart, such as enchancing the resistance of heart against acute hypoxia/re-oxygen or acute ischemia/reperfusion (I/R) injury, reducing infarct size, limiting cardiac ultrastructure damage and anti-arrhythmia. The candidate mechanisms involved enhancement of antioxidation, increase of myocardium capillary desity and coronary flow, overexpression of heat shock protein, increase of NO production, anti-apoptosis, stabilization of mitochondria and function of handling calcium, prolongation of action potential duration and effective refractory period. It was reported that the cardioprotective effect of CIHH against I/R was related to anti-calcium overload and preservation of calcium homeostasis. However, it was not clear about the role of CIHH on calcium homeostasis and calcium overload in postnatal developing hearts. The objective of the study was to explore the effects of CIHH on calcium homeostasis and underling cellular and molecular mechanisms on postnatal developing rat using functional, cellular, molecular biology and electrophysiological methods.Our study consists of four parts: (1) To observe cardioprotective effects of CIHH on cardiac function and infart size using Langendorff perfusion technique. (2) To examine effects of CIHH on intracellular free calcium concentration in developing rat ventricular myocytes using laser confocal microscope system. (3) To investigate the effect of chronic intermittent hypobaric hypoxia on the expression of calcium handling protein in developing rats using molecular biology technique. (4) To examine of effects of CIHH on calcium currents and Na+/Ca2+ exchange currents in ventricular myocytes of developing rat by using whole-cell patch clamp techenique, and to explore the ionic mechanism of CIHH cardiac protection against IR- induced injury and calcium overload.I The cardiac protective effect of chronic intermittent hypobaric hypoxia on developing rat heartsObjective: to observe the effect of CIHH on cardiac function and infart size during ischemia/reperfusion in developing rat hearts.Methods: In this part of experiment, age- and body weight-matched postnatal male rats were divided into four groups: 28-day CIHH group (CIHH28), 42-day CIHH group (CIHH42), 28-day control group (CON28) and 42-day control group (CON42). For CIHH group, neonatal rats with the maternals were exposed to a hypobaric chamber, enduring CIHH mimicking 3000 m altitude (PB = 525 mmHg, PO2 = 108.8 mmHg, 5 hrs/day) for 28days and 42days, respectively. The isolated hearts were perfused in the langendorff apparatus, undergoing 30min global ischemia and 60min reperfusion(I/R). Parameters of cardiac function including left ventricular developing pressure (LVDP), left ventricular end-diastolic pressure (LVEDP), maximal velocity of left ventricular pressure (LVdp/dtmax), coronary flow (CF) were recorded, respectively. The infart size was measured at the end of the experiment.Results:1 Under the basic condition,the coronary flow (CF) in CIHH rats was significant higher than that in CON rats(P>0.05), while other parameters of cardiac function and infart size were not changed significantly(P<0.05). The recovery of cardiac function from IR in CIHH rats was much better than that in CON rats, including the increase of LVDP and±LVdP/dt, the decrease of LVEDP (P<0.05). Also, the myocardial infart size of was significantly smaller in CIHH rats than that in CON rats(P<0.05).2 The cardiac protection of CIHH rats was abolished by Bay K8644 (a L-type calcium channel agonist, 0.5μM) or ryanodine (a SR ryanodine receptor agonist, 10 nM) pretreatment (P<0.05). Conclusion: CIHH has significant cardiac protection, enhancing the resistance against ishchemia/reperfusion injury in developing rat heart, which might be related with preservation of calcium hoeostasis of cardiomyocytes during I/R. The cardiac protection could be abolished by increase of calcium influx via L-type calcium channel and release from SR in cardiomyocytes.ⅡEffects of chronic intermittent hypobaric hypoxia on intracellular free calcium concentration in developing rat ventricular myocytesObjective: To observe the effects of CIHH on intracellular free calcium concentration ([Ca2+]i) in ventricular myocytes of developing rat and the possible mechanisms involved.Methods:In this part of experiment, age- and body weight-matched postnatal male rats were divided into four groups: 28-day CIHH group (CIHH28), 42-day CIHH group (CIHH42), 28-day control group (CON28) and 42-day control group (CON42). For CIHH group, neonatal rats with the maternals were exposed to a hypobaric chamber, enduring CIHH mimicking 3000 m altitude (PB = 525 mmHg, PO2 = 108.8 mmHg, 5 hrs/day) for 28days and 42days, respectively. Isolated ventricular myocytes were were obtained from ventricles by enzymatic dissociation method and incubated with Fluo3-AM solution at 37°C for 60 min. Fluorescence intensity was recorded and images were scanned using laser confocal microscope system during pre-ischemia, simulated ischemia and reperfusion.Results:1 There was no difference of [Ca2+]i of ventricular myocytes under normal Tyrode's solution between CON and CIHH rats. [Ca2+]i in ventricular myocytes was increased after 5 min simulated ischemia and 10 min reperfusion, but the increasing of [Ca2+]i was blunted in CIHH28 and CIHH42 rats (P<0.05 ).2 The effect of CIHH28 and CIHH42 on [Ca2+]i in ventricular myocytes during I/R was abolished by Bay K8644 (a L type calcium channel agonist), thapsigargin (a inhibitor of Ca2+ ATPase in SR), and chelerythrine chloride (a PKC inhibitor) (P<0.05).Conclusion: CIHH can inhibite the elevation of [Ca2+]i during I/R in developing rat ventricular myocytes. The the effect of CIHH on [Ca2+]i during I/R can be abolished by Bay K8644, thapsigargin, and chelerythrine chloride, suggesting the cardioprotective effect of CIHH is related with anti-calcium overload effect and PKC activation.III Effect of chronic intermittent hypobaric hypoxia on the expression of calcium handling proteins in developing ratsObjective: To investigate the effect of CIHH on the expression of calcium handling proteins in developing rats using Western blot method.Methods:In this part of experiment, age- and body weight-matched postnatal male rats were divided into four groups: 28-day CIHH group (CIHH28), 42-day CIHH group (CIHH42), 28-day control group (CON28) and 42-day control group (CON42). For CIHH group, neonatal rats with the maternals were exposed to a hypobaric chamber, enduring CIHH mimicking 3000 m altitude (PB = 525 mmHg, PO2 = 108.8 mmHg, 5 hrs/day) for 28days and 42days, respectively. The heart tissues were homogenated and SR and cell sarcolemmal membrane protein were extracted. The protein expressions of ryanodine receptors (RyR2), Ca2+ ATPase in the sarcoplasmic reticulum (SERCA2) and Na+/Ca2+ exchanger (NCX1) in the sarcolemmal membrane were assessed during I/R using Western blot method.Results:The protein expression of NCX1,RyR2, and SERCA2 were decreased markedly at 30 min of ischemia (I30), 5 min (R5) and 60 min (R60) of reperfusion in CON rats (P<0.05), but the inhibition of NCX1, RyR2, and SERCA2 expression during I/R were significantly attenuated in CIHH rats compared with CON rats (P<0.05).Conclusion: CIHH can effectively antagonize the depression of the protein expression of NCX1, RyR2 and SERCA2 in myocardium during I/R, suggesting that CIHH may maintain Ca2+ homeostasis and suppress I/R-induced cytosolic Ca2+ overload by reserving the protein expression of NCX1, RyR2 and SERCA2.IV Effect of chronic intermittent hypobaric hypoxia on L-type Ca2+ currents and Na+-Ca2+ exchange currents in cardiomyocytes of developing ratObjective: To explore the effect of CIHH on L-type Ca2+ currents (ICaL) and Na+-Ca2+ exchange currents (INa/Ca) in ventricular cardiomyocyte of developing rat by using whole-cell patch clamp technique, and the ionic mechanism for CIHH cardioprotection and anti-calcium overload.Methods: In this part of experiment, age- and body weight-matched postnatal male rats were divided into four groups: 28-day CIHH group (CIHH28), 42-day CIHH group (CIHH42), 28-day control group (CON28) and 42-day control group (CON42). For CIHH group, neonatal rats with the maternals were put into a hypobaric chamber to get CIHH mimicking 3000-m altitude (PB = 525 mmHg, PO2 = 108.8 mmHg, 5 hrs/day) for 28days and 42days, respectively. Isolated myocytes were obtained from ventricles by enzymatic dissociation method. ICaL and Ni2+ sensitive INa/Ca were recorded before and during simulated ischemia as well as reperfusion in CON and CIHH groups.Results:1 ICaL1.1 There were no significant differences of peak current and current-voltage relationship curve of ICaL between CON and CIHH cardiomyocytes under normal extracullar solution (P>0.05). The peak amplitude of ICaL was decreased and I-V relationship curve was shifted upward during simulated ischemia and reperfusion in CON28 and CON42 cardiomyocytes (P<0.05). However, the peak amplitude and I–V relationship of ICaL in CIHH28 and CIHH42 cardiomyocytes was not changed significantly during simulated ischemia and reperfusion (P>0.05).1.2 There were no significant differences of steady-state activation curve and steady-state inactivation curve of ICaL under normal extracullar solution in all cardiomyocytes (P>0.05). The steady-state inactivation curve of ICaL was not changed significantly under normal extracullar solution condition in all cardiomyocytes (P>0.05), but was shifted to the left in CON28 and CON42 (P<0.05), not in CIHH28 and CIHH42 (P>0.05), cardiomyocytes during simulated ischemia and reperfusion.2 INa/Ca2.1 There was no significant difference of Ni2+ sensitive Na+-Ca2+ exchange current (INa/Ca) between all cardiomyocytes(P>0.05). During simulated ischemia and reperfusion, INa/Ca in CON28 and CON42 cardiomyocytes was decreased(P<0.05), the decreasing of inward current part in INa/Ca was more significant than that of outward current part. But INa/Ca in CIHH28 and CIHH42 cardiomyocytes was not changed signifcantly during simulated ischemia (P>0.05).2.2 The reversal potential of INa/Ca in CON28 and CON42 cardiomyocytes was shifted toward negative potential during simulated ischemia (P<0.05), but the reversal potential of INa/Ca in CIHH28 and CIHH42 cardiomyocytes was changed significantly during simulated ischemia(P>0.05).Conclusion: CIHH has no effect on ICaL and INa/Ca of ventricular myocytes in developing rat under basic condition, but effectively antagonizes the inhibition of ICaL and INa/Ca in ventricular myocytes during simulated ischemia and reperfusion, which might be one of ionic mechanism of CIHH cardiac protection against I/R injury and anti-calcium overload.
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