ROCK promotes hypoxia-induced cardiomyocyte apoptosis by inhibiting PI3K/Akt pathway

AIM: To investigate the expression of Rho-associated coiled-coil protein kinase (ROCK) at different hypoxic phases and to explore its role in myocardial cell apoptosis. METHODS: The rat cardiomyocytes were primarily cultured and identified by an antibody targeting α-actin of striated muscle. The myocardial cell hypoxic model was established by exposing the cells in hypoxic liquid for 1 h, 3 h, 6 h and 9 h. The cell apoptotic rate was assessed by flow cytometry. The cell survival rate was determined by MTT assay. The protein levels of ROCK-1, ROCK-2, caspase-3 activation fragment, PI3K and p-PI3K at different hypoxia phases were determined by Western blotting.RESULTS: After exposed to hypoxic liquid for 1 h, 3 h, 6 h and 9 h, the apoptotic rates of the cardiomyocytes were （8.76±1.51）%, （15.36±2.34）%, （26.50±3.43）% and （41.96±4.22）%, respectively, significantly higher than those in control group ［(2.60±0.34）%, P<0.01］. The survival rates were （93.20±4.12）%, （86.14±3.10）%, （75.53±7.25）%and （60.21±6.75）%, respectively, signficantly lower than those in control group ［（97.60±1.12）%, P<0.05］. After 1 h of hypoxic exposure, the levels of ROCK-1 and ROCK-2 began to rise, reached its peak at 3～6 h, and began to decrease after 9 h, which were significantly higher than those in control group (P<0.05). After 1 h of hypoxic exposure, the caspase-3 activation fragment began to rise, which was sustained in a high level at following observed time points as compared with control group (P<0.01). No difference of the PI3K expression in the course of hypoxia was observed. However, after 1 h of hypoxic exposure, the p-PI3K level began to rise, reached its peak at 3 h, began to decrease at 6 h, and was almost undetectable at 9 h. CONCLUSION: Hypoxia stimulates the cardiomyocytes to increase the expression of ROCK-1 and ROCK-2, and is in parallel with the cardiomyocyte apoptosis. ROCKs may play an important role in the process of hypoxia-induced cardiomyocyte apoptosis by inhibiting the p-PI3K pathways.     

Metabolic scope, swimming performance and the effects of hypoxia in the mulloway, Argyrosomus japonicus (Pisces: Sciaenidae)

The culture of the mulloway (Argyrosomus japonicus), like many other Sciaenidae fishes, is rapidly growing. However there is no information on their metabolic physiology. In this study, the effects of various hypoxia levels on the swimming performance and metabolic scope of juvenile mulloway (0.34 ± 0.01 kg, mean ± SE, n = 30) was investigated (water temperature = 22 °C). In normoxic conditions (dissolved oxygen = 6.85 mg l^− 1), mulloway oxygen consumption rate (M·o₂) increased exponentially with swimming speed to a maximum velocity (U_crit) of 1.7 ± < 0.1 body lengths s^− 1 (BL s^− 1) (n = 6). Mulloway standard metabolic rate (SMR) was typical for non-tuna fishes (73 ± 8 mg kg^− 1 h^− 1) and they had a moderate scope for aerobic metabolism (5 times the SMR). Mulloway minimum gross cost of transport (GCOT_min, 0.14 ± 0.01 mg kg^− 1 m^− 1) and optimum swimming velocity (U_opt, 1.3 ± 0.2 BL s^− 1) were comparable to many other body and caudal fin swimming fish species. Energy expenditure was minimum when swimming between 0.3 and 0.5 BL s^− 1. The critical dissolved oxygen level was 1.80 mg l^− 1 for mulloway swimming at 0.9 BL s^− 1. This reveals that mulloway are well adapted to hypoxia, which is probably adaptive from their natural early life history within estuaries. In all levels of hypoxia (75% saturation = 5.23, 50% = 3.64, and 25% = 1 .86 mg l^− 1), M·o₂ increased linearly with swimming speed and active metabolic rate (AMR) was reduced (218 ± 17, 202 ± 14 and 175 ± 10 mg kg^− 1 h^− 1 for 75%, 50% and 25% saturation respectively). However, U_crit was only reduced at 50% and 25% saturation (1.4 ± < 0.1 and 1.4 ± < 0.1 BL s^− 1 respectively). This demonstrates that although the metabolic capacity of mulloway is reduced in mild hypoxia (75% saturation) they are able to compensate to maintain swimming performance. GCOT_min (0.09 ± 0.01 mg kg^− 1 m^− 1) and U_opt (0.8 ± 0.1 BL s^− 1) were significantly reduced at 25% dissolved oxygen saturation. As mulloway metabolic scope was significantly reduced at all hypoxia levels, it suggests that even mild hypoxia may reduce growth productivity.     

Clinical Characteristics of 53 Dogs with Doppler-Derived Evidence of Pulmonary Hypertension: 1992–1996

Pulmonary hypertension occurs as a primary or secondary disorder of the pulmonary vasculature. Doppler echocardiography provides a noninvasive tool for the estimation of pulmonary arterial pressure when tricuspid regurgitation or pulmonic insufficiency is present. The cardiology database at Colorado State University was reviewed, and echocardiographic records from cases diagnosed with pulmonary hypertension were evaluated. Application of the modified Bernoulli equation to the maximal instantaneous velocity of a right-sided regurgitant jet provided evidence of pulmonary hypertension in 53 dogs over a 4-year period. Tricuspid regurgitant velocity > or = 2.8 m/second or pulmonic insufficiency velocity > or = 2.2 m/second was considered abnormal and indicative of pulmonary hypertension. Tricuspid regurgitant gradients in 51 dogs ranged from 32 to 145 mm Hg (mean, 63.0 mm Hg; median, 57.0 mm Hg; 25th-75th percentiles, 45.2-76.5 mm Hg). Pulmonic insufficiency gradients in 8 dogs ranged from 20 to 100 mm Hg (mean, 59.5 mm Hg; median, 61.5 mm Hg; 25th-75th percentiles, 32.0-84.5 mm Hg). Affected dogs ranged in age from 2 months to 16 years. Clinical signs were characteristic of cardiopulmonary disease, but a relatively high frequency of syncope was noted (12 of 53 dogs, 23%). Pulmonary hypertension was probably due to increased pulmonary vascular resistance in 23 dogs, pulmonary overcirculation in 2 dogs, and pulmonary venous hypertension in 23 dogs. Five dogs lacked a clinically recognizable cardiopulmonary cause of pulmonary vascular disease. Our results suggest that pulmonary hypertension can occur as a complication of commonly encountered cardiopulmonary diseases, and that Doppler echocardiography can facilitate recognition of this condition.     

饲料中淀粉和糊精对红鳍东方鲀生长和低氧耐受性的影响

在水产养殖鱼粉鱼油供应相对紧缺的背景下,充分挖掘碳水化合物(糖类)的营养功能具有重要意义。低氧是水产养殖中一种常见的环境应激。大量研究表明,鱼类在低氧条件下主要利用糖来提供能量,但饲料糖源对红鳍东方鲀(Takifugu rubripes)的生长和急性低氧耐受性尚未见报道。本研究分别在饲料中添加玉米淀粉或糊精(玉米淀粉水解物)作为糖源,养殖8周后,测定生长、急性低氧存活率、代谢产物和低氧诱导因子(HIF)信号通路等指标。结果显示,与淀粉组相比,糊精虽然不会影响红鳍东方鲀的生长性能和鱼体成分,但显著提高了其在急性低氧条件下的存活率。在常氧条件下,淀粉组和糊精组肝脏的糖原和乳酸含量均无显著差异(P>0.05),但在低氧条件下,糊精组肝脏的乳酸含量和乳酸脱氢酶基因表达量均显著高于淀粉组(P<0.05)。同时,糊精组肝脏hif-1α (hypoxia inducible factor 1 subunit alpha)和hif-3α (hypoxia inducible factor 1 subunit alpha, like)、肌肉hif-1α和vegfa (vascular endothelial growth factor A)的表达量均显著高于淀粉组(P<0.05)。在低氧条件下,糊精组血清甘油三酯(TG)含量显著升高,但淀粉组血清和肝脏的TG含量在低氧后却显著下降(P<0.05)。与常氧组相比,糊精组在低氧后的肌肉总蛋白含量、肝脏蛋白激酶B (v-akt murine thymoma viral oncogene homolog 1, akt1)和哺乳动物雷帕霉素靶蛋白(mechanistic target of rapamycin kinase, mtor)基因表达量均显著降低,但淀粉组肝脏的mtor基因表达在低氧后却显著升高(P<0.05)。综上所述,用易消化的糊精替代玉米淀粉激活了HIF信号通路介导的无氧糖酵解来为鱼体提供更多的能量,同时还可以抑制脂肪的分解和蛋白质的合成,减少了O2的消耗,从而提高了红鳍东方鲀对急性低氧的耐受性。本研究结果为鱼类耐低氧饲料的设计和健康养殖提供了重要参考。     

克氏原螯虾PcCRCN-L基因的克隆鉴定及在低氧胁迫下的表达响应

虾青蛋白(Crustacyanin,CRCN)在甲壳动物脂类代谢及低氧胁迫应激调控等方面具有重要的功能。为获取虾青蛋白在克氏原螯虾(Procambarus clarkii)性腺发育和低氧胁迫应答中的作用,本研究在克氏原螯虾肝胰腺组织中鉴定出1个类虾青蛋白PcCRCN-L基因的c DNA序列,分析了PcCRCN-L基因的结构特征和进化模式,研究了PcCRCN-L基因在不同组织与性腺不同发育时期的表达特征,探讨了Pc CRCN-L在低氧–复氧胁迫下的表达响应模式。结果显示,PcCRCN-L基因c DNA序列长2 700 bp,其开放阅读框(ORF)长度为1 587 bp,编码528个氨基酸残基;DNA序列长6 130 bp,位于克氏原螯虾基因组的第12号染色体,包含5个外显子和4个内含子,内含子/外显子剪接方式符合GT-AG规则。Pc CRCN-L具有1个完整的lipocalin结构域,包含典型的序列保守区Ⅰ(SCR1)序列G-X-W、保守区Ⅱ(SCR2)序列T-D-Y和保守区Ⅲ(SCR3)序列精氨酸R。多序列比对与系统进化分析结果显示,PcCRCN-L独立于传统虾青蛋白A和C亚族,单独聚...     

斑石鲷低氧耐受能力及血液生理生化指标变化研究

为阐明斑石鲷(Oplegnathus punctatus)低氧耐受能力及低氧胁迫过程中血液生理生化指标的变化规律，本研究通过生理学和生态学方法，查明2种规格斑石鲷在临界氧分压(critical oxygen tension, Pcrit)和失去平衡点(loss equilibrium, LOE)时的溶解氧浓度，观察自然耗氧条件下其呼吸行为变化，分析低氧胁迫和恢复溶解氧过程中血液皮质醇和葡萄糖含量、血红蛋白(hemoglobin, Hb)浓度、红细胞(red blood cell, RBC)和白细胞(white blood cell, WBC)数目、红细胞积压(hematocrit, HCT)变化。结果显示，在水温(23.0±0.5)℃、氨氮浓度<0.5 mg/L、亚硝酸盐浓度0~0.05 mg/L、盐度为30、pH为7.80的条件下，200和50 g斑石鲷的Pcrit值分别为(4.05±0.09)和(3.15±0.12) mg/L，LOE值分别为(1.16±0.08)和(0.93±0.11) mg/L，且50 g斑石鲷到达LOE值的时间(t=480 min)比200 g斑石鲷(t=110 min)更长；自然耗氧过程中，2种规格斑石鲷呼吸频率均呈先升高后降低的变化趋势，且均在Pcrit值处显著升高，达到最大值(P<0.05)。低氧胁迫导致血浆葡萄糖和皮质醇含量显著升高，在LOE值处达到最大值(P<0.05)，但50 g斑石鲷的葡萄糖和皮质醇含量上升幅度显著低于200 g斑石鲷(P<0.05)。同时，低氧引起斑石鲷的WBC和RBC数目、Hb浓度和HCT显著升高，50和200 g斑石鲷分别在Pcrit和LOE值处达到最大值(P<0.05)。恢复正常溶解氧24 h后，2种规格斑石鲷均恢复正常呼吸运动，上述生理生化指标均与对照组无显著差异(P>0.05)。综上所述，2种规格的斑石鲷相比较，50 g斑石鲷的低氧耐受能力更强，200 g斑石鲷对低氧胁迫的应激反应更敏感，2种规格斑石鲷均可通过增加呼吸频率、提高血液WBC和RBC数、皮质醇、葡萄糖和Hb浓度，增强对溶解氧的吸收利用，应对低氧胁迫导致的生理状态改变，相关结果为斑石鲷高效养殖提供了基础数据。