Onishi Junji, Browne Vaughn A, Kono Satoshi, Stiffel Virginia M, Gilbert Raymond D
Center for Perinatal Biology, Department of Physiology and Obstetrics and Gynecology, Loma Linda University School of Medicine, Loma Linda, California, USA.
J Soc Gynecol Investig. 2004 Jan;11(1):1-8. doi: 10.1016/j.jsgi.2003.07.003.
We studied the effects of long-term high-altitude hypoxia and protein kinase A (PKA) phosphorylation on calcium (Ca2+) responses of skinned cardiac papillary muscles from fetal and adult sheep.
Fetal and nonpregnant adult sheep were exposed to high-altitude (3820 m), long-term (approximately 110 days) hypoxia. Papillary muscles were isolated and mounted in well-oxygenated, temperature-controlled baths. After the papillary muscles were stimulated electrically to establish the diastolic tension that produced the maximum active contraction, the electrical stimulation was stopped, and the muscles were skinned with 1% vol/vol Triton-X-100. In protocol 1, the skinned muscles were exposed to activating solutions containing different calcium concentrations (pCa; from pCa 8.0 to pCa 4.0), which were prepared by varying the Ca-EGTA/EGTA ratio, and the steady-state tension was measured at each pCa. In protocol 2, the skinned muscles were contracted with activating solution containing a pCa of 5.0. After equilibration, the solution in some baths was changed to activating solution at the same pCa of 5.0 but also containing the catalytic subunit of PKA. The other baths were exchanged with activating solution at a pCa of 5.0 containing no PKA. We then measured the degree of tension reduction caused by PKA until tension reached a new steady state.
In the long-term hypoxic fetal heart, the maximum tension response of right, but not left, ventricular skinned papillary muscle to Ca2+ was significantly less than that in control muscles. In the long-term hypoxic adult heart, the left ventricle, but not the right ventricle, displayed an increased maximum tension response to Ca2+ compared with control. Phosphorylation of troponin I (TnI) with PKA reduced active tension in both fetal ventricles of the long-term hypoxic group more than in hearts from control fetuses. In the adult, phosphorylation with PKA resulted in a larger decrease in tension in the left ventricle and a smaller decrease in tension in the right ventricle in the long-term hypoxic group, although the differences were small.
In the long-term hypoxic fetal right ventricle, the decreased maximum tension response to Ca2+ is consistent with the decrease in myofibrillar magnesium-activated adenosine triphosphatase activity observed previously. The larger decrease in tension after PKA phosphorylation of TnI in the long-term hypoxic fetal left ventricle indicates a larger reduction in Ca2+ binding to troponin C.
我们研究了长期高原缺氧和蛋白激酶A（PKA）磷酸化对胎儿和成年绵羊皮肤化心脏乳头肌钙（Ca2+）反应的影响。
将胎儿和未怀孕的成年绵羊暴露于高海拔（3820米）、长期（约110天）缺氧环境中。分离乳头肌并安装在充分供氧、温度可控的浴槽中。在对乳头肌进行电刺激以建立产生最大主动收缩的舒张张力后，停止电刺激，并用1%体积/体积的 Triton-X-100 对肌肉进行皮肤化处理。在方案1中，将皮肤化的肌肉暴露于含有不同钙浓度（pCa；从pCa 8.0到pCa 4.0）的激活溶液中，这些溶液通过改变Ca-EGTA/EGTA比例制备，并在每个pCa下测量稳态张力。在方案2中，将皮肤化的肌肉用含有pCa为5.0的激活溶液收缩。平衡后，一些浴槽中的溶液换成含有相同pCa为5.0但也含有PKA催化亚基的激活溶液。其他浴槽换成含有pCa为5.0且不含PKA的激活溶液。然后我们测量PKA引起的张力降低程度，直到张力达到新的稳态。
在长期缺氧的胎儿心脏中，右心室而非左心室皮肤化乳头肌对Ca2+的最大张力反应明显低于对照肌肉。在长期缺氧的成年心脏中，与对照相比，左心室而非右心室对Ca2+的最大张力反应增加。长期缺氧组胎儿的两个心室中，用PKA使肌钙蛋白I（TnI）磷酸化导致的主动张力降低比对照胎儿心脏更大。在成年人中，长期缺氧组中用PKA磷酸化导致左心室张力下降幅度更大，右心室张力下降幅度更小，尽管差异较小。
在长期缺氧的胎儿右心室中，对Ca2+的最大张力反应降低与先前观察到的肌原纤维镁激活三磷酸腺苷酶活性降低一致。长期缺氧胎儿左心室中TnI经PKA磷酸化后张力下降幅度更大，表明Ca2+与肌钙蛋白C的结合减少幅度更大。
