Zajdel R W, Dube D K, Lemanski L F
Department of Anatomy and Cell Biology, SUNY Health Science Center at Syracuse, Syracuse, New York, 13210, USA.
Exp Cell Res. 1999 May 1;248(2):557-66. doi: 10.1006/excr.1999.4419.
Hearts from cardiac mutant Mexican axolotl, Ambystoma mexicanum, do not form organized myofibrils and fail to beat. Though previous biochemical and immunohistochemical experiments showed a possible reduction of cardiac tropomyosin it was not clear that this caused the lack of organized myofibrils in mutant hearts. We used cationic liposomes to introduce both rabbit and chicken tropomyosin protein into whole hearts of embryonic axolotls in whole heart organ cultures. The mutant hearts had a striking increase in the number of well-organized sarcomeric myofibrils when treated with rabbit or chicken tropomyosin. FITC-labeled rabbit tropomyosin was used to examine the kinetics of incorporation of the exogenous protein into mutant hearts and confirmed the uptake of exogenous protein by the cells of live hearts in culture. By 4 h of transfection, both normal and mutant hearts were found to incorporate FITC-labeled tropomyosin into myofibrils. We also delivered an anti-tropomyosin antibody (CH 1) into normal hearts to disrupt the existing cardiac myofibrils which also resulted in reduced heartbeat rates. CH1 antibody was detected within the hearts and disorganization of the myofibrils was apparent when compared to normal controls. Introduction of a C-protein monoclonal antibody (ALD 66) did not result in a disruption of organized myofibrils. The results show clearly that chicken or rabbit tropomyosin could be incorporated by the mutant hearts and that it was sufficient to overcome the factors causing a lack of myofibril formation in the mutant. This finding also suggests that a lack of organized myofibrils is caused primarily by either inadequate levels of tropomyosin or endogenous tropomyosin in mutant hearts is unsuitable for myofibril formation, which we were able to duplicate with the introduction of tropomyosin antibody. Furthermore, incorporation of a specific exogenous protein or antibody into normal and mutant hearts of the Mexican axolotl in whole heart organ culture offers an unique model to evaluate functionalroles of contractile proteins necessary for cardiac development and differentiation.
来自心脏突变型墨西哥钝口螈（Ambystoma mexicanum）的心脏无法形成有组织的肌原纤维，且无法跳动。尽管先前的生化和免疫组化实验表明心肌肌钙蛋白可能减少，但尚不清楚这是否导致突变心脏中缺乏有组织的肌原纤维。我们使用阳离子脂质体将兔和鸡的肌钙蛋白引入胚胎钝口螈全心脏器官培养物的整个心脏中。当用兔或鸡的肌钙蛋白处理时，突变心脏中组织良好的肌节肌原纤维数量显著增加。用异硫氰酸荧光素（FITC）标记的兔肌钙蛋白来检测外源蛋白掺入突变心脏的动力学，并证实培养的活心脏细胞摄取了外源蛋白。转染4小时后，发现正常心脏和突变心脏均将FITC标记的肌钙蛋白掺入肌原纤维中。我们还将抗肌钙蛋白抗体（CH 1）导入正常心脏以破坏现有的心肌肌原纤维，这也导致心率降低。在心脏中检测到CH1抗体，与正常对照相比，肌原纤维的紊乱明显。引入C蛋白单克隆抗体（ALD 66）并未导致有组织的肌原纤维破坏。结果清楚地表明，突变心脏可以掺入鸡或兔的肌钙蛋白，并且足以克服导致突变体中缺乏肌原纤维形成的因素。这一发现还表明，缺乏有组织的肌原纤维主要是由于肌钙蛋白水平不足或突变心脏中的内源性肌钙蛋白不适用于肌原纤维形成，我们通过引入肌钙蛋白抗体能够重现这一情况。此外，在全心脏器官培养中将特定的外源蛋白或抗体掺入墨西哥钝口螈的正常和突变心脏中，提供了一个独特的模型来评估心脏发育和分化所需的收缩蛋白的功能作用。
