Champion Hunter C, Georgakopoulos Dimitrios, Haldar Saptarsi, Wang Lili, Wang Yibin, Kass David A
Division of Cardiology, Department of Medicine, 600 N Wolfe St, Halsted 500, Johns Hopkins Hospital, Baltimore, Md 21287, USA.
Circulation. 2003 Dec 2;108(22):2790-7. doi: 10.1161/01.CIR.0000096487.88897.9B. Epub 2003 Nov 24.
Viral gene transfer to the whole heart in vivo has been achieved in several mammalian species but remained difficult to accomplish in murine hearts. We postulated that a key impediment derives from the use of proximal aortic occlusion during virus injection, because this eliminates coronary perfusion gradients in mice as aortic root and left ventricle pressures equalize.
Pressure-volume analysis confirmed these mechanics. In contrast, descending aortic occlusion with whole-body cooling (20 degrees C) preserved transmyocardial perfusion gradients and allowed for sustained (>10-minute) dwell times in an upper-body perfusion circuit. This approach yielded robust cardiac transfection with adenovirus (AdV) and adeno-associated virus (AAV) injected into the left ventricle cavity or more simply via a central vein. Cardio-specific expression was achieved with a myocyte-specific promotor. Optimal AdV transfection required 9-minute aortic occlusion, versus 5-minute occlusion for AAV. Using this method, we examined the in vivo function of phospholamban (PLB) by stably transfecting PLB-null mice with AAV encoding PLB (AAV(PLB)). AAV(PLB) restored PLB protein to near control levels that colocalized with SERCA2A in cardiomyocytes. At baseline, PLB-null hearts exhibited enhanced systolic and diastolic function, but frequency-dependent reserve was blunted versus wild-type controls. These properties, particularly the frequency response, returned toward control 3 months after AAV(PLB) transfection.
The new simplified approach for murine whole-heart viral transfection should assist molecular physiology studies.
在几种哺乳动物体内已实现向整个心脏进行病毒基因转移，但在鼠类心脏中仍难以完成。我们推测一个关键障碍源于病毒注射过程中使用近端主动脉阻断，因为随着主动脉根部和左心室压力平衡，这会消除小鼠的冠状动脉灌注梯度。
压力 - 容积分析证实了这些力学原理。相比之下，通过全身冷却（20摄氏度）进行降主动脉阻断可保留跨心肌灌注梯度，并允许在上半身灌注回路中持续（>10分钟）停留。这种方法通过将腺病毒（AdV）和腺相关病毒（AAV）注入左心室腔或更简单地通过中心静脉，实现了强大的心脏转染。使用心肌细胞特异性启动子可实现心脏特异性表达。最佳的AdV转染需要9分钟的主动脉阻断，而AAV则需要5分钟。使用这种方法，我们通过用编码磷酸受纳蛋白（PLB）的AAV（AAV(PLB)）稳定转染PLB基因缺失小鼠，研究了PLB在体内的功能。AAV(PLB)将PLB蛋白恢复到与心肌细胞中肌浆网Ca²⁺ - ATP酶2A（SERCA2A）共定位的接近对照水平。在基线时，PLB基因缺失的心脏表现出增强的收缩和舒张功能，但与野生型对照相比，频率依赖性储备减弱。在AAV(PLB)转染3个月后，这些特性，特别是频率反应，恢复到对照水平。
这种用于鼠类全心病毒转染的新简化方法应有助于分子生理学研究。
