MICROINJECTION OF ANTIBODIES AND EXPRESSION VECTORS INTO LIVING MYOCARDIAL-CELLS - DEVELOPMENT OF A NOVEL-APPROACH TO IDENTIFY CANDIDATE GENES THAT REGULATE CARDIAC GROWTH AND HYPERTROPHY

Background. Microinjection approaches in the cardiac cell context have allowed delivery of various calcium dyes and monitoring of short-term physiological responses. However, unlike other cell types, it has proved difficult to microinject myocardial cells without the concomitant loss of long-term cell viability. Methods and Results. An analysis of experimental variables was conducted to adapt microinjection techniques to the neonatal rat ventricular cell context. Among the variables optimized were the selection of culture dishes, plating substrate, microinjection parameters, and a variety of maneuvers to inhibit myocyte hypercontracture, injury, and consequent death after micropuncture. With the modified technique, the percentage of injected cells that maintained long-term viability (48 hours) increased from <1% to 30%. Similarly, an increased efficiency of gene transfer and expression (measured as the percentage of injected cells that express the delivered gene) was obtained after either cytoplasmic or nuclear injection of a beta-galactosidase expression vector into cardiac myocytes. Microinjection of marker immunoglobulin G does not interfere with the induction of the hypertrophic response or the expression of a coinjected atrial natriuretic factor promoter-luciferase reporter fusion gene construct. Conclusions. To the best of our knowledge, this study provides the first description of the efficient microinjection of neonatal cardiac muscle cells with maintenance of long-term cell viability. The microinjection technique is now a viable approach to examine cause-and-effect relations between specific gene products and any defined feature or response of cardiac myocytes that can be assayed at a single-cell level.