Enhancement of adenoviral gene transfer to adult rat cardiomyocytes in vivo by immobilization and ultrasound treatment of the heart.
Direct injection of adenoviral vectors into ventricular myocardium in vivo produces local transfection of cells including cardiomyocytes. The use of vectors coexpressing GFP with the gene of interest allows subsequent identification of transfected myocytes isolated from the heart some days later, and examination of their function in cell bath experiments. We have injected vectors for antisense to phospholamban, or a control virus for expression of GFP only, into adult rat heart in vivo and then removed the heart and isolated ventricular myocytes 7 days later. Brief immobilization of the ventricle during and after injection using a haemoclip increased the number of transfected rod-shaped, viable myocytes from 1.7 +/- 0.8% (n = 8) to 5.6 +/- 0.8% (n = 9). This was further increased to 13.2 +/- 1.1% (n = 8) by the application of ultrasound pulses to the site before and after injection. Phospholamban antisense increased contraction amplitude and accelerated myocyte relengthening or decline of the Ca(2+) transient in transfected myocytes, while GFP control did not. Qualitative and quantitative effects of phospholamban downregulation were comparable between in vivo and in vitro transfections. This technique will have a number of uses, including production of transfected myocytes without the problem of culture-induced changes in contractility.
Sato
M
M
Department of Cardiac Medicine, NHLI Division, Imperial College London, UK.
O'Gara
P
P
Harding
S E
SE
Fuller
S J
SJ
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D016375:Antisense Elements (Genetics); D002135:Calcium-Binding Proteins / Q000235:genetics; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D007103:Immobilization; D008297:Male; D009200:Myocardial Contraction; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection; D014465:Ultrasonics; D000099211:Phospholamban
D016375:Antisense Elements (Genetics); D002135:Calcium-Binding Proteins; D000099211:Phospholamban