Recombinant Sindbis virus allows expression and precise targeting of proteins of the contractile apparatus in cultured cardiomyocytes.
Expression of epitope-tagged sarcomeric proteins in cardiomyocytes is a powerful approach for the characterization of interacting domains. Here, we report a new strategy for the study of the targeting of contractile proteins in cardiomyocytes by Sindbis virus (SIN)-mediated gene transfer. Two recombinant SIN were generated, one encoding the myosin-light chain MLC3f-eGFP fusion protein (SINrep5/MLC3f-eGFP), and the other encoding the alpha-actinin-DsRed fusion protein (SINrep5/alpha-actinin-DsRed). After infection of long-term cultured neonatal and adult rat cardiomyocytes with SINrep5/MLC3f-eGFP, the exogenous MLC3f-eGFP fusion protein localized to the sarcomeres. Freshly isolated rod-shaped ventricular cardiomyocytes infected with SINrep5/alpha-actinin-DsRed exhibited a correct incorporation of the newly synthesized alpha-actinin-DsRed fusion protein at the Z-band of the sarcomere. This allows the assumption that the exogenous protein is assembled into myofibrils in living cardiomyocy-tes using the same molecular interactions equally to the endogenous counterpart. It has been thus demonstrated that the SIN expression system makes possible the straightforward analysis of the localization of sarcomeric proteins in cultured cardiomyocytes and may offer new possibilities for the characterization of mutant proteins involved in hypertrophic cardiomyopathies.
Dätwyler
D A
DA
Institute of Cell Biology, ETH-Hoenggerberg, Zurich, Switzerland.
Magyar
J P
JP
Busceti
V
V
Hirschy
A
A
Perriard
J C
JC
Bailey
J E
JE
Eppenberger
H M
HM
D000185:Actinin / Q000235:genetics; D000818:Animals; D002478:Cells, Cultured; D003285:Contractile Proteins / Q000235:genetics*; D005260:Female; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000502:physiology*; D009200:Myocardial Contraction / Q000502:physiology; D009206:Myocardium / Q000166:cytology*; D018994:Myosin Light Chains / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics; D012845:Sindbis Virus / Q000235:genetics*; D014162:Transfection / Q000379:methods*
D003285:Contractile Proteins; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D018994:Myosin Light Chains; D011994:Recombinant Proteins; C413663:fluorescent protein 583; D000185:Actinin; D049452:Green Fluorescent Proteins