Differential Protein Localization in Sarcomeric and Nonsarcomeric Contractile Structures of Cultured Cardiomyocytes

Abstract

The use of cardiomyocyte cell culture models allows the identification of various cell mediators that bring about changes in subcellular structures and gene expression associated with hypertrophy. The effects of insulin-like growth factor-I (IGF-I), basic fibroblast growth factor (bFGF), and triiodothyronine (T3) on gene expression and on the structural organization of myofibrillar and cytoskeletal proteins were compared in adult atrial (aARC) and ventricular (vARC) as well as in neonatal ventricular rat cardiomyocytes (vNRC) in long-term culture. Structural changes were evaluated by confocal microscopy and correlated to biochemical alterations. In vARC, IGF-I enhanced myofibrillar growth, whereas bFGF or T3 restricted sarcomere assembly to the central cell area, forming a sharp boundary in more than 50% of the cells. However, myosin occurred both in the cross-striated myofibrillar structures and in patches running along the nonsarcomeric fibrillar structures (also called stress fiber-like structures) in the cell periphery. In cells treated with either bFGF or T3, the expression of α-smooth muscle actin (α-sm actin) was greatly increased. This actin isoform was incorporated mainly into the nonsarcomeric contractile structures outside the area where myofibrils ended abruptly. α-sm actin protein increased up to 14- to 17-fold while the mRNA showed a moderate increase of 2- to 4-fold. This suggests that α-sm actin is mainly regulated at the translational or posttranslational level. In contrast, the cytoskeletal proteins α-actinin and vinculin increased only moderately (less than 2-fold) but also showed a relocalization in cells with restricted myofibrils. In aARC and in vNRC, α-sm actin was only moderately upregulated by bFGF or T3 and no drastic morphological changes were observed. In conclusion, IGF-I, bFGF, and T3 induced characteristic structural phenotypes depending on the type of cardiomyocyte. Large amounts of α-sm actin as expressed in bFGF and T3 treated vARC seem to be incompatible with sarcomere assembly.