Figure 3
Epinephrine treatment induced sarcomere assembly through MLC2v phosphorylation.
Original magnification, ×1,000 (A–C and E–G). (A–D) Polymerized actin stained with rhodamine-phalloidin (A) as well as phosphorylated MLC2v labeled with p-s15MLC (B) exhibited regular patterns of striation. (C) Merged image of A and B. (D) Higher magnification of boxed area in C revealed that rhodamine-phalloidin predominantly stained the I-band, whereas phosphorylated MLC2v (p-MLC2v) was localized in the A-band. Original magnification, ×4,000 (D). (E–G) Cardiac-MLCK (cMLCK) labeled with RcMK showed a diffuse cytosolic labeling pattern. (H) Cultured cardiomyocytes were stimulated with 0.2–20 μM epinephrine (Epi), which upregulated MLC2v phosphorylation in a dose-dependent manner. (I) Cultured cardiomyocytes were stimulated with 2 μM epinephrine for the indicated time periods. Epinephrine-induced phosphorylation of MLC2v in cultured cardiomyocytes was observed as early as 5 minutes after stimulation; maximal phosphorylation was obtained after approximately 30 minutes. (J–L) Cardiomyocytes cultured with serum contained organized patterns of striation and a moderate level of MLC2v phosphorylation. Middle panels show higher magnification of boxed regions in top panels. Cardiomyocytes cultured in serum-free conditions were incubated in the absence (K) or presence (L) of 2 μM epinephrine. (K) Cardiomyocytes cultured under serum-free conditions contained disorganized, punctuated actin staining with a reduced level of MLC2v phosphorylation. (L) Stimulation with epinephrine provoked rapid sarcomere reassembly and augmented MLC2v phosphorylation. Original magnification, ×1,000 (J–L, upper and lower panels); ×3,000 (J–L, middle panels).
