Cardiac myofibrillogenesis inside intact embryonic hearts

Abstract

How proteins assemble into sarcomeric arrays to form myofibrils is controversial. Immunostaining and transfections of cultures of cardiomyocytes from 10-day avian embryos led us to propose that assembly proceeded in three stages beginning with the formation of premyofibrils followed by nascent myofibrils and culminating in mature myofibrils. However, premyofibril and nascent myofibril arrays have not been detected in early cardiomyocytes examined in situ in the forming avian heart suggesting that the mechanism for myofibrillogenesis differs in cultured and uncultured cells. To address this question of in situ myofibrillogenesis, we applied non-enzymatic procedures and deconvolution imaging techniques to examine early heart forming regions in situ at 2- to 13-somite stages (beating begins at the 9-somite stage), a time span of about 23 h. These approaches enabled us to detect the three myofibril stages in developing hearts supporting a three-step model of myofibrillogenesis in cardiomyocytes, whether they are present in situ, in organ cultures or in tissue culture. We have also discovered that before titin is organized the first muscle myosin filaments are about half the length of the 1.6 mum filaments present in mature A-bands. This supports the proposal that titin may play a role in length determination of myosin filaments.

Figure 1. Arrangement of sarcomeric alpha-actinin

Arrangement of sarcomeric alpha-actinin (A, E, red in C, D, G, H) and non-muscle myosin IIB (B, F, green in C, D, G, H) early in myofibrillogenesis in cardiomyocytes fixed in situ (6-somite stage, HH stage 8+) (A–D), and in the spreading margin of cells cultured for three days after isolation from 10-day embryonic hearts (E–H). Graph and high magnification view of premyofibrils below each set illustrate the alternating pattern of the two proteins that can be resolved in cells in situ (D) and that is clearer in the flat cultured cardiomyocytes (H). The sizes and spacing of the two different types of bands are the same in the two types of cardiomyocytes. Bars = 5 µm A–C and E–G. Bars = 2 µm in D and H.

Figure 2. Premyofibrils and mature myofibrils

Premyofibrils and mature myofibrils in the same cardiomyocytes in situ (A–F) (9 somite stage, HH stage 9+). Two different focal planes of the same cardiomyocytes in situ stained with sarcomeric alpha-actinin antibody (A, D, red in C, F) and non-muscle myosin IIB antibody (B, E, green in C, F). In a plane near the middle of a cell marked at either end with asterisks (A–C), sarcomeric alpha-actinin is in Z-bands of myofibrils (A, red in C), and non-muscle myosin II is in small bands around the cell periphery (B, green in C). In a plane near the top of the same cell (asterisks, D–F), both sarcomeric alpha-actinin (D) and non-muscle myosin II (E) are in small bands suggesting that the assembly of myofibrils is initiated in proximity to the cell surface. Intermediate stages of assembly can be seen in the same cell. Bar = 5 µ.

Figure 3. Premyofibrils and Z-bands at same level of cell

Premyofibrils and mature myofibril in the same plane of a cardiomyocyte fixed in situ. (9 somite stage, HH stage 9+) stained with DAPI (blue in C) and antibodies to sarcomeric alpha-actinin (A and red in C) and non-muscle myosin IIB (B and green in C). (A) Sarcomeric alpha-actinin is present in both Z-bands (arrowheads) of a mature myofibril and z-bodies of a premyofibril (arrows) along the boundary of the same cell. (B) Non-muscle myosin IIB is localized with sarcomeric alpha-actinin in the premyofibril (arrows). (C) Non-muscle myosin IIB is not localized in bands between the Z-bands as expected. Bar = 5 µ.

Figure 4. Assembly of A-bands in cardiomyocytes in situ

Assembly of A-bands in cardiomyocytes in situ. The embryos were stained with antibodies against muscle myosin II and non-muscle myosin IIB at three developmental stages. (A–C) Early (6-somite), (D–F) middle (7-somite), and (G–I) late (12-somite) stages of myofibril assembly showed decreasing association of muscle-specific myosin II (A, D, G, red in C, F, I) with non-muscle myosin IIB (B, E, H, green in C, F, I). Before muscle myosin II was organized in A-bands (arrows D, F and G, I), it was present in bundles that varied in length (arrowheads A, C and D, F). Non-muscle myosin IIB was in small bands that often appeared to be coincident with fibrils of muscle-specific myosin in early stages (arrows, A–C); adjacent to myofibrils in middle stages (double arrows, E, F); and finally absent among mature myofibrils (G–H). Inserts show low magnification views of the cells and nuclei stained with DAPI (blue). Bars = 10 µm.

Figure 5. Intracellular asynchrony of myofibrillogenesis

Intracellular asynchrony of myofibrillogenesis in situ with nascent and mature myofibrils in the same cells (12-somite stage, HH stage 10+). Two focal planes (A–C and D–F) of the same cardiomyocytes in situ stained with the two different myosin II antibodies: muscle-specific myosin II (A, D red in C, F); non-muscle myosin IIB (B, D green in C, F). There was a greater amount of non-muscle myosin IIB in the plane just beneath the upper layer of tissue (B, C) than in a lower plane (E, F), whereas organized A-bands of muscle-specific myosin II were numerous in the lower plane (D, F) but few A-bands were present in the upper plane (A, C). In some cells the two myosins were in the same fibrils (arrows, A–C: double arrows point to area enlarged in insets), but they were not precisely colocalized (insets A–F). Non-muscle myosin IIB was not associated with the muscle myosin II in the A-bands, i.e., mature myofibril (arrowheads D, F). Bars in C, F = 10 µm. Bar in inset = 5 µm.

Figure 6. Division in situ

Cardiomyocyte in an embryo fixed in situ undergoing cytokinesis surrounded by interphase cells (12-somite stage, HH stage 10+). DAPI stains the nuclei of the interphase cells and the condensed chromatin (c) of the dividing cardiomyocyte. The cells have been stained with non-muscle myosin IIB (green) and muscle myosin II (red). Non-muscle myosin IIB is in a punctate arrangement in the cleavage furrow (between the arrowheads) and in fibrils in the interphase cells (small arrows). The muscle myosin II in the interphase cells is in an overlapping pattern (large arrow), but is not organized in the dividing cell. Bar = 5 µm.

Figure 7. Division in culture

Dividing cardiomyocyte in tissue culture from 10-day quail embryo hearts co-stained with antibodies to non-muscle myosin IIB (A, green in C) and sarcomeric alpha-actinin (B, red in C). Both proteins are arranged in a punctate pattern in the cleavage furrow (arrowhead). As expected, there is no non-muscle myosin IIB staining between the Z-bands of the mature myofibril (arrows in A to C) in an adjacent interphase cardiomyocyte. Bar = 5 µm.

Figure 8. Titin and muscle myosin II filaments

Titin and muscle myosin II filaments. Embryos at two developmental stages (2 somites A–F; 5 somites G–I) stained with either of two different muscle myosin II antibodies (MF20, A and red in C; Pepe myosin II antibody, D, G, and red in F, I) and titin (9D10, A–I junction epitope, B, E, H and green in C, F, I). At the 2 somite stage short rodlets of muscle myosin ( A, D, and red in C, F) were scattered in the cytoplasm, and titin staining was present as a few dots in some cells (B and green in C). The myosin rodlets were labeled uniformly with MF20 antibody (inset in A and C), whereas the staining with the Pepe myosin antibody revealed a bare zone in the middle of the rodlets (inset in D [arrows] and F). Similar myosin rodlets were present along with small aggregates of titin in the spreading margins of a cardiomyocyte in culture for three days after isolation from 10-day embryonic hearts (G–I). Box in G–I is shown enlarged in J–L with a myofibril from the cell at the same enlargement. Many of the myosin rodlets had a bare midzone (J arrows). The titin had no regular pattern of localization with respect to the myosin rodlets (K, L arrows). Bars = 5 µm.

Figure 9. Lengths of Muscle Myosin Filaments

(A) The distribution of lengths of muscle myosin “filaments” or rodlets (2 somite stage) and A-bands (13 somite stage) in cardiomyocytes fixed in situ. The myosin “filaments” at the 2 somite stage with an average length of 0.8 µm are much shorter than the 1.6 µm average myosin filament lengths measured in A-bands. (B) The distribution of lengths of muscle myosin “filaments” in the spreading edges of cultured cardiomyocytes and in A-bands in the mature myofibrils in the same cells. The myosin “filaments” or rodlets at the spreading edges with an average length of about 0.7 µm are much shorter than the 1.5 µm lengths measured in the A-bands.

Figure 10. Titin (9D10 antibody) and muscle myosin

Titin (9D10 antibody) and muscle myosin (Pepe antibody) localization during A-band assembly. Early (A–C), middle (D–F), and late (G–I) stages of A-band assembly show muscle specific myosin (A, D, G, red in C, F, I) progressing from long bundles of myosin filaments (A) to emerging A-bands (D) to ordered, aligned A-bands in myofibrils (G). Regions in small boxes A–F are shown at higher magnification in larger insets A–F. Before A-bands formed, muscle myosin was present in bundles of varying lengths (A, and red in C), and the 9D10 titin epitope was in small puncta (B, and green in C) that sometimes appeared as beads along the myosin bundles (large inset A–C). (5 somite stage) At later stages when A-bands began to form, the titin 9D10 epitope localized at the ends of the assembling A-bands (arrowheads in D–I). Bars in large insets A–F = 2µm. All other bars = 10µm.