Multiple influences of blood flow on cardiomyocyte hypertrophy in the embryonic zebrafish heart

Abstract

Cardiomyocyte hypertrophy is a complex cellular behavior involving coordination of cell size expansion and myofibril content increase. Here, we investigate the contribution of cardiomyocyte hypertrophy to cardiac chamber emergence, the process during which the primitive heart tube transforms into morphologically distinct chambers and increases its contractile strength. Focusing on the emergence of the zebrafish ventricle, we observed trends toward increased cell surface area and myofibril content. To examine the extent to which these trends reflect coordinated hypertrophy of individual ventricular cardiomyocytes, we developed a method for tracking cell surface area changes and myofibril dynamics in live embryos. Our data reveal a previously unappreciated heterogeneity of ventricular cardiomyocyte behavior during chamber emergence: although cardiomyocyte hypertrophy was prevalent, many cells did not increase their surface area or myofibril content during the observed timeframe. Despite the heterogeneity of cell behavior, we often found hypertrophic cells neighboring each other. Next, we examined the impact of blood flow on the regulation of cardiomyocyte behavior during this phase of development. When blood flow through the ventricle was reduced, cell surface area expansion and myofibril content increase were both dampened, and the behavior of neighboring cells did not seem coordinated. Together, our studies suggest a model in which hemodynamic forces have multiple influences on cardiac chamber emergence: promoting both cardiomyocyte enlargement and myofibril maturation, enhancing the extent of cardiomyocyte hypertrophy, and facilitating the coordination of neighboring cell behaviors.

Figure 1. Myofibril maturation proceeds during cardiac chamber emergence

(A–F) Ventral views of dissected wild-type hearts, arterial pole at the top, depict localization of phalloidin (A–C) and α-actinin (D–F) at 24, 36, and 48 hpf. As the ventricular chamber emerges, the striated pattern of myofibril Z-bands becomes increasingly apparent. (G–O) Higher magnification views of the regions marked by red dashed rectangles in A–C indicate the apparent progression of cardiomyocyte surface area expansion (G–I) and increased myofibril organization (J–L). The red dashed polygon outlines an individual cardiomyocyte in each panel. (M–O) Merged view of phalloidin and α-actinin localization reveals the appearance of myofibrils across the cell interior (N, arrow). Scale bars are 10 μm.

Figure 2. Method for quantification of myofibril content

(A) Schematic depicts a three-dimensional model of a cylindrical myofibril (blue cylinder), with a two-dimensional projection of its Z-bands (black rectangle). Sarcomere organization is illustrated on the side of the cylindrical myofibril. The green rings represent Z-bands, which are equidistant and intersected with thin and thick filaments (yellow lines) perpendicularly. In our confocal images, we view a two-dimensional projection of this type of three-dimensional object, such that Z-bands appear as line segments (green lines on black rectangle). In the two-dimensional projection, the length of a myofibril is represented by the number of parallel Z-bands, and the thickness of a myofibril is represented by the size of each Z-band. (B, C) Localization of cortical actin and Z-bands in the ventricle at 48 hpf, visualized by phalloidin and α-actinin antibody staining (B), can be converted into measurable objects (C). Red polygons, tracing cell boundaries, are used to quantify cell surface area. Green line segments, tracing Z-bands, are used to quantify Z-band size. Scale bar is 10 μm. See Materials and Methods and Figure S1 for additional details regarding our imaging and measurement methods.

Figure 3. Concomitant increases in cell size and myofibril content during chamber emergence

(A) Bar graph indicates mean cell surface area measurements in μm² for ventricular cardiomyocytes at the indicated stages; error bars indicate standard error. An asterisk indicates a statistically significant difference from the prior timepoint (p<0.05). Cardiomyocytes expand in size continuously as chambers form between 24 and 48 hpf. (B) Bar graph indicates mean measurements of total Z-band size in μm per ventricular cardiomyocyte at the indicated stages; error bars indicate standard error. An asterisk indicates a statistically significant difference from the prior timepoint (p<0.05). Myofibril content per cell increases continuously as chambers form between 24 and 48 hpf. See Materials and Methods for details regarding measurements.

Figure 4. Live imaging demonstrates hypertrophic behavior of cardiomyocytes

(A, B) Lateral views, arterial pole at the top, of the ventricular outer curvature in a live embryo at 40 hpf (A) and 45 hpf (B); expression of Tg(myl7:mkate-caax) highlights cardiomyocyte cell contours. Selected cells from the central portion of the imaged territory were selected for analysis when they were clearly rendered in the plane of imaging and had adequately intense expression of both transgenes. Numbered cells were compared at 40 and 45 hpf; individual cells could be tracked between timepoints based on the configuration of cell contacts and the landmarks provided by variation in levels of transgene expression. For example, the position of cell 2 is unambiguous because of its relationships to cells 1, 3, and 4, all of which exhibit lower levels of transgene expression. (C, D) Lateral view of the ventricular outer curvature in the same live embryo at 40 hpf (C) and 45 hpf (D); expression of Tg(myl7:actn3b-egfp) highlights Z-bands. (E, F) Overlay of the fluorescent signals from both transgenes reveals changes in cell size and myofibril content between 40 and 45 hpf. The hypertrophic cell outlined in white is cell #3 from panels A and B; its surface area increased 25.6% over the imaged time interval and its Z-bands became larger and more numerous, resulting in a 103.4% change in total Z-band size. See Materials and Methods for additional details regarding our imaging and measurement methods. Scale bars are 10 μm.

Figure 5. Heterogeneity of cardiomyocyte behavior during chamber emergence

(A) Scatter plot depicts observed behaviors of individual cardiomyocytes analyzed by live imaging at 40 and 45 hpf. Each symbol represents an individual cardiomyocyte, plotted with respect to its percent change in cell surface area (x-axis) and its percent change in total Z-band size (y-axis). Cell behaviors are subdivided into four categories, according to the observed changes in cell surface area and total Z-band size. We consider a change of greater than 1% in surface area or a change of greater than 5% in total Z-band size to represent a substantial increase in either of these parameters over the observed 5-hour timeframe. Red symbols indicate cells that substantially increased both their size and myofibril content. Yellow symbols indicate cells that substantially increased only their myofibril content. Blue symbols indicate cells that substantially increased only their size. Green symbols indicate cells that did not increase either their size or their myofibril content. The percentage of the population in each category is indicated in the appropriate corner of the scatter plot. (B–E) Bar graphs depict the frequencies with which cells in a particular category shared a cell boundary with a cell in each of the four behavioral categories (colored bars), compared to the observed frequencies of occurrence of each behavior in our data set (grey bars). Asterisks indicate statistically significant differences between the calculated frequency of neighboring a cell from a specific category (colored bar) and the theoretical frequency of randomly encountering a cell from that category (grey bar) (p<0.05; Fisher’s exact test). See Materials and Methods for details of calculations.

Figure 6. Myofibril maturation is diminished in wea mutant embryos

(A–F) Ventral views, arterial pole at the top, of dissected hearts at 28, 38, and 50 hpf depict localization of Actn3b-egfp (green) in Z-bodies and Z-bands and Dm-grasp (red) at cell boundaries. Actn3b-egfp and Dm-grasp localization indicate the dynamic progression of cell size and myofibril content during chamber emergence. Wild-type (wt) and wea mutant ventricular cardiomyocytes are indistinguishable at 28 hpf (A, D), but cell size expansion and myofibril growth and organization do not progress normally in wea mutants (B, C, E, F). Scale bar is 10 μm. (G) Bar graph indicates mean cell surface area measurements in μm² for ventricular cardiomyocytes at the indicated stages; error bars indicate standard error. Asterisks indicate statistically significant differences between wild-type and wea (p<0.05); these differences become apparent by 38 hpf. (H) Bar graph indicates mean measurements of total Z-band size in μm per ventricular cardiomyocyte at the indicated stages; error bars indicate standard error. The asterisk indicates a statistically significant difference between wild-type and wea at 50 hpf (p<0.05). The carat indicates a less striking difference between wild-type and wea at 38 hpf (p=0.06).

Figure 7. Blood flow influences the degree and spatial organization of cardiomyocyte hypertrophy

(A) Scatter plot depicts observed behaviors of individual cardiomyocytes analyzed by live imaging at 40 and 45 hpf in wea morphant embryos. Each symbol represents an individual cardiomyocyte, plotted with respect to its percent change in cell surface area (x-axis) and its percent change in total Z-band size (y-axis). Cell behaviors are subdivided into four categories, based on the criteria described for Figure 5A and color-coded as in Figure 5A. Notably, in comparison to our wild-type data set (Fig. 5A), we found fewer hypertrophic cells (red), more cells that did not increase either their size or myofibril content (green), and fewer cells that only increase their size but not their myofibril content (blue). (B) Scatter plot compares the hypertrophic cells found in wild-type and wea morphant hearts in terms of their absolute increases in cell surface area (x-axis) and total Z-band size (y-axis). The average extent of the increase in both parameters is significantly dampened in wea morphants. Scatter plots depicting the absolute amounts of change for all observed cells in both wild-type and wea morphant embryos are included in Figure S5. (C–F) Bar graphs depict the frequencies with which wea morphant cells in each behavioral category were neighbors of cells from each of the four categories, as in Figure 5B–E. Notably, we found no statistically significant differences between any of the calculated frequencies (colored bars) and theoretical frequencies (grey bars) in our wea morphant data set. For example, unlike in wild-type hearts, the hypertrophic cells in wea morphant hearts do not exhibit a particular tendency to neighbor each other.