Coupling to substrate adhesions drives the maturation of muscle stress fibers into myofibrils within cardiomyocytes

Figures

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  FIGURE 1:. Three-dimensional organization of myofibrils and DSFs. (A) SIM of actin filaments (magenta) and paxillin (green) in hiCM plated for 24 h. (B) SIM showing the layers of actin filaments in the axial (Z) dimension from the box in A. Arrowheads denote different DSFs. (C) Three-dimensional color projection of the data in (B). Lines denote the different DSFs denoted in B. (D, E) Focused laser-mediated cutting of DSF attached to an MSF (D) or a myofibril (E) in hiCM expressing Lifeact-mApple at 16 h postplating. Green arrowheads denote DSFs and blue arrowheads denote MSF or myofibril. Yellow line denotes cut site. Temporal color-coded overlays and image subtractions show the change in positioning of the MSF or myofibril following cutting of the DSF. See Supplemental Figure S1C for uncropped views of cells.

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  FIGURE 2:. Dynamics of adhesions and myofibril maturation. (A) Time montage of actin labeled with Lifeact-mEmerald and paxillin-mCherry during myofibril assembly. The first frame (top) and last frame (bottom) are separated by a time montage of the regions shown in the boxes (dotted lines). The purple arrowhead denotes an MSF becoming a myofibril. The green arrowhead denotes a growing adhesion. The yellow arrowhead denotes an extending DSF. (B) Representative SIM images of endogenous paxillin and α-actinin-2 in hiCMs plated from 6 h, 24 h, or 1 wk. (C) Quantification of Z-line lengths and adhesion area in hiCMs plated for 6 h, 24 h, and 1 wk. Z-line measurements: 6H: n = 58 cells, N = 4 independent experiments; 24H: n = 75 cells, N = 4 independent experiments; 1 Week: n = 81 cells, 3 independent experiments. Adhesion measurements: 6H; n = 38 cells, N = 5 independent experiments; 24H: n = 48 cells, N = 5 independent experiments; 1 Week: n = 28 cells, N = 3 independent experiments.

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  FIGURE 3:. Knockdown of vinculin results in attenuation of myofibril maturation. (A) Representative Western blot for vinculin knockdown using siRNA. Tubulin was used as a loading control. Knockdown efficiency was calculated from four independent experiments. (B) Paxillin localization (single optical section) in hiCMs treated with either scrambled or vinculin siRNA. (C) Quantification of adhesion area. si-Scr: n = 54 cells, N = 6 independent experiments; si-Vinculin: n = 30 cells, N = 4 independent experiments. (D) α-Actinin-2 localization (maximum Z-projection) in hiCMs treated with either scrambled or vinculin siRNA. (E) Quantification of average length of Z-lines. si-Scr: n = 131 cells, N = 6 independent experiments; si-Vinculin: n = 69 cells, N = 5 independent experiments. (F) Localization of titin in hiCMs treated with either scrambled or vinculin siRNA. (G) Quantification of distance of titin localization from the cell edge. si-Scr: n = 19 cells, N = 3 independent experiments; si-Vinculin: n = 21 cells, N = 3 independent experiments. Exact p values are stated in the graphs.

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  FIGURE 4:. Knockdown of vinculin results in faster translocation of MSFs. (A) Control (scrambled siRNA-treated) cell expressing Lifeact-mApple 16 h postplating. (B) Kymograph generated using yellow line in A. Yellow arrow denotes MSFs translocating away from the edge (dotted white line). (C) Vinculin-depleted cell expressing Lifeact-mApple 16 h postplating. (D) Kymograph generated using yellow line in C. Yellow arrow denotes MSFs translocating away from the edge (dotted white line). (E) Quantification of MSF translocation rates in control vs. vinculin-depleted cells. si-Scr: n = 30 MSFs from 19 cells; si-Vinculin: n = 22 MSFs from 14 cells; N = 3 independent experiments. Exact p values are stated in the graphs.

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  FIGURE 5:. Expression of talin head domain results in attenuation of myofibril maturation. (A) Representative hiCM expressing talin head-mEGFP vs. nonexpressing hiCM. (B) Paxillin localization (single optical section) in hiCM overexpressing talin head-mEGFP vs. a nonexpressing hiCM. (C) Quantification of adhesion area. Nonexpressing: n = 21 cells; talin head-mEGFP: n = 22 cells, N = 3 independent experiments each. (D) α-Actinin-2 localization (maximum Z-projection) in hiCM overexpressing talin head-mEGFP vs. a nonexpressing hiCM. (E) Quantification of average Z-lines length. Nonexpressing: n = 43 cells; talin head-mEGFP: n = 36 cells; N = 3 independent experiments each. (F) Localization of titin in hiCM overexpressing talin head-mEGFP vs. a nonexpressing hiCM. (G) Quantification of distance of titin localization from the cell edge. Nonexpressing: n = 19 cells; Talin head: n = 21 cells, N = 3 independent experiments each. (H) Quantification of MSF translocation rates in control hiCMs vs. hiCMs overexpressing talin head = mEGFP. Control: n = 25 MSFs from 16 cells; talin head-mEGFP: n = 26 MSFs from 17 cells; N = 3 independent experiments. Exact p values are stated in the graphs.

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  FIGURE 6:. Knockdown of FAK results in attenuation of myofibril maturation. (A) Representative Western blot for FAK knockdown using siRNA. Tubulin was used as a loading control. Knockdown efficiency was calculated from three independent experiments. (B) Paxillin localization (single optical section) in hiCMs treated with either scrambled or FAK siRNA. (C) Quantification of adhesion area in FAK-depleted cells. si-FAK: n = 36 cells, N = 4 independent experiments. The si-Scr dataset is the same as in Figure 3C. (D) α-Actinin-2 localization (maximum Z-projection) in hiCMs treated with either scrambled or FAK siRNA. (E) Quantification of average length of Z-lines in FAK-depleted cells. si-FAK: n = 106 cells, N = 4 independent experiments. The si-Scr dataset is the same as in Figure 3E. (F) Localization of titin in hiCMs treated with either scrambled or FAK siRNA. (G) Quantification of distance of titin localization from the cell edge. si-FAK: n = 12 cells, N = 3 independent experiments. The si-Scr dataset is the same as in Figure 3F. (H) Quantification of MSF translocation rates in FAK-depleted cells vs. scrambled siRNA-treated hiCMs. si-FAK: n = 25 MSFs from 16 cells; N = 3 independent…

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  FIGURE 7:. Inhibition of FAK kinase activity results in precocious myofibril maturation. (A) Localization of pFAK Y397 in control or 3 µM PF-228-treated hiCMs 24 h postplating. (B) Paxillin localization (single optical section) in control vs. PF-228-treated hiCMs 6 h postplating. (C) Quantification of adhesion area in PF-228-treated cells. Control: n = 38 cells, N = 5 independent experiments. PF-228: n = 44 cells, N = 4 independent experiments. (D) α-actinin-2 localization (maximum Z-projection) in control vs. PF-228-treated hiCMs 6 h postplating. (E) Quantification of average length of Z-lines in PF-228-treated hiCMs. Control: n = 58 cells, N = 4 independent experiments; PF-228: n = 107 cells, N = 4 independent experiments. (F) Localization of titin in control vs. PF–228-treated hiCMs after 6 h of spreading. (G) Quantification of distance of titin localization from the cell edge. Control: n = 23 cells; PF-228: n = 23 cells; N = 3 independent experiments each. (H) Quantification of MSF translocation rates in PF–228-treated cells vs. control. PF-228: n = 24 MSFs from 17 cells; N = 3 independent experiments. The control data set is the same is in Figure 5H. Exact p values are stated in the…

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  FIGURE 8:. Increasing fibronectin concentration results in precocious myofibril maturation. (A) Paxillin localization (single optical section) in hiCMs plated on either 10 µg/ml or 50 µg/ml fibronectin. (B) Quantification of adhesion area in hiCMs plated on either 10 µg/ml or 50 µg/ml fibronectin; 50 µg/ml: n = 47 cells, N = 5 independent experiments. The 10 µg/ml dataset is the same as Figure 8C. (C) α-Actinin-2 localization (maximum Z-projection) in hiCMs plated on either 10 µg/ml or 50 µg/ml fibronectin. (D) Quantification of average length of Z-lines in hiCMs plated on either 10 µg/ml or 50 µg/ml fibronectin; 50 µg/ml: n = 201 cells, N = 6 independent experiments. The 10 µg/ml dataset is the same as Figure 8E. (E) Localization of titin in hiCMs plated on either 10 µg/ml or 50 µg/ml fibronectin. (F) Quantification of distance of titin localization from the cell edge; 50 µg/ml: n = 23 cells, N = 3 independent experiments. The 10 µg/ml dataset is the same as Figure 8F. (G) Quantification of MSF translocation rates in hiCMs plated on either 10 µg/ml or 50 µg/ml fibronectin; 50 µg/ml: n = 20 MSFs from 12 cells; N = 3 independent experiments. The 10 µg/ml dataset is the same as in Figure 5H.…

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  FIGURE 9:. Knockdown of DIAPH1 results in attenuation of myofibril maturation. (A) Total internal reflection microscopy (TIRF) image showing the ventral section of an hiCM spread for 24 h. Actin filaments (phalloidin) and DIAPH1-mEmerald are shown in inverted gray. Note the colocalization of DIAPH1 with actin filaments associated with focal adhesions. (B) Representative Western blot for DIAPH1 knockdown using siRNA. Tubulin was used as a loading control. Knockdown efficiency was calculated from three independent experiments. (C) Three-dimensional SIM of actin filaments in a si-DIAPH1 KD hiCM at 24 h postplating. Note the lack of prominent DSFs. (D) Paxillin localization (single optical section) in hiCMs treated with either scrambled or DIAPH1 siRNA. (E) Quantification of adhesion area in DIAPH1- depleted cells. si-DIAPH1: n = 33 cells, N = 4 independent experiments. The si-Scr dataset is the same as Figure 3C. (F) α-Actinin-2 localization (maximum Z-projection) in hiCMs treated with either scrambled or DIAPH1 siRNA. (G) Quantification of average length of Z-lines in DIAPH1-depleted cells. si-DIAPH1: n = 58 cells, N = 3 independent experiments. The si-Scr dataset is the same as Figure 3E.…

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  FIGURE 10:. Working model for the role of substrate adhesion in myofibril maturation. Increasing cell–ECM adhesion results in greater mechanical coupling of MSFs to the ECM through DSFs. This is accompanied by slowing down of MSFs as they translocate away from the edge. Greater substrate coupling leads to the transition of the MSF to a myofibril, possibly by increased mechanical forces. Decreasing cell–ECM adhesion leads to reduced mechanical coupling, accompanied by faster translocation of MSFs and an inability to mature into force-generating myofibrils.