Independent regulation of Z-lines and M-lines during sarcomere assembly in cardiac myocytes revealed by the automatic image analysis software sarcApp
- Department of Cell and Developmental Biology, Vanderbilt University School of Medicine Basic Sciences, United States
- Sorbonne Université, INSERM, Institut de Myologie, Centre de Recherche en Myologie, France
Figures
Figure 1 with 1 supplement
yoU-Net follows a U-Net architecture to binarize immunofluorescence images.
(A) Representative images of α-actinin-2 and actin filaments (phalloidin) in a human induced pluripotent stem cell-derived cardiac myocyte (hiCM). The α-actinin-2 binary was thresholded in FIJI …
Figure 1—figure supplement 1
Details of yoU-Net architecture and trained model generation.
(A) U-Net architecture details used for this manuscript (details can be adjusted in yoU-Net). (B) Dice coefficient formula, used for validating trained models. (C) Representative fluorescence image …
Figure 2 with 1 supplement
Quantifying sarcomere and myofibril organization using α-actinin-2 binaries.
(A) Z-Lines and myofibrils identified by sarcApp. Each line denotes a Z-Line, and each different color represents a different myofibril. (B) Quantification scheme for myofibrils and Z-Lines. Details …
Figure 2—figure supplement 1
Details of geometric calculations used in sarcApp.
(A) How Z-Line spacing is calculated in sarcApp. In short, for each pair of Z-Lines, a line is measured from the centroid of one Z-Line to the next Z-Line using a line drawn perpendicularly to the …
Figure 3 with 3 supplements
Blebbistatin treatment ablates Z-Line formation in human induced pluripotent stem cell-derived cardiac myocytes (hiCMs).
(A) Representative images and insets of α-actinin-2 and F-actin in hiCMs treated with DMSO, 50 µM Blebbistatin, or 100 µM Blebbistatin. (B) Representative platinum replica EM image of a control hiCM …
Figure 3—figure supplement 1
α-actinin-2 quantification and organization in Blebbistatin-treated human induced pluripotent stem cell-derived cardiac myocytes (hiCMs) 6 hr post-plating.
(A) Myofibrils per cell in cells treated with DMSO or 50/100 µM Blebbistatin. N = 3 biological replicates, 73 control cells, 54 50 µM Blebbistatin cells, and 72 100 µM Blebbistatin cells. Myofibrils …
Figure 3—figure supplement 2
α-actinin-2 quantification and organization in Blebbistatin-treated human induced pluripotent stem cell-derived cardiac myocytes (hiCMs) 12 hr post-plating.
(A) Myofibrils per cell in cells treated with DMSO or 50/100 µM Blebbistatin. N = 3 biological replicates, 95 control cells, 77 50 µM Blebbistatin cells, and 80 100 µM Blebbistatin cells. Myofibrils …
Figure 3—figure supplement 3
α-actinin-2 quantification and organization in Blebbistatin-treated human induced pluripotent stem cell-derived cardiac myocytes (hiCMs) 24 hr post-plating.
(A) Myofibrils per cell in cells treated with DMSO or 50/100 µM Blebbistatin. N = 4 biological replicates, 118 control cells, 108 50 µM Blebbistatin cells, and 93 100 µM Blebbistatin cells. Note …
Figure 4
sarcApp uses titin binaries to identify myofibrils and precursor ring structures.
(A) Representative image of titin and α-actinin-2 in a control human induced pluripotent stem cell-derived cardiac myocyte (hiCM). Arrow: an α-actinin-2-positive Z-Body with titin localized in a …
Figure 5 with 1 supplement
Blebbistatin affects myofibril orientation and the morphology of titin structures.
(A) Representative images of titin and F-actin in human induced pluripotent stem cell-derived cardiac myocytes (hiCMs) treated with DMSO, 50 µM Blebbistatin, and 100 µM Blebbistatin. (B) Myofibrils …
Figure 5—figure supplement 1
Titin quantification and organization in Blebbistatin-treated human induced pluripotent stem cell-derived cardiac myocytes (hiCMs) 24 hr post-plating.
(A) Myofibrils per cell in cells treated with DMSO or 50/100 µM Blebbistatin. Myofibrils defined as having four or more doublets in a row. N = 3 biological replicates, 107 DMSO control cells, 95 50 …
Figure 6 with 1 supplement
sarcApp uses myomesin binaries to identify myofibrils and M-Lines in human induced pluripotent stem cell-derived cardiac myocytes (hiCMs).
(A) Schematic showing myomesin localization at the M-Line. (B) Representative image of myomesin and F-actin in a hiCM. The myomesin binary was predicted using yoU-net as described in the …
Figure 6—figure supplement 1
Myomesin quantification and organization in Blebbistatin-treated human induced pluripotent stem cell-derived cardiac myocyte (hiCMs) 24 hr post-plating.
(A) Myofibrils per cell in cells treated with DMSO or 50/100 µM Blebbistatin. Myofibrils defined as having three or more M-Lines in a row. N = 3 biological replicates, 112 DMSO control cells, 90 50 …
Figure 7 with 7 supplements
Knockdown of α or β cardiac myosin II reduces but does not eliminate sarcomeres.
(A) Schematic showing cardiac myosin localization in a sarcomere. (B) Representative western blot showing α cardiac myosin (MYH6) knockdown in hiCMs. (C) Representative images of α-actinin-2, titin, …
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Figure 7—source data 1
Original 16-bit image file for anti-MYH6 western blot.
See Figure 7-figure supplement 7A and Figure 7B.
- https://cdn.elifesciences.org/articles/87065/elife-87065-fig7-data1-v1.zip
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Figure 7—source data 2
Original 16-bit image file for anti-tubulin western blot.
See Figure 7-figure supplement 7A and Figure 7B.
- https://cdn.elifesciences.org/articles/87065/elife-87065-fig7-data2-v1.zip
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Figure 7—source data 3
Original 16-bit image file for anti-MYH7 western blot.
See Figure 7-figure supplement 7B and Figure 7H.
- https://cdn.elifesciences.org/articles/87065/elife-87065-fig7-data3-v1.zip
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Figure 7—source data 4
Original 16-bit image file for anti-tubulin western blot.
See Figure 7-figure supplement 7B and Figure 7H.
- https://cdn.elifesciences.org/articles/87065/elife-87065-fig7-data4-v1.zip
Figure 7—figure supplement 1
α-actinin-2 quantification and organization in MYH6 knockdown human induced pluripotent stem cell-derived cardiac myocytes (hiCMs).
(A) Myofibrils per cell in two groups of siCon (scramble)-treated hiCMs and two separate MYH6 siRNA-treated hiCMs (sequences 1 and 2). N = 3 biological replicates, 81 siCon cells and 78 siMYH6 (1) …
Figure 7—figure supplement 2
Titin quantification and organization in MYH6 knockdown human induced pluripotent stem cell-derived cardiac myocytes (hiCMs).
(A) Myofibrils per cell in siCon (scramble)-treated hiCMs and two separate MYH6 siRNA-treated hiCMs (sequences 1 and 2). Myofibrils were defined as having four or more doublets in a row. N = 3 …
Figure 7—figure supplement 3
Myomesin quantification and organization in MYH6 knockdown cells.
(A) Myofibrils per cell in siCon (scramble)-treated human induced pluripotent stem cell-derived cardiac myocytes (hiCMs) and two separate MYH6 siRNA-treated hiCMs (sequences 1 and 2). Myofibrils …
Figure 7—figure supplement 4
α-actinin-2 quantification and organization in MYH7 knockdown human induced pluripotent stem cell-derived cardiac myocytes (hiCMs).
(A) Myofibrils per cell in two groups of siCon (scramble)-treated hiCMs and two separate MYH7 siRNA-treated hiCMs (pools 1 and 2). N = 3 biological replicates, 86 siCon cells and 66 siMYH7 (1) …
Figure 7—figure supplement 5
Titin quantification and organization in MYH7 knockdown human induced pluripotent stem cell-derived cardiac myocytes (hiCMs).
(A) Myofibrils per cell in siCon (scramble)-treated hiCMs and two separate MYH7 siRNA-treated hiCMs (pools 1 and 2). Myofibrils defined as having four or more doublets in a row. N = 3 biological …
Figure 7—figure supplement 6
Myomesin quantification and organization in MYH7 knockdown cells.
(A) Myofibrils per cell in siCon (scramble)-treated human induced pluripotent stem cell-derived cardiac myocytes (hiCMs) and two separate MYH7 siRNA-treated hiCMs (pools 1 and 2). Myofibrils defined …
Figure 7—figure supplement 7
MYH6 and MYH7 knockdown western blots.
Figure 8 with 3 supplements
Myomesin knockdown alters titin and cardiac myosin II localization, but not α-actinin-2.
(A) Representative images of sarcomeric proteins α-actinin-2 and titin in myomesin (MYOM) knockdown human induced pluripotent stem cell-derived cardiac myocytes (hiCMs). (B) Representative western …
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Figure 8—source data 1
Original 16-bit image file for anti-MYH6 western blot.
See Figure 8—figure supplement 3A and Figure 8B.
- https://cdn.elifesciences.org/articles/87065/elife-87065-fig8-data1-v1.zip
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Figure 8—source data 2
Original 16-bit image file for anti-tubulin western blot.
See Figure 8—figure supplement 3A and Figure 8B.
- https://cdn.elifesciences.org/articles/87065/elife-87065-fig8-data2-v1.zip
Figure 8—figure supplement 1
α-actinin-2 quantification and organization in myomesin knockdown human induced pluripotent stem cell-derived cardiac myocytes (hiCMs).
(A) Myofibrils per cell in siCon (scramble)-treated hiCMs and two separate myomesin siRNA-treated hiCMs (sequences 1 and 2). Myofibrils defined as having four or more Z-Lines in a row. N = 4 …
Figure 8—figure supplement 2
Titin quantification and organization in myomesin knockdown human induced pluripotent stem cell-derived cardiac myocytes (hiCMs).
(A) Myofibrils per cell in siCon (scramble)-treated hiCMs and two separate MYOM siRNA-treated hiCMs (sequences 1 and 2). Myofibrils defined as having four or more doublets in a row. N = 3 biological …
Figure 8—figure supplement 3
MYOM knockdown western blots.
(A) Full image for the western blots shown in in Figure 8B. Individual lanes are denoted by numbers. Protein knockdown using siRNA targeted to MYOM. Individual lanes are denoted by numbers. (B) …
Author response image 1
Uncropped images and merges from Figures 2, 4 and 6, respectively.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Anti-alpha actinin 2 (mouse monoclonal) | Sigma | A7811 | IF, 1:200 |
| Antibody | Anti-Titin (mouse monoclonal) | DHSB | 9D10 | IF, 1:2 |
| Antibody | Mouse anti-Myomesin | DHSB | MYOM | IF, 1:2 |
| Antibody | Mouse anti-MYH | DHSB | A4.591 | IF, 1:2 |
| Antibody | Rabbit anti-MYH6 | ProteinTech | 22281-1-AP | WB, 1:500 |
| Antibody | Rabbit anti-MYH7 | ProteinTech | 22280-1-AP | WB, 1:500 |
| Antibody | Goat anti-mouse 488 | Life Technologies | A11001 | IF, 1:100 |
| Antibody | Goat anti-rabbit 488 | Life Technologies | A11034 | IF, 1:100 |
| Antibody | Goat anti-mouse 568 | Life Technologies | A11004 | IF, 1:100 |
| Antibody | Goat anti-rabbit 568 | Life Technologies | A11036 | IF, 1:100 |
| Antibody | Goat anti-mouse 647 | Life Technologies | A32728 | IF, 1:100 |
| Antibody | Goat anti-rabbit 647 | Life Technologies | A32733 | IF, 1:100 |
| Biological sample (Bos taurus) | Bovine serum albumin | RPI | A30075-100 | |
| Chemical compound, drug | Phalloidin Alexa Fluor 488 | Invitrogen | A12379 | |
| Chemical compound, drug | Phalloidin Alexa Fluor 568 | Invitrogen | A12380 | |
| Chemical compound, drug | Phalloidin Alexa Fluor 647 | Invitrogen | A22287 | |
| Chemical compound, drug | PBS, 10×, Ca2+/Mg2+ free | Life Technologies | 70011-044 | |
| Chemical compound, drug | PBS, 10×, with Ca2+/Mg2+ | Corning | 46-013CM | |
| Chemical compound, drug | PFA, 16% | Electron Microscopy Sciences | 15710 | |
| Chemical compound, drug | 0.5% Trypsin | Life Technologies | 15400-054 | |
| Chemical compound, drug | 0.1% Gelatin | Sigma | ES-006-B | |
| Chemical compound, drug | Dimethyl sulfoxide | Sigma | 276855 | |
| Chemical compound, drug | Vectashield with DAPI | Vector | H-1200 | |
| Chemical compound, drug | Blebbistatin | Sigma | B0560 | |
| Chemical compound, drug | Fibronectin | Corning | 354008 | |
| Chemical compound, drug | Lipofectamine RNAiMAX | Thermo Fisher | LMRNA015 | |
| Chemical compound, drug | TBS, 10× | Corning | 46-012CM | |
| Chemical compound, drug | Tween 20 | Sigma | P7949 | |
| Cell line (Homo sapiens) | iCell cardiomyocytes^2 kit | Fujifilm International | CMC-100-012-000.5 | |
| Sequence-based reagent | SMART Pool siRNA against human MYH7 (13–16) | Horizon Discovery | A-011086 | |
| Sequence-based reagent | SMART Pool siRNA against human MYH7 (13, 14) | Horizon Discovery | A-011086-13, A-011086-14 | |
| Sequence-based reagent | siRNA against human MYH6 3’ UTR (13) | Horizon Discovery | A-012645-13-0005 | |
| Sequence-based reagent | siRNA against human MYH6 CDS (14) | Horizon Discovery | A-012645-14-0005 | |
| Sequence-based reagent | siRNA against human myomesin 3’UTR (13) | Horizon Discovery | A-006342-13-0005 | |
| Sequence-based reagent | siRNA against human myomesin CDS (16) | Horizon Discovery | A-006342-16-0005 | |
| Software, algorithm | FIJI | NIH | ||
| Software, algorithm | sarcApp | This study | ||
| Software, algorithm | yoU-Net | This study |
