The formin Fhod3 is required for sarcomere reassembly after mitosis in postnatal cardiomyocytes

Figures

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  Fig. 1. MCK-Cre mediated deletion of Fhod3 in primary culture of cardiomyocytes. A Preparation of primary culture from Fhod3 cKO (Fhod3flox/–; MCK-CreTg) and control littermate (Fhod3flox/+) P0 neonates. Partially created with BioRender.com (https://www.biorender.com). B PCR analyses of genomic DNAs prepared from cultured cardiomyocytes at DIV8. Small black arrows “a”– “d” indicate primers used. The PCR product from the wild-type allele (Fhod3+) or the null allele (Fhod3–) was 329 bp in length (#3). The PCR product from the floxed allele (Fhod3flox) was 387 bp in length (#2). The 549-bp fragment was produced by Cre-mediated recombination (#1). C Detection of the Fhod3 protein by immunoblot analysis. Proteins prepared from cultured cardiomyocytes of Fhod3 cKO (Fhod3flox/–; MCK-CreTg) and control littermate (Fhod3flox/+) P0 neonates at DIV10 were analyzed by immunoblot with the anti-Fhod3-(873–974). The loading amounts were verified by immunoblot with anti-GAPDH antibody. D Confocal fluorescence micrographs of cultured cardiomyocytes from Fhod3 cKO (Fhod3flox/–; MCK-CreTg) and control littermate (Fhod3flox/+) neonates. Cardiomyocytes at DIV8 were stained with the anti-α-actinin (red) and…

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  Fig. 2. Long-term monitoring experiment of Fhod3-depleted cardiomyocytes with MCK-Cre. A Primary cultures prepared from Fhod3 cKO (Fhod3flox/–; MCK-CreTg) and control littermate (Fhod3flox/+) P0 neonates were monitored every 3 h with the incubation monitoring system CM30 housed within a standard CO2 incubator for 7–15 days. Partially created with BioRender.com (https://www.biorender.com). B The percentage of cardiomyocytes that are positive for Fhod3 expression (left) and that are positive for well-organized sarcomeres (right) determined from immunofluorescent images at the end of culture on DIV 7–15 (experiment #1, DIV15; experiment #2, DIV7; experiment #3, DIV12). The criteria for positivity of Fhod3 and sarcomere were determined as described under “Materials and Methods.” For each genotype, the ratios of positive cells were calculated from a total of 43–101 cells in a single monitoring experiment, and the mean ± SEM was obtained from three independent monitoring experiments with cardiomyocytes prepared separately. *, p < 0.05. C Representative immunofluorescent images of cultured cardiomyocytes from control littermate (Fhod3flox/+) P0 neonates at DIV7 (left panels) and the…

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  Fig. 3. Time-lapse imaging reveals the temporal relationship between mitosis and sarcomere disruption in Fhod3-depleted cardiomyocytes. A–B Representative time-lapse images of mitotic events in cardiomyocytes prepared from control littermate (Fhod3flox/+) A and Fhod3 cKO (Fhod3flox/–; MCK-CreTg) B P0 neonates (left panels) and the final frame of monitoring images and corresponding immunofluorescent images at the end of culture (right panel). Scale bars, 20 μm. Yellow arrowheads indicate nuclear division. C–D The temporal relationship between the timing of mitosis and sarcomere disruption. Cultured cardiomyocytes from control littermate (Fhod3flox/+) C and Fhod3 cKO (Fhod3flox/–; MCK-CreTg) D neonates were classified into two groups based on the presence or absence of Fhod3 signals in immunofluorescence images at the end of culture: Fhod3-positive and Fhod3-negative groups (upper and lower). Subsequently, each cardiomyocyte was plotted against the timing of mitotic events (DIV), with color-coded levels of sarcomere organization: deep blue, excellent; sky blue, high; pink, low; red, poor. The criteria for levels of sarcomere organization were determined as described under “Materials and…

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  Fig. 4. 4-OHT-induced deletion of Fhod3 in primary cultured cardiomyocytes. A Preparation of primary culture from Fhod3flox/flox; αMHC-MerCreMerTg/Tg P0 neonates. Partially created with BioRender.com (https://www.biorender.com). B PCR analyses of genomic DNAs prepared at the indicated DIV from Fhod3flox/flox; αMHC-MerCreMerTg/Tg cardiomyocytes treated with DMSO or 4-OHT. The PCR product from the floxed allele (Fhod3flox) was 387 bp in length (#2). The 549-bp fragment was produced by Cre-mediated recombination (#1). Primers used are shown in Fig. 1B. C Detection of the Fhod3 protein by immunoblot analysis. Proteins prepared at the indicated DIV from cultured cardiomyocytes of Fhod3flox/flox; αMHC-MerCreMerTg/Tg P0 neonates were analyzed by immunoblot with the anti-Fhod3-(873–974). The loading amounts were verified by immunoblot with anti-GAPDH antibody. D Representative immunofluorescent images of cultured cardiomyocytes from Fhod3flox/flox; αMHC-MerCreMerTg/Tg P0 neonates. Cardiomyocytes were treated with 0.5 μM 4-OHT from DIV1, then fixed at DIV10 and stained with the anti-troponin T (green) and anti-Fhod3-(650–802) (green) antibodies and Hoechst (blue). Scale bars, 20 μm

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  Fig. 5. Effect of varying the timing of 4-OHT treatment on sarcomeric structure. (A-B) Representative immunofluorescent images of cultured cardiomyocytes from Fhod3flox/flox; αMHC-MerCreMerTg/Tg P0 neonates. Cardiomyocytes were treated with 0.5 μM 4-OHT from DIV1 A and from DIV9 B, then fixed at DIV22 and stained with the anti-α-actinin (red) and anti-Fhod3-(650–802) (green) antibodies and Hoechst (blue). Scale bars, 20 μm. C Cardiomyocytes prepared from Fhod3flox/flox; αMHC-MerCreMerTg/Tg P0 neonates were treated with 0.5 μM 4-OHT from DIV1 or from DIV9, then fixed and immunostained at the indicated DIV. D-E The percentage of cardiomyocytes that are positive for Fhod3 expression D and that are positive for well-organized sarcomeres E determined from immunofluorescent images at the indicated DIV. The evaluation of images was conducted by the researcher blinded to conditions, with the criteria described in “Materials and Methods.” The ratios of positive cells were calculated from a total of 55–197 (119 ± 36) cells in a single experiment for each condition (total 7,154 cells), and the mean ± SEM was obtained from three independent experiments with cardiomyocytes prepared separately

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  Fig. 6. Long-term monitoring experiment of Fhod3-depleted cardiomyocytes with αMHC-MerCreMer. A-B Primary cultures prepared from Fhod3flox/flox; αMHC-MerCreMerTg/Tg P0 neonates were monitored every 3 h with the incubation monitoring system CM30 housed within a standard CO2 incubator for 17–22 days. Partially created with BioRender.com (https://www.biorender.com). C-D The percentage of cardiomyocytes that are positive for Fhod3 expression C and that are positive for well-organized sarcomeres D determined from immunofluorescent images at the end of culture on DIV 17–22 (experiment #1, DIV22; experiment #2, DIV18; experiment #3, DIV17). The criteria for positivity of Fhod3 and sarcomere were determined as described under “Materials and Methods.” For each condition, the ratios of positive cells were calculated from a total of 4–60 cells in a single monitoring experiment, and the mean ± SEM was obtained from three independent monitoring experiments with cardiomyocytes prepared separately. *, p < 0.05; **, p < 0.01; ns, not significant. E–F The temporal relationship between the timing of mitosis and sarcomere disruption in cardiomyocytes treated with DMSO or 0.5 μM 4-OHT from DIV1 E and from…

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  Fig. 7. Effect of blocking the cell cycle in the G2/M phase on sarcomere disassembly in Fhod3-depleted cardiomyocytes. A Preparation of primary culture from Fhod3 cKO (Fhod3flox/–; MCK-CreTg) P0 neonates. Partially created with BioRender.com (https://www.biorender.com). B Cardiomyocytes were treated with 10 μM RO3306 or DMSO for 16 h before the fixation at DIV3. C The percentage of binucleated cardiomyocytes determined from immunofluorescent images at the end of culture on DIV3. D–F The percentage of cardiomyocytes that are positive for Fhod3 expression D, that are positive for well-organized sarcomeres E, and that are positive for well-organized sarcomeres in Fhod3 negative cardiomyocytes F, determined from immunofluorescent images at the end of culture on DIV3. The criteria for positivity of Fhod3 and sarcomere were determined as described under “Materials and Methods.” For each condition, the ratios of positive cells were calculated from a total of 67–92 cells in a single monitoring experiment, and the mean ± SEM was obtained from four independent monitoring experiments with cardiomyocytes prepared separately. **, p < 0.01; ns, not significant. G-H Representative immunofluorescent…

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  Fig. 8. Schematic representation of the involvement of Fhod3 in the final mitosis after birth. In the presence of Fhod3, sarcomere reassembly after mitosis is successful. The depletion of Fhod3 after the last mitosis demonstrates minimal effect on sarcomeres, whereas depletion before the last mitosis results in a failure of sarcomere reassembly. Partially created with BioRender.com (https://www.biorender.com)