CAP2 is a regulator of actin pointed end dynamics and myofibrillogenesis in cardiac muscle

Fig. 1. CAP2 localizes to the pointed ends of thin filaments. Subcellular assembly of CAP2 in chick embryonic cardiomyocytes was investigated by a immunostaining for endogenous CAP2 and by b expressing GFP-CAP2. Anti-α-actinin (red) antibodies were used to mark the Z-discs of the sarcomeres. GFP-CAP2-expressing cells were stained for c Tmod1, or d myomesin (red) to mark thin filament pointed ends or M-lines, respectively, and subjected to super resolution-structured illumination microscopy (SR-SIM). The intensity profiles of stained proteins reveal that GFP-CAP2 resolves both into singlets (yellow arrowheads) and into doublets at the pointed ends (red arrowheads) based on the contractile state. The line intensity profile of GFP-CAP2 doublets (green lines) colocalize with the profile of Tmod1, but not with that of myomesin (red lines). Scale bar = 1 μm.

Fig. 2. The levels of CAP2 protein decrease in neonatal rat cardiomyocytes during myofibrillogenesis in culture. a Immunoblot analysis of proteins from whole cell lysates on days 1, 3, and 5 after plating. Quantification of immunoblots show compared to day 1, b decreasing CAP2 levels (**p = 0.0066, *p = 0.0112, F = 14.37), c increasing Lmod2 levels (n.s.: p = 0.4078, *p = 0.0236, F = 6.985) and d unchanged Tmod1 levels (n.s.: p = 0.1499, n.s.: p = 0.3140, F = 2.588) as cardiomyocytes mature (mean ± SD, n = 3 cultures, one-way ANOVA, df = 2, n.s.: not significant).

Fig. 3. Depletion of actin monomers disrupts the assembly of CAP2, but not Tmod1 in cardiomyocytes. Neonatal rat cardiomyocytes were treated with dimethyl sulfoxide (DMSO, control) or latrunculin A (Lat A) and fixed. Immunostaining was done using: a anti-CAP2 (green), or b anti-Tmod1 (green) and anti-α-actinin (red) antibodies, and phalloidin to probe for F-actin. Scale bar = 10 μm. c, d % of cells showing consistent CAP2 (black dots) or Tmod1 (red dots) pointed end striations, respectively (mean ± SD, 30 cells/culture, n = 3 cultures, ****p < 0.0001, t = 25.71; n.s.: p = 0.8975, t = 0.1373, Student’s t-test, df = 4, n.s.: not significant).

Fig. 4. CAP2 depolymerizes actin filaments but stabilization by tropomyosin prevents CAP2-mediated filament disassembly. a Immunoblot analysis for GFP, globular (G-actin, G) and filamentous (F-actin, F) cardiac actin (cActin) in neonatal rat cardiomyocytes (NRCMs) transduced with GFP or GFP-CAP2 adenovirus. b Quantification of immunoblots demonstrates that the percentage of cardiac F-actin (red dots) was significantly lower in GFP-CAP2-expressing cells (49.5 ± 3.4%) compared to GFP-expressing control cells (60.6 ± 3.7%) (mean ± SD, n = 3 cultures, *p = 0.0105, two-way ANOVA F = 28.78, df = 1). c Immunoblot analysis for G-actin or F-actin from NRCMs treated with 0.01% dimethyl sulfoxide (DMSO), 2 μM Latrunculin A (Lat A), or 0.15 μM jasplakinolide (Jasp.) overnight. d Quantification of immunoblots demonstrates that Lat A treatment results in less (71.5 ± 7.0 to 18.5 ± 5.6%), whereas jasplakinolide (Jasp.) treatment leads to more F-actin (92.3 ± 3.4%, red dots), respectively (mean ± SD, n = 3 cultures, ****p < 0.0001, **p = 0.0038, two-way ANOVA F = 280.3, df = 2). e Actin filaments (5 μM) in the absence or presence of 5 μM striated muscle α-tropomyosin (Tpm1.1) were diluted five-fold and…

Fig. 5. CAP2 inhibits actin dynamics by delaying actin incorporation to thin filaments in cardiomyocytes. a Representative images of fluorescence recovery after photobleaching (FRAP) of GFP-cardiac actin (cActin) in neonatal rat cardiomyocytes transduced with mCherry or mCherry-CAP2. Barbed (yellow arrowheads) and pointed (blue arrowheads) ends of actin filaments are marked. b Mean relative recovery at thin filament pointed ends after photobleaching over time in mCherry (black dots) or mCherry-CAP2-expressing cells (red dots). Rate constants (k) for the c fast and d slow phases of association of GFP-cActin in mCherry (black dots) or mCherry-CAP2-expressing cells (red dots) are indicated as mean ± SEM (n = 11, 7, 13, 10 cells from three independent cultures; n.s.: p = 0.9671, ***p = 0.0006, F = 6.330; n.s.: p = 0.8908, *p = 0.0204, F = 19.10, two-way ANOVA, df = 1, n.s.: not significant). e Representative images of FRAP of GFP-cActin in neonatal WT or Cap2-KO mouse cardiomyocytes. Barbed (yellow arrow) and pointed (blue arrowheads) ends of actin filaments are marked. f Mean relative recovery at thin filament pointed ends after photobleaching over time in WT (black dots) or Cap2-KO…

Fig. 6. Cap2-KO cells exhibit thin filament disorganization with higher levels of α-smooth muscle and α-skeletal muscle actin and a delay in maturation. Cardiomyocytes were isolated from hearts of neonatal WT or Cap2-KO mice and fixed a 1 day or b 5 days after plating for immunofluorescence analysis. Immunostaining of WT or Cap2-KO cells was performed using anti-α-actinin (red) antibody, and phalloidin (green) to probe for F-actin. Immunostaining of WT or Cap2-KO cardiomyocytes 5 days after plating with c anti-α-smooth muscle actin (α-SMA, green) d anti-cardiac actin (cActin, green) or e anti-skeletal muscle actin (α-SKA, green) antibody, and phalloidin (red) to probe for F-actin. Scale bar = 5 μm. f Immunoblot analysis for whole cell lysates of WT (black dots) or Cap2-KO cells (red dots) collected 5 days after plating. Quantification of immunoblots shows unchanged levels of cActin but increased levels of α-SMA and α-SKA (~2-fold) in Cap2-KO cells (mean ± SD, n = 3–4 cultures, n.s.: p = 0.1780, t = 1.567, df = 5; **p = 0.0044, t = 4.436, df = 6; *p = 0.0467, t = 2.844, df = 4, Student’s t-test, n.s.: not significant).

Fig. 7. Thin filaments from Cap2-KO mice are composed of a heterogenous population of actin isoforms. Immunoblot analysis from left ventricular tissue of WT (black dots) or Cap2-KO mice (red dots) at postnatal days (PD) a 15 (**p = 0.0092, t = 3.102, df = 12; **p = 0.0012, t = 4.328, df = 11; ****p < 0.0001, t = 8.962, df = 11, n.s.: p = 0.9505, t = 0.06343, df = 12), b 60 (**p = 0.0032, t = 3.667, df = 12; ****p < 0.0001, t = 10.64, df = 12, **p = 0.0018, t = 3.993, df = 12, n.s.: p = 0.7179, t = 0.3699, df = 12) and c 120 (*p = 0.0116, t = 2.974, df = 12; ****p < 0.0001, t = 10.75, df = 12; ****p < 0.0001, t = 6.788, df = 12; n.s.: p = 0.6945, t = 0.40339, df = 11). Quantification of immunoblots show down-regulation of cardiac actin (cActin), up-regulation of α-smooth muscle actin (α-SMA) and α-skeletal muscle actin (α-SKA) but unchanged levels of total α-actin in Cap2-KO mice (mean ± SD, n = 6–7 mice, Student’s t-test, n.s.: not significant). Representative immunofluorescence images of PD 120 WT or Cap2-KO mice stained with d anti-cActin, e anti-α-SMA or anti-α-SKA antibodies. f Line scan and plot profile analyses reveal shorter lengths for cActin striations in Cap2-KO (red dots) mice…

Fig. 8. The levels of CAP2 protein decrease in mice with age. a Immunoblot analysis of proteins from left ventricular lysate of WT mice on postnatal days (PD) 15, 60, 120. Quantification of immunoblots show that b CAP2 (***p = 0.0004, ****p < 0.0001, F = 45.87) and c α-smooth muscle actin (α-SMA) levels (*p = 0.0363, *p = 0.0337, F = 6.085) decrease with age, whereas d α-skeletal muscle actin (α-SKA) follows a biphasic expression pattern (***p = 0.0005, *p = 0.0250, F = 20.61) and e cActin is stably expressed (n.s.: p = 0.2429, n.s.: p = 0.5915, F = 1.529) compared to PD 15 (mean ± SD, n = 3–4 mice, one-way ANOVA, df = 2, n.s.: not significant).