Profilin modulates sarcomeric organization and mediates cardiomyocyte hypertrophy

Abstract

Aims: Heart failure is often preceded by cardiac hypertrophy, which is characterized by increased cell size, altered protein abundance, and actin cytoskeletal reorganization. Profilin is a well-conserved, ubiquitously expressed, multifunctional actin-binding protein, and its role in cardiomyocytes is largely unknown. Given its involvement in vascular hypertrophy, we aimed to test the hypothesis that profilin-1 is a key mediator of cardiomyocyte-specific hypertrophic remodelling.

Methods and results: Profilin-1 was elevated in multiple mouse models of hypertrophy, and a cardiomyocyte-specific increase of profilin in Drosophila resulted in significantly larger heart tube dimensions. Moreover, adenovirus-mediated overexpression of profilin-1 in neonatal rat ventricular myocytes (NRVMs) induced a hypertrophic response, measured by increased myocyte size and gene expression. Profilin-1 silencing suppressed the response in NRVMs stimulated with phenylephrine or endothelin-1. Mechanistically, we found that profilin-1 regulates hypertrophy, in part, through activation of the ERK1/2 signalling cascade. Confocal microscopy showed that profilin localized to the Z-line of Drosophila myofibrils under normal conditions and accumulated near the M-line when overexpressed. Elevated profilin levels resulted in elongated sarcomeres, myofibrillar disorganization, and sarcomeric disarray, which correlated with impaired muscle function.

Conclusion: Our results identify novel roles for profilin as an important mediator of cardiomyocyte hypertrophy. We show that overexpression of profilin is sufficient to induce cardiomyocyte hypertrophy and sarcomeric remodelling, and silencing of profilin attenuates the hypertrophic response.

Keywords: Cardiac hypertrophy; Cardiomyocyte; Profilin-1; Sarcomere remodelling; chickadee.

Figure 1

Increased expression of profilin-1 in hypertrophic cardiomyocytes. (A) Pressure-overload following TAC resulted in elevated levels of profilin-1 in mouse hearts. Protein levels were corrected for total gel loading (see Supplementary material online, Figure S1A). Representative actin bands from Direct Blue 71-stained membranes are shown. A significant increase in profilin-1/total protein was observed in the myocardium of the TAC vs. Sham group (n = 5–10, *P < 0.05; Student's t-test). (B) A hypertrophic/HF mouse model overexpressing wild-type Gαq showed higher levels of cardiac profilin-1 compared with control (n = 3–7, *P < 0.05; Student's t-test). Protein levels were corrected for total gel loading (see Supplementary material online, Figure S1A). Representative actin bands from Direct Blue 71-stained membranes are shown. (C) PE treatment in NRVMs increased Pfn1 mRNA (n = 6, *P < 0.05; Student's t-test) and profilin-1 (n = 6, ***P < 0.001; Student's t-test) content. (D) Representative confocal images of control murine cardiac tissue show profilin-1 repetitively associates with myofibrils in a striated manner. Scale bar, 10 μm.

Figure 2

Cardiomyocyte-specific overexpression of profilin in Drosophila induces cardiomyopathy. (A) Representative M-mode kymograms generated from high-speed videos of beating control, Pfn_1, and Pfn_2 heart tubes. DD, diastolic diameter; SD, systolic diameter; HP, heart period. (B) Semi-automated optical heartbeat analysis from flies overexpressing profilin via the HG4 cardiac-specific driver revealed significant reductions in heart period and increased cardiac dimensions relative to control (n = 28–30, *P < 0.05, **P < 0.01 and ***P < 0.001; Kruskal–Wallis test with Dunn's post hoc test for HP and SD analysis; one-way ANOVA with the Bonferroni post hoc test for DD analysis).

Figure 3

Overexpression of profilin in Drosophila IFM impairs muscle function and ultrastructure. (A) Western blot analysis showed increased profilin in whole Mef2 > Pfn_1 and Mef2 > Pfn_2 transgenic flies (top) (n = 5, *P < 0.05, **P < 0.01; one-way ANOVA with the Bonferroni post hoc test). Actin/myosin heavy chain ratios remained unchanged in flies with muscle-restricted profilin overexpression compared with control (bottom) (n = 5). (B) Two-day-old Mef2 > Pfn_1 and Mef2 > Pfn_2 flies were unable to fly and demonstrated significantly reduced climbing ability (n = 35–64, ***P < 0.001; Kruskal–Wallis test with Dunn's post hoc test). (C) UH3-GAL4-mediated overexpression of profilin significantly diminished flight ability (n = 54–83, ***P < 0.001; one-way ANOVA with the Bonferroni post hoc test). (D) Representative electron micrographs of transverse sections of Mef2 > Pfn_1 IFMs (top) show that the double hexagonal lattice of myofilament arrangement was less ordered and thin and thick filaments were missing on the outer edges of the myofibril (inset) relative to control. Moreover, there was Z-band buckling and M-line distortion in longitudinal sections (bottom). Single arrowheads delineate an M-line and double arrowheads a Z-line. MT, mitochondrion; MF, myofibril. Scale bars, 500 nm and 250 nm for longitudinal and transverse sections, respectively, and 50 nm in the inset.

Figure 4

Elongated thin filaments and sarcomeres in flies overexpressing profilin. (A) Left: increased IFM thin filament (n = 252–255, ***P < 0.001; one-way ANOVA with the Bonferroni post hoc test) and sarcomere lengths (n = 106–116, ***P < 0.001; one-way ANOVA with the Bonferroni post hoc test) were measured in flies with Mef2-GAL4-driven profilin overexpression. Right: typical IFM sarcomeres for control and profilin-overexpressing flies (red, TRITC-phalloidin-stained thin filaments; yellow, anti-α-actinin-stained Z-lines). (B) Averaged composite images of consecutive IFM sarcomeres from flies with elevated profilin levels revealed localization at the Z-line and at the thin filament pointed end/H-zone, whereas controls predominantly showed profilin localization at the Z-line. The M-line/H-zone was labelled using an MHC antibody that recognizes the centre of thick filaments along IFM myofibrils. (C) Based on normalized fluorescence intensity, Mef2 > Pfn_1 and Mef2 > Pfn_2 transgenic flies had significantly more profilin at the M-lines/H-zones, proximal to the thin filament pointed ends, relative to that at the Z-lines compared with controls (n = 123–143, ***P < 0.001; one-way ANOVA with the Bonferroni post hoc test).

Figure 5

Adenoviral-mediated overexpression of profilin-1 induces a hypertrophic response in NRVMs. (A) Transcript levels of Pfn1 in NRVMs were significantly higher than control following adenoviral-mediated transfection (n = 12, ***P < 0.001; Student's t-test). (B) Pfn1 overexpression resulted in significantly increased levels of profilin-1 (n = 6, ***P < 0.001; Student's t-test). (C) Elevated Pfn1 expression resulted in increased transcript levels of the hypertrophic markers ANP and BNP (n = 12, *P < 0.05; Student's t-test). (D) NRVMs exhibited significantly larger cellular areas in response to Pfn1 overexpression (n = 33, **P < 0.01; Student's t-test). (E) Representative profilin-1 and α-actinin antibody-stained confocal images of Adv-Control- and Adv-Profilin-1-transfected NRVMs. Scale bar, 15 µm. (F) A ×3.1 zoom of confocal images of Adv-Profilin-1-transfected cells (white box in the merged image). Scale bar, 5 µm.

Figure 6

Silencing of Pfn1 attenuates hypertrophic signalling in NRVMs. (A) Representative confocal images of control and PE-stimulated NRVMs. NRVMs were treated with control siRNA or Pfn1 siRNA. Nuclei were stained with DAPI (blue). Profilin-1 was dramatically reduced in response to Pfn1 siRNA. Scale bar, 10 μm. (B) Western blot analysis showed significantly decreased profilin-1 levels after the treatment of NRVMs with Pfn1 siRNA (n = 3, ***P < 0.001; two-way ANOVA with the Bonferroni post hoc test). (C) Transcript levels of Pfn1 in PE-stimulated NRVMs were increased compared with control, and treatment with Pfn1 siRNA significantly reduced mRNA levels (n = 4, *P < 0.05, **P < 0.01, and ***P < 0.001; two-way ANOVA with the Bonferroni post hoc test). Cell surface area increased significantly upon treatment with PE and was diminished upon profilin-1 silencing (n = 20–29, *P < 0.05 and ***P < 0.001; two-way ANOVA with the Bonferroni post hoc test). Transcription of the hypertrophic markers ANP, BNP, and skeletal muscle actin was significantly reduced after Pfn1 silencing in PE treated cells (n = 4, *P < 0.05, **P < 0.01, and ***P < 0.001; two-way ANOVA with the Bonferroni post hoc test). (D) NRVMs that were treated with Pfn1 siRNA and stimulated with ET1 exhibited significantly reduced ANP, BNP, and skeletal α-actin mRNA levels compared with control siRNA-treated cells (n = 3, *P < 0.05, **P < 0.01, and ***P < 0.001; two-way ANOVA with the Bonferroni post hoc test).

Figure 7

Profilin-1 is involved in the ERK1/2 signalling pathway. (A) The transcriptional activity of RCAN and MEF2 significantly increased when cells were stimulated with PE. Activity did not decrease upon silencing of profilin-1 (n = 5, **P < 0.01 and ***P < 0.001; two-way ANOVA with the Bonferroni post hoc test). (B) Phosphorylation levels of JNK (corrected for total JNK) and p38 (corrected for total p38) were unaltered by Pfn1 silencing and PE treatment (n = 3, two-way ANOVA with the Bonferroni post hoc test). (C) Phosphorylation of ERK1/2 (corrected for total ERK1/2) and Raf (corrected for GAPDH) was significantly increased in hypertrophic NRVMs and reduced upon diminished profilin-1 expression (n = 3, *P < 0.05 and **P < 0.01; two-way ANOVA with the Bonferroni post hoc test). (D) PE-increased mRNA levels of IL-6 and CTGF, effector genes of the ERK1/2 signalling cascade, were significantly reduced upon silencing of Pfn1 (n = 3, *P < 0.05, **P < 0.01, and ***P < 0.001; two-way ANOVA with the Bonferroni post hoc test).