Myopalladin knockout mice develop cardiac dilation and show a maladaptive response to mechanical pressure overload

Figures

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  Figure 1.. Myopalladin (MYPN) directly binds to titin in the Z-line. (A) Yeast two-hybrid (Y2H) assay showing binding of the C-terminal region of MYPN and its homologue palladin (PALLD) to the titin Ig domains Z4-Z5 (titin Ig Z4-Z5) in the Z-line. A culture plate with different combinations of Y2H co-transformations is shown as indicated in the table. (B) Microscale thermophoresis (MST) analysis of 450 nM labeled titin Ig Z4-Z5 incubated with increasing concentrations of MYPN and PALLD C-terminal domains. Due to precipitation, signal saturation was not reached. The dose/response curve is representative of two independent experiments (n = 17 and n = 16 runs). Fnorm, normalized fluorescence. (C) MST analysis of 400 nM labeled MYPN C-terminal domain incubated with increasing concentrations of titin Ig Z4-Z5. The dose/response curve is representative of four independent experiments (n = 7 runs). Figure 1—source data 1.Microscale thermophoresis binding (MST) analysis of myopalladin (MYPN)/titin interaction.

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  Figure 2.. Histological and electron microscopy analyses of hearts from wild-type (WT) and myopalladin knockout (MKO) mice. (A) Hematoxylin and eosin (H&E) (top) and Picro Sirius Red (bottom) stainings showing no morphological abnormalities or fibrosis in the heart of 6-month-old MKO male mice compared to WT mice (n = 3 per group). (B) Histograms and Gaussian distribution curves showing similar adult cardiomyocyte (CMC) length in 4-month-old WT and MKO male mice (n = 1292 CMCs from three WT mice and 1740 CMCs from three MKO mice). (C) Average CMC length in WT and MKO male mice. Data are represented as mean ± standard error of the mean (SEM). (D) Histograms and Gaussian distribution curves showing reduced adult CMC width in 4-month-old MKO male mice compared to WT mice (n = 1373 CMCs fromthree WT mice and 1193 CMCs from three MKO mice). (E) Average CMC width in WT and MKO male mice. Data are represented as mean ± SEM. ***p < 0.001; unpaired Student’s t-test. (F) Body weight to tibia length ratio (BW/TL) of 10-week-old WT and MKO male mice. Data are represented as mean ± SEM (n = 12–13 per group). ***p < 0.001; unpaired Student’s t-test. (G–H) Electron micrographs of papillary muscle from…

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  Figure 2—figure supplement 1.. Cardiomyocyte (CMC) size in 4-month-old wild-type (WT) and myopalladin knockout (MKO) male mice. (A) Histograms and Gaussian distribution curves showing reduced adult CMC size in MKO mice compared to WT mice (n = 1274 CMCs from 3 WT mice and 1587 CMCs from 4 MKO mice). (B) Average CMC size in WT and MKO mice. Data are represented as mean ± SEM. ***p < 0.001; Student’s t-test.

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  Figure 3.. Echocardiographic analyses and sarcomere-length-tension relationship in cardiac myofibrils from wild-type (WT) and myopalladin knockout (MKO) mice. (A–H) Echocardiographic analysis of WT and MKO male mice at 10 weeks (10 W), 4 months (4 M), and 6 months (6 M) of age. LVID, left ventricular inner diameter; FS, fractional shortening; EF, ejection fraction; LVPW, left ventricular posterior wall thickness; IVS, interventricular septum thickness; RWT, relative wall thickness ((LVPWd + IVSd)/LVIDd); CO, cardiac output; d, diastole; s, systole. Data are represented as mean ± standard error of the mean (SEM) (n = 16–42 per group). *p < 0.05; **p < 0.01; ***p < 0.001; two-way analysis of variance (ANOVA) with Bonferroni’s multiple comparison test. (I) Representative echocardiographic short-axis M-mode images from hearts of 6-month-old WT and MKO male mice. (J–L) Body weight (BW) (J), heart weight (HW) (K), and heart weight to body weight ratio (HW/BW) (L) of WT and MKO male mice at 10 W, 10 M, and 6 M of age. Data are represented as mean ± SEM (n = 12–24 per group). *p < 0.05; **p < 0.01; ***p < 0.001; two-way ANOVA with Bonferroni’s multiple comparison test. (M) Ratio of tension measured…

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  Figure 3—figure supplement 1.. Titin isoform expression and phosphorylation in the left ventricle of 4-month-old wild-type (WT) and myopalladin knockout (MKO) male mice. Titin (TTN) isoform expression was determined by SDS-PAGE and Coomassie blue staining. The TTN N2B isoform is the principal isoform in mouse heart. Titin phosphorylation was determined by Western blot analysis using anti-phosphoserine/threonine antibody (pTTN-Ser/Thr) and titin phosphosite-specific antibodies against Ser3991, Ser4080, and Ser12742. Total protein was determined by Coomassie blue staining of each blot.

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  Figure 4.. Echocardiographic analyses of wild-type (WT) and myopalladin knockout (MKO) mice following transaortic constriction (TAC). (A–H) Echocardiography analyses of WT and MKO male mice under basal conditions and 1, 2, and 4 weeks after TAC. Pressure gradient >70 mmHg. LVID, left ventricular inner diameter; FS, fractional shortening; EF, ejection fraction; LVPW, left ventricular posterior wall thickness; IVS, interventricular septum thickness; RWT, relative wall thickness ((LVPWd + IVSd)/LVIDd); CO, cardiac output; d, diastole; s, systole. Data are represented as mean ± standard error of the mean (SEM) (n = 8–19 per group). **p < 0.01; ***p < 0.001; linear mixed model (LMM) with Bonferroni’s multiple comparison test. (I) Representative echocardiographic short-axis M-mode images from hearts of WT and MKO male mice under basal conditions and 1 and 4 weeks after TAC. (J–L) Body weight (BW) (J), heart weight (HW) (K), and heart weight to body weight ratio (HW/BW) (L) of WT and MKO male mice 4 weeks after TAC or SHAM surgery. Data are represented as mean ± SEM (n = 9–11 per group). **p < 0.01; ***p < 0.001; two-way analysis of variance (ANOVA) with Bonferroni’s multiple comparison test.…

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  Figure 5.. Histological and transmission electron microscopy analyses of hearts from wild-type (WT) and myopalladin knockout (MKO) male mice 4 weeks after transaortic constriction (TAC) or SHAM. (A) Top, Hematoxylin and eosin (H&E) stainings of hearts from WT and MKO mice subjected to TAC or SHAM. Middle, Representative Picro Sirius Red stainings of the left ventricle, showing fibrosis in MKO mice after TAC. Bottom, Representative wheat germ agglutinin (WGA) stainings of the left ventricle (red). Nuclei are visualized by DAPI (blue). (B) Percent area of interstitial fibrosis in the left ventricle. Data are represented as mean ± standard error of the mean (SEM) (n = 3 per group). **p < 0.01; ***p < 0.001; two-way analysis of variance (ANOVA) with Bonferroni’s multiple comparison test. (C) Gaussian distribution curves from histograms of cardiomyocyte (CMC) size in the left ventricle of WT and MKO mice subjected to TAC or SHAM (n = 333 CMCs from three WT mice and 809 CMCs from three MKO mice subjected to SHAM; 423 CMCs from three WT mice and 665 CMCs from three MKO mice subjected to TAC). (D) Average CMC size in WT and MKO mice subjected to TAC or SHAM. Data are represented as mean ± SEM. **p…

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  Figure 6.. Quantitative real-time PCR (qRT-PCR) and Western blot analyses on wild-type (WT) and myopalladin knockout (MKO) mice at basal conditions and following transaortic constriction (TAC). (A) qRT-PCR analysis for Mypn, Palld, and markers of cardiac remodeling on left ventricular RNA from WT and MKO male mice at basal conditions and 4 days (D) and 4 weeks (W) after TAC. B2m was used for normalization. Data are represented as mean ± standard error of the mean (SEM) (n = 3–8 per group performed in triplicate). *p < 0.05, **p < 0.01, **p < 0.001; two-way analysis of variance (ANOVA) with Bonferroni’s multiple comparison test. (B) Western blot analysis on left ventricular lysate from WT and MKO male mice at basal conditions and 4 days (D) and 4 weeks (W) after TAC. Representative blots are shown (n = 3–4 per group). (C) Densitometric analysis for proteins that were significantly altered in MKO mice. Normalization was performed to total protein content as assessed on TGX Stain-Free gels (Bio-Rad Laboratories). Data are represented as mean ± SEM (n = 3–4 per group). *p < 0.05, **p < 0.01, ***p < 0.001; two-way ANOVA with Bonferroni’s multiple comparison test. Figure 6—source data…

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  Figure 6—figure supplement 1.. Immunofluorescence stainings of left ventricular tissue from the heart of 4-month-old wild-type (WT) and myopalladin knockout (MKO) male mice for palladin (PALLD) and myopalladin (MYPN)-interacting proteins. Nuclei are visualized by DAPI (blue).

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  Figure 7.. Functional analyses on adult cardiomyocytes (CMCs) from wild-type (WT) and myopalladin knockout (MKO) male mice 7 days after transaortic constriction (TAC) or SHAM surgery. (A–C) Sarcomere shortening, time to 90% peak, and time to 50% relaxation (n = 62 cells from nine WT mice and 71 cells from seven MKO mice). (D–F) Amplitude of Ca2+ transient, time to 90% peak of Ca2+ transient, and time to 50% decay of Ca2+ transient (n = 29 cells from eight WT mice and 16 cells from six MKO mice). (G) Representative Ca2+ spark images from WT and MKO male mice subjected to TAC or SHAM for 7 days. (H–M) Ca2+ spark frequency, amplitude, velocity of Ca2+ release (derivative; dF/dt), time to peak, full width at half maximum (FWHM), and tau (n = 355 sparks from 33 cells from three WT SHAM mice, 403 sparks from 27 cells from three MKO SHAM mice, 462 sparks from 31 cells from three WT TAC mice, and 429 sparks from 33 cells from three MKO TAC mice). Data are represented as mean ± standard error of the mean (SEM) as determined by hierarchical analysis (Sikkel et al., 2017). *p < 0.05, **p < 0.01, ***p < 0.001; two- or three-level hierarchical testing with Bonferroni correction, as appropriate. Figure…

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  Figure 8.. Overview figure illustrating the phenotype of myopalladin knockout (MKO) mice under basal conditions and following transaortic constriction (TAC). M, month; LV, left ventricle; SL, sarcomere length, ICD, intercalated disc.

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  Author response image 1..