Ablation of palladin in adult heart causes dilated cardiomyopathy associated with intercalated disc abnormalities

Figures

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  Figure 1.. Palladin (PALLD) binds to cardiac ankyrin repeat protein (CARP/Ankrd1) and formin homology 2 domain containing 1 (FHOD1) and is localized in the Z-line, I-band, intercalated disc (ICD), and nucleus in cardiomyocytes (CMCs). (A) Schematic representation of the domain structure of PALLD. Immunoglobulin (Ig) domains are shown in blue and proline-rich regions are indicated in red. Binding sites for known and novel interaction partners are shown with the ones in common with myopalladin (MYPN) in red. In a yeast two-hybrid screening, the N-terminal region of PALLD, comprising two Ig domains, was found to bind to CARP and FHOD1, as illustrated. The interacting region in FHOD1 was narrowed down to a region within the C-terminal region of FHOD1 (residue 965–1052), as indicated. GBD, GTPase-binding domain; DID, diaphanous inhibitory domain; FH1, formin homology 1 domain; FH2, formin homology 2 domain; DAD, diaphanous autoregulatory domain. (B) Confirmation of PALLD-CARP and PALLD-FHOD1 interactions in NanoBRET protein interaction assays with CARP-NanoLuc or FHOD1-NanoLuc as donor and fluorescently labeled PALLD-HaloTag as acceptor. Data are represented as mean ± standard error of the mean…

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  Figure 1—figure supplement 1.. Schematic representation of known murine PALLD protein isoforms and their domain organization. Blue boxes indicate immunoglobulin (Ig) domains, while red boxes indicate proline-rich regions. The regions against which the PALLD antibodies were raised are indicated above. Known molecular weights of isoforms are indicated.

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  Figure 1—figure supplement 2.. Schematic representation of the murine Palld gene structure with known transcripts indicated below. Exon numbers are indicated above the exons. Exon 14 was floxed. Regions marked in blue encode immunoglobulin domains, whereas regions marked in red encode proline-rich regions. Arrows above the different splice variants indicate the start and end of translated regions, which are marked in gray.

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  Figure 1—figure supplement 3.. Yeast-two hybrid (Y2H) assays showing the interaction of the N-terminal region of palladin (PALLD) with cardiac ankyrin-repeat protein (CARP/Ankrd1) and formin homology 2 domain containing 1 (FHOD1). (A) Culture plate with different combinations of Y2H cotransformations to narrow down the interaction size between PALLD and CARP as indicated in the table. PALLD’s N-terminal region binds to CARP. (B) Culture plate with different combinations of Y2H cotransformations to narrow down the interaction size between PALLD and FHOD1 as indicated in the table. FHOD1 residue 965–1052 is sufficient for binding to the N-terminal region of both PALLD and its homologue myopalladin (MYPN).

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  Figure 1—figure supplement 4.. Confocal fluorescence microscopy showing the presence of palladin (PALLD) in the nucleus of cardiomyocytes both from wild-type (WT) mice and cardiac ankyrin repeat protein (CARP/Ankrd1) knockout (CKO) mice.

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  Figure 2.. Echocardiographic and histological analyses of cardiomyocyte-specific palladin (cPKO) mice as well as cPKO and myopalladin (MYPN) double knockout (cPKO/MKO dKO) mice. (A) Quantitative real-time PCR (qRT-PCR) for transcripts encoding the most common PALLD isoform on left ventricular (LV) RNA from 2-mo-old cPKO (Palldfl/fl;Myh6Cre/+) and control (Palldfl/fl) male mice (n = 3–4 biological replicates and three technical replicates per group). Data were normalized to Gapdh. ***p<0.001; two-way analysis of variance (ANOVA) with Šidák’s multiple comparison test. (B) Western blot analysis for PALLD (PALLD 622 antibody) on LV lysate from cPKO and control male mice. (C) Densitometric analysis for the 90 kDa PALLD isoform using GAPDH for normalization (n = 4–6 per group). ***p<0.001; Student’s t-test. (D) Echocardiography analyses of cPKO and control male mice under basal conditions and 1, 2, and 4 wk after transaortic constriction (TAC). Pressure gradient >70 mmHg. LVID, left ventricular inner diameter; LVPW, left ventricular posterior wall thickness; IVS, interventricular septum thickness; FS, fractional shortening; EF, ejection fraction; d, diastole; s, systole (n = 4–14 per group).…

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  Figure 2—figure supplement 1.. Generation of Palld floxed mice. (A) Targeting strategy for the generation of Palld floxed mice containing LoxP sites flanking exon 14. A restriction map of the relevant genomic region of Palld is shown on the top, the targeting construct is shown in the middle, and the mutated locus after recombination is shown at the bottom. Arrowheads indicate loxP sites and black boxes indicate Flpase Recognition Target (FLP) sites. Neo, neomycin resistance gene; DTA, diptheria toxin A fragment gene. (B) Detection of wild-type (WT) and targeted alleles by Southern blot analysis following digestion with EcoRV using the probe shown in (A). (C) Genotyping of WT and Palld floxed mice after removal of the neo cassette using Palld-specific primers. All data are represented as mean ± standard error of the mean (SEM).

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  Figure 2—figure supplement 2.. Echocardiographic and histological analyses of cardiomyocyte-specific palladin knockout (cPKO) male mice. (A) Echocardiographic analysis of cPKO and control male mice at 3 and 6 mo (M) of age. LVID, left ventricular inner diameter; LVPW, left ventricular posterior wall thickness; IVS, interventricular septum thickness; FS, fractional shortening; EF, ejection fraction; d, diastole; s, systole (n = 7–17 per group). *p<0.05; linear mixed model with Tukey’s multiple comparisons test. (B) Hematoxylin and eosin (H&E) and Picro Sirius Red stainings of heart sections from 6-mo-old cPKO and control male mice. The areas indicated with a box are shown at higher magnification below. Examples of vessels are shown on the bottom. (C) Percent area of interstitial fibrosis (left) and perivascular fibrosis (right) in the left ventricle (n = 5–6 per group). No statistical differences by one-way analysis of variance (ANOVA) with Tukey' multiple comparisons test. All data are represented as mean ± standard error of the mean (SEM). Figure 2—figure supplement 2—source data 1.Echocardiographic parameters of 3- and 6-mo-old cardiomyocyte-specific palladin knockout (cPKO) male mice compared to controls…

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  Figure 3.. Echocardiographic and functional analyses of inducible cardiomyocyte-specific palladin knockout (cPKOi) mice. (A) Quantitative real-time PCR (qRT-PCR) for transcripts encoding the most common palladin (PALLD) isoforms on left ventricular (LV) RNA from cPKOi (Palldfl/fl;Myh6MCM/+ TAM) and control (Palldfl/fl TAM) mice 8 wk after tamoxifen (TAM) induction (n = 4 biological replicates and three technical replicates per group). Data were normalized to Gapdh. ***p<0.001; two-way analysis of variance (ANOVA) with Šidák’s multiple comparisons test. (B) Western blot analysis for PALLD (PALLD 622 antibody) on adult cardiomyocytes (CMCs) from cPKOi and control mice 8 wk after TAM induction. (C) Densitometric analysis for the 90 kDa PALLD isoform using GAPDH for normalization (n = 6–7). ***p<0.001; Student’s t-test. (D) Echocardiographic analysis of cPKOi and control male mice 0, 8, and 24 wk after TAM induction. See (D) for abbreviations. RWT, relative wall thickness ((LVPWd + IVSd)/LVIDd); HW, heart weight, BW, body weight (n = 4–44 per group). *p<0.05; **p<0.01; ***p<0.001; linear mixed model with Tukey’s multiple comparisons test. (E) Representative echocardiographic short-axis M-mode…

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  Figure 3—figure supplement 1.. Echocardiographic analysis of cardiomyocyte-specific palladin (cPKO) and control male mice 8 wk after tamoxifen (TAM) injection. LVID, left ventricular inner diameter; LVPW, left ventricular posterior wall thickness; IVS, interventricular septum thickness; FS, fractional shortening; EF, ejection fraction; d, diastole; s, systole (n = 7 per group). No differences using linear mixed model with Tukey’s multiple comparisons test. Figure 3—figure supplement 1—source data 1.Echocardiographic parameters of cardiomyocyte-specific palladin knockout (cPKO) male mice compared to controls 8 weeks after tamoxifen (TAM) injection. Figure 3—figure supplement 1—source data 2.Echocardiographic analysis on cardiomyocyte-specific palladin knockout (cPKO) and control male mice 8 wk after tamoxifen (TAM) injection.

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  Figure 3—figure supplement 2.. Sarcomere-length tension relationship in cardiac myofibrils from the left ventricle of inducible cardiomyocyte-specific palladin knockout (cPKOi) and control male mice 8 wk after tamoxifen (TAM) induction. Each data point is represented as mean ± standard error of the mean (SEM) from 4 to 12 myofibrils from three Palldfl/fl TAM mice, from 11 to 19 myofibrils from three Myh6MCM/+ TAM mice, and from 5 to 15 myofibrils from three Palldfl/fl;Myh6MCM/+ TAM mice. The figure is related to the data displayed in Table 1. Figure 3—figure supplement 2—source data 1.Data for sarcomere-length tension relationship in cardiac myofibrils from the left ventricle of inducible cardiomyocyte-specific palladin knockout (cPKOi) and control male mice 8 wk after tamoxifen (TAM) induction.

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  Figure 4.. Histological and molecular analyses of cardiomyocyte-specific palladin knockout (cPKOi) mice. (A) Hematoxylin and eosin (H&E) and Picro Sirius Red stainings of heart sections from cPKOi and control male mice 24 wk after tamoxifen (TAM) induction. The areas indicated with a box are shown at higher magnification. Examples of vessels are shown on the bottom. (B) Percent area of interstitial fibrosis (left) and perivascular fibrosis (right) in the left ventricle (LV) (n = 3–5 per group). **p<0.01; one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test. (C−E) Left, frequency distributions of cardiomyocyte (CMC) size (n = 830 CMCs from three Palldfl/fl mice and 1079 CMCs from three Palldfl/fl;Myh6MCM/+ TAM mice) (C), length (n = 877 CMCs from three Palldfl/fl mice and 1576 CMCs from three Palldfl/fl;Myh6MCM/+ TAM mice) (D), and width (n = 842 CMCs from three Palldfl/fl mice and 1565 CMCs from three Palldfl/fl;Myh6MCM/+ TAM mice) (E) in cPKOi and control male mice 12 wk after TAM induction. Right, average CMC size (C), length (D), and width (E) in the same mice. *p<0.05; ***p<0.001; Student’s t-test. (F) Quantitative real-time PCR (qRT-PCR) analysis on LV RNA from…