Fig 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). *p<0.05; **p<0.01; linear mixed model with Tukey’s multiple comparisons test. (E) Hematoxylin and eosin (H&E) and Picro Sirius Red stainings of heart sections from cPKO and control male mice 4 wk after TAC or SHAM. The areas indicated with a box are shown at higher magnification below. Examples of vessels are shown on the bottom. (F, G) Percent area of interstitial fibrosis (F) and perivascular fibrosis (G) in the LV (n = 3 per group). No statistical difference by one-way ANOVA with Tukey’s multiple comparisons test. (H) Echocardiographic analysis of cPKO/MKO dKO vs. single KO and control male mice at 2 and 4 mo (M) of age. See (D) for abbreviations (n = 6–42 per group). *p<0.05; **p<0.01; ***p<0.001; linear mixed model with Tukey’s multiple comparisons test. All data are represented as mean ± standard error of the mean (SEM). Figure 2—source data 1.Quantitative real-time PCR (qRT-PCR) and densitometry of western blots on cardiomyocyte-specific palladin (cPKO) and control male mice. Figure 2—source data 2.Uncropped western blots for Figure 2B. Figure 2—source data 3.Echocardiographic parameters of 2-mo-old cardiomyocyte-specific palladin knockout (cPKO) male mice compared to controls before and 4 wk after mechanical pressure overload induced by transaortic constriction (TAC). Figure 2—source data 4.Echocardiographic analysis on cardiomyocyte-specific palladin (cPKO) and control male mice subjected to transaortic constriction (TAC) or SHAM. Figure 2—source data 5.Quantification of interstitial and vascular fibrosis in the left ventricle of cardiomyocyte-specific palladin (cPKO) and control male mice subjected to transaortic constriction (TAC) or SHAM. Figure 2—source data 6.Echocardiographic parameters of 3- and 6-mo-old cardiomyocyte-specific palladin and myopalladin double knockout (cPKO/MKO dKO) mice subjected to transaortic constriction (TAC) or SHAM. Figure 2—source data 7.Echocardiographic analysis on 2- and 4-mo-old cardiomyocyte-specific palladin (cPKO) and myopalladin (MYPN) double knockout and control male mice.

Fig 3: 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 (SEM) (n = 3–4). **p<0.01; Student’s t-test. (C) Immunofluorescence analysis of sectioned heart by STED microscopy, showing localization of both PALLD and CARP in the sarcomeric I-band. (D) Confocal fluorescence microscopy showing the presence of PALLD in the nucleus of CMCs. Both the PALLD 622 antibody and the PALLD Proteintech antibody showed nuclear localization of PALLD in most CMCs (an example of a CMC without nuclear staining for PALLD is shown on the bottom), while the PALLD 4D10 antibody did not stain the nucleus. Nuclei are visualized by DAPI (blue). (E) Confocal fluorescence microscopy of sectioned heart, showing colocalization of PALLD and FHOD1 at the ICD and the Z-line. (F) Confocal fluorescence microscopy showing colocalization of PALLD and Sorbin and SH3 domain-containing 2 (SORBS2) at the ICD in CMCs. Figure 1—source data 1.Confirmation of PALLD-CARP and PALLD-FHOD1 interactions in NanoBRET protein interaction assays.

Fig 4: 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 images of hearts from cPKOi and control mice 24 wk after TAM induction. (F) IonOptix analysis of ventricular CMC contractility and Ca2+ transients in cPKOi male mice vs. control mice 12 wk after TAM induction. Top, Sarcomere shortening, time to 90% peak, and time to 50% relaxation (n = 33 cells from 4 Palldfl/fl TAM mice, n = 34 cells from five MCM+/0 TAM mice, and n = 52 cells from six Palldfl/fl;Myh6MCM/+ TAM mice). Bottom, amplitude of Ca2+ transient, time to 90% peak of Ca2+ transient, and time to 50% decay of Ca2+ transient (n = 39 cells from four Palldfl/fl TAM mice, n = 18 cells from three MCM+/0 TAM mice, and n = 58 cells from six Palldfl/fl;Myh6MCM/+ TAM mice). *p<0.05; **p<0.01; ***p<0.001; two-level hierarchical testing with Bonferroni correction (Sikkel et al., 2017). All data are represented as mean ± standard error of the mean (SEM). Figure 3—source data 1.Quantitative real-time PCR (qRT-PCR) and densitometry of western blots on inducible cardiomyocyte-specific palladin (cPKOi) and control male mice. Figure 3—source data 2.Uncropped western blots for Figure 3B. Figure 3—source data 3.Echocardiographic parameters of inducible cardiomyocyte-specific palladin knockout (cPKOi) male mice compared to controls 8 and 24 wk after tamoxifen (TAM) induction. Figure 3—source data 4.Echocardiographic analysis on inducible cardiomyocyte-specific palladin (cPKOi) and control male mice 8 and 24 wk after tamoxifen (TAM) induction. Figure 3—source data 5.Heart weight to body weight ratio (HW/BW) measurements on inducible cardiomyocyte-specific palladin (cPKOi) and control male mice 8 and 24 wk after tamoxifen (TAM) induction. Figure 3—source data 6.Analysis of sarcomere shortening and Ca2+ transients in cardiomyocytes from inducible cardiomyocyte-specific palladin (cPKOi) and control male mice 12 wk after tamoxifen (TAM) induction.