Palladin (PALLD) is an actin-associated protein, which, together with myopalladin (MYPN) and myotilin (MYOT), belongs to a small protein family of immunoglobulin (Ig) domain-containing proteins in the Z-line associated with the actin cytoskeleton (reviewed in Otey et al., 2009). While MYPN and MYOT are specifically expressed in striated and skeletal muscle, respectively, PALLD is ubiquitously expressed in various tissues, including the heart, where it is expressed at high levels (Wang and Moser, 2008). Mutations in the MYPN gene have been associated with dilated (DCM), hypertrophic (HCM), and restrictive cardiomyopathy (Bagnall et al., 2010; Duboscq-Bidot et al., 2008; Meyer et al., 2013; Purevjav et al., 2012), while MYOT gene mutations can cause various skeletal muscle disorders (reviewed in Otey et al., 2009). Mutations in the PALLD gene have been linked to pancreatic cancer (Liotta et al., 2021; Pogue-Geile et al., 2006; Slater et al., 2007), and PALLD levels have been shown to correlate with increased invasiveness of cancer cells (Gilam et al., 2016; Goicoechea et al., 2009; von Nandelstadh et al., 2014). However, the role of PALLD in the heart has remained unknown. Whereas MYPN and MYOT are expressed as single isoforms, PALLD exists in many different isoforms due to the presence of four alternative promoters, alternative splicing, and early termination as illustrated in Figure 1—figure supplement 1 (Goicoechea et al., 2010; Wang and Moser, 2008). The longest 200 kDa isoform of PALLD is predominantly expressed in striated muscle and highly homologous in structure to MYPN, sharing five Ig domains and a central proline-rich region (Otey et al., 2009). Furthermore, PALLD contains an additional proline-rich region, not shared with MYPN. The 140 kDa and 90 kDa isoforms containing four and three Ig domains, respectively, are also expressed in the heart, but have a more ubiquitous expression pattern (Wang and Moser, 2008). Molecular weights and expression profiles of the other isoforms have not been determined in detail (Goicoechea et al., 2010; Wang and Moser, 2008). Within the cardiac Z-line, PALLD and MYPN share several interaction partners, including α-actinin (Bang et al., 2001; Rönty et al., 2004), nebulette (Bang et al., 2001), titin (Filomena et al., 2021), and members of the PDZ-LIM protein family (cypher/ZASP/LDB3, ALP/PDLIM3, CLP36/PDLIM1, RIL/PDLIM4) (von Nandelstadh et al., 2009) as illustrated in Figure 1A. Furthermore, PALLD and MYPN bind to myocardin-related transcription factors (MRTFs) (Filomena et al., 2020; Jin et al., 2010) as well as bind and bundle F-actin (Dixon et al., 2008; Filomena et al., 2020; Gurung et al., 2016), stabilizing the actin cytoskeleton and consequently promoting serum response factor (SRF) signaling. Additionally, PALLD binds to sorbin and SH3 domain-containing 2 (SORBS2) (Rönty et al., 2005), localized at the intercalated disc (ICD) and Z-line of the heart, as well as various proteins expressed in non-muscle cells, including actin-associated (Boukhelifa et al., 2006; Boukhelifa et al., 2004; Goicoechea et al., 2006; Jin et al., 2007; Mykkänen et al., 2001; Rachlin and Otey, 2006) and signaling/adaptor (Lai et al., 2006; Rönty et al., 2007) proteins.

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Confirmation of PALLD-CARP and PALLD-FHOD1 interactions in NanoBRET protein interaction assays.

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Consistent with the interaction of PALLD with F-actin and numerous actin binding proteins, PALLD is associated with various actin-based structures, including focal adhesions, stress fibers, cell-cell junctions, and Z-lines, and is important for the assembly, organization, and maintenance of the actin cytoskeleton (reviewed in Jin, 2011; Otey et al., 2009). PALLD is well-known for its involvement in cell motility and smooth muscle cell differentiation, implicating it in cancer (Jin, 2011), while its role in the heart has remained elusive, in part due to embryonic lethality of PALLD knockout mice (Luo et al., 2005). Thus, to provide insights into the specific role of PALLD in cardiac muscle, we generated conditional (cPKO) and inducible (cPKOi) cardiomyocyte (CMC)-specific knockout mice for the most common PALLD isoforms. While conditional knockout of PALLD in the heart did not cause any pathological cardiac phenotype, PALLD knockdown in adult heart resulted in left ventricular (LV) dilation and systolic dysfunction, associated with fibrosis and ICD abnormalities. Furthermore, we identified cardiac ankyrin repeat protein (CARP/Ankrd1) and formin homology 2 domain containing 1 (FHOD1) as novel interaction partners of the N-terminal region of PALLD. Quantitative real-time PCR (qRT-PCR) analyses on myocardial tissue from DCM and ischemic cardiomyopathy (ICM) patients showed increased transcript levels of MYPN and the PALLD long isoform in DCM patients, while total transcript levels of PALLD were reduced in both DCM and ICM patients. On the other hand, no changes in the protein levels of MYPN and PALLD were found.

In this study, we show that in addition to its known localization in the sarcomeric Z-line, PALLD is located in the I-band, the ICD, and the nucleus of CMCs. The PALLD C-terminal region was previously found to target to the nucleus in podocytes (Endlich et al., 2009) and smooth muscle cells (SMCs) (Jin et al., 2010), while the N-terminal region was shown to be required for nuclear export (Endlich et al., 2009). Consistently, immunofluorescence stainings with antibodies towards all PALLD isoforms or the PALLD C-terminal region showed the localization of PALLD in the nucleus of CMCs. However, surprisingly, antibodies against the second proline-rich region present in the major isoforms did not stain the nucleus. This suggests that only isoforms without the second proline-rich region (indicated in Figure 1—figure supplement 1) are present in the nucleus, which is consistent with immunohistochemical stainings of human biopsies from pancreas (Goicoechea et al., 2010). Thus, PALLD isoforms may have different cellular functions depending on the presence or absence of the second proline-rich region. As shown in Figure 1A, many interaction partners of PALLD bind to its second proline-rich region, including SORBS2, which is present in the ICD and Z-line of the heart, so one possibility could be that the second proline-rich region tethers PALLD to the actin cytoskeleton, preventing it from going to the nucleus.

In a yeast two-hybrid screening, we identified CARP and FHOD1 as novel interaction partners of the PALLD N-terminal region for which no interaction partners have previously been identified. Since CARP is a well-known interaction partner of the MYPN N-terminal region (Bang et al., 2001), structurally homologous to the PALLD N-terminal region, it is not surprising that also PALLD binds to CARP. CARP and MYPN are known to form a complex in the I-band, where CARP is linked to the titin N2A region (Bang et al., 2001; Miller et al., 2003). Consistently, we found that also PALLD is located in the I-band but hypothesize that only the 200 kDa PALLD isoform containing the N-terminal CARP-binding region is targeted to the I-band. Titin is a largest known protein, stretching from the Z-line to the M-line and functioning as a molecular spring responsible for the passive stiffness of striated muscle, implicating it in force transmission and mechanosensing (Loescher et al., 2022). Thus, based on their link to titin and dual localization in the sarcomere and the nucleus, CARP and MYPN were previously proposed to form a complex involved in the transduction of stretch-induced signaling from the sarcomere to the nucleus (Laure et al., 2010; Miller et al., 2003). To determine whether CARP may be responsible for the targeting of PALLD to the nucleus, we performed immunofluorescence stainings of CMCs from CARP knockout mice. However, CARP ablation did not affect the localization of PALLD in the nucleus of CMCs, questioning the hypothesis of a role of PALLD and MYPN in mechanosensing. Consistent with targeting experiments in podocytes and SMCs, it is therefore likely that the PALLD C-terminal region is responsible for its targeting to the nucleus also in CMCs. In particular, based on the binding of the PALLD Ig3 domain to the transcriptional cofactor MRTF-A (Filomena et al., 2020; Jin et al., 2010), which cycles between the cytosol and nucleus to regulate SRF signaling, it is tempting to speculate that MRTF-A is responsible for the targeting of PALLD to the nucleus. Another possibility could be that PALLD contains a nuclear localization signal (NLS), as previously predicted by sequence analysis (Endlich et al., 2009). On the other hand, no NLSs were predicted in MYPN, which is also present in the nucleus. Based on the high homology between the two proteins, we therefore consider it most likely that PALLD and MYPN are transported to the nucleus through binding to proteins that translocate to the nucleus, such as MRTF-A.

The interaction of PALLD with FHOD1 is consistent with its location in the ICD, where it also binds to SORBS2 (Rönty et al., 2005). FHOD1 has been reported to be localized at the ICD and costamere of the heart (Al Haj et al., 2015; Dwyer et al., 2014) consistent with our findings. In contrast, Sanematsu et al., showed that FHOD1 is dispensable for normal cardiac development and function in mouse and failed to detect FHOD1 in the heart (Sanematsu et al., 2019). The reason for this discrepancy is unclear as several different FHOD1 antibodies were tested in each of the studies. Nevertheless, the low expression level of FHOD1 in the heart and absence of a pathological cardiac phenotype of FHOD1-deficient mice questions the relevance of the PALLD-FHOD1 interaction in CMCs. In contrast, FHOD1 is highly expressed in SMCs and other cell types, where also PALLD is present, so the interaction is likely to be important in other tissues. FHOD1 suppresses actin polymerization by inhibiting nucleation and elongation but stabilizes actin filaments by capping the actin barbed ends, protecting them from depolymerization, while simultaneously mediating F-actin bundling by connecting them to adjacent actin filaments (Schönichen et al., 2013). In line with this, FHOD1 silencing was shown to reduce migration and invasion of breast cancer cells by inhibiting the nuclear translocation of the SRF coactivator MRTF-A, which is sequestered in the cytoplasm by G-actin and regulated by changes in actin dynamics (Jurmeister et al., 2012). PALLD directly binds to MRTF-A (Filomena et al., 2020; Jin et al., 2010) and stabilizes the actin cytoskeleton by promoting actin polymerization and preventing actin depolymerization, consequently promoting MRTF-mediated activation of SRF signaling (Dixon et al., 2008; Filomena et al., 2020; Gurung et al., 2016). Thus, it is possible that PALLD and FHOD1 may cooperate in regulating actin dynamics and SRF signaling.

Global knockout of PALLD in mouse was reported to result in embryonic lethality before E15.5 due to multiple defects, including failure of body walls to close, resulting in exencephaly and herniation of abdominal organs (Luo et al., 2005). Furthermore, analysis of PALLD knockout embryos revealed a key role of PALLD in the induction of SMC differentiation in the developing vasculature (Jin et al., 2010) and fibroblasts derived from knockout embryos showed defects in stress fiber formation, cell adhesion, and motility (Luo et al., 2005). Based on its similarity to MYPN, associated with different forms of cardiomyopathy (Bagnall et al., 2010; Duboscq-Bidot et al., 2008; Meyer et al., 2013; Purevjav et al., 2012), as well as high expression levels of specific PALLD isoforms in cardiac muscle both during development and at adult stage, we hypothesized that PALLD plays an important role also in the heart (Wang and Moser, 2008).

To determine the role of PALLD in the heart in vivo, we floxed Palld exon 14 (see Figure 1—figure supplement 2), allowing us to generate conditional (cPKO) and inducible (cPKOi) CMC-specific knockout mice for all PALLD isoforms except for two predicted isoforms not containing exon 14. Due to extensive differential splicing of PALLD, it was not possible to target all isoforms. Unexpectedly, cPKO mice exhibited no pathological cardiac phenotype at basal levels and showed a normal hypertrophic response to TAC. In contrast, cPKOi mice induced at adult stage developed cardiac dilation and systolic dysfunction within 8 wk after TAM induction, associated with interstitial fibrosis and upregulation of cardiac stress markers. Consistently, reduced sarcomere shortening and Ca²⁺ transient amplitude were observed in CMCs, and decreased maximum Ca²⁺-activated isometric tension was found in cPKOi myofibrils, while the kinetics of contraction were unaffected. As biomechanical properties of myofibril preparations from the LV were measured at Ca²⁺ concentrations that cannot be reached under the physiological conditions of cardiac contraction, we cannot exclude that our observations might not fully recapitulate in vivo mechanisms. The heart weight to body weight ratio was unaltered, although CMC size was increased, mainly due to increased CMC width.

At the ultrastructural level, ICDs in cPKOi mice were disorganized and more convoluted compared to those of control mice. The ICD is a highly complex structure joining together adjacent CMCs in the heart and is made up of three major complexes: adherens junctions, where actin filaments are anchored and connect the sarcomere to the cell membrane; desmosomes, which tether the cell membrane to the intermediate filament network; and gap junctions, which permit the passage of ions and small molecules between neighboring CMCs, allowing for electrical and metabolic coupling between cells (reviewed in Vermij et al., 2017). In contrast to previous belief, rather than functioning independently, the three junctions cooperate via crosstalk between their components in mixed-type junctions termed ‘area composita’ (Borrmann et al., 2006; Franke et al., 2006). Thus, the ICD can be considered as a single functional unit important for mechanical coupling and transmission of contractile force and electrical signals between adjacent CMCs. PALLD plays a well-known role in the organization of the actin cytoskeleton (Jin, 2011; Otey et al., 2009) and binds directly to F-actin (Dixon et al., 2008), ALP (von Nandelstadh et al., 2009), and SORBS2 (Rönty et al., 2005), which are associated with the adherens junction (Ding et al., 2020; Pashmforoush et al., 2001). Thus, destabilization of the adherens junction in cPKOi mice may be responsible for the structural alterations at the ICD, also affecting nearby sarcomeres, as evident by Z-line abnormalities and sarcomere disruption in the vicinity of the adherens junction in high-amplitude ICDs. PALLD also interacts with several components of the transitional junction, where the contractile apparatus is connected the ICD to allow force transmission between adjacent CMCs (Bennett, 2012). In particular, titin (Filomena et al., 2021), α-actinin (Bang et al., 2001; Rönty et al., 2004), and cypher (von Nandelstadh et al., 2009), all interaction partners of PALLD, are associated with the transitional junction, suggesting a possible effect of PALLD on the attachment of the myofilament to the ICD and the stability of the junction. Immunofluorescence stainings and western blot analyses did not show any changes in the localization of ICD proteins, including the PALLD interaction partners FHOD1 and SORBS2. However, the protein level of SORBS2 was ~1.6-fold upregulated in cPKOi heart. SORBS2, also known as Arg kinase-binding protein 2 (ArgBP2), is a scaffold protein localized at the adherens junction of the ICD as well as the Z-line at lower levels (Ding et al., 2020). SORBS2 overexpression in mouse heart was reported to lead to upregulation of β-tubulin, a direct interaction partner of SORBS2, and consequent microtubule densification, resulting in junctophilin 2 translocation, T-tubule disorganization, defective excitation-contraction coupling, and systolic dysfunction (Li et al., 2020). Furthermore, SORBS was found to be upregulated in patients with left ventricular non-compaction (LVNC), but not HCM and arrhythmogenic cardiomyopathy, and may play a role in the progression of heart failure in LVNC patients (Li et al., 2020). Thus, increased SORBS2 expression in cPKOi mice may contribute to ICD instability and cardiac dysfunction. ICD alterations, such as altered expression of ICD proteins and increased, more variable ICD amplitude, are commonly associated with DCM both in human and mouse (Wilson et al., 2014). In addition, mutations in several genes (VCL,DSP,DSG2, DES) encoding ICD proteins have been associated with DCM and several knockout mouse models of adherens junction and desmosomal proteins develop DCM (Bang et al., 2022; Hirschy et al., 2010; Kostetskii et al., 2005; Li et al., 2012; Norgett et al., 2000; Sheikh et al., 2006; Zemljic-Harpf et al., 2007). Thus, it is plausible to suggest that structural alteration of the ICD in the absence of PALLD is the primary event leading to DCM and systolic dysfunction in cPKOi mice. Our data has shown that, in addition to the ICD, PALLD localizes to other cellular compartments, including the nucleus. Therefore, we cannot rule out the possibility that ultrastructural changes observed in cPKOi cardiomyocytes might be a consequence of LV dilation rather than the primary cause of the observed phenotype. Interestingly, MKO mice, which developed a mild form of DCM under basal conditions, also showed an increase in ICD fold amplitude, although only by ~27% and without other obvious ICD abnormalities (Filomena et al., 2021), suggesting that PALLD plays a more important structural role in the ICD than MYPN.

The absence of a pathological cardiac phenotype in cPKO mice is surprising based on the rather severe cardiac phenotype of cPKOi mice induced at adult stage. Due to the structural homology of PALLD to MYPN, we hypothesized that MYPN can compensate for the absence of PALLD. However, the phenotype of cPKO/MKO dKO mice was indistinguishable from that of MKO mice, strongly suggesting that MYPN does not compensate for the loss of PALLD in cPKO mice. Another possibility is that the small PALLD isoforms not targeted by our knockout approach can compensate for the absence of the other PALLD isoforms when absent during development. However, these isoforms were not upregulated in cPKO mice and do not contain binding sites for most of the PALLD interaction partners, so we consider this rather unlikely. We hypothesize that when PALLD is knocked out during embryonic development, the mice are able to adapt through activation of compensatory mechanisms, whereas this is not anymore possible when PALLD is knocked out in adult mice. This phenomenon has also been observed in other genetic models (reviewed in El-Brolosy and Stainier, 2017).

Analysis of MYPN and PALLD mRNA levels in LV biopsies from human ICM and DCM patients showed an ~2.9-fold upregulation of MYPN and an ~1.9-fold upregulation of the structurally similar 200 kDa PALLD isoform in DCM hearts, whereas the overall transcript level of all PALLD isoforms except for potential truncated N-terminal transcript(s) was ~2.8- and ~3.0-fold reduced in DCM and ICM patients, respectively. Similarly, in two RNA-Seq studies, PALLD was reported to be downregulated in both DCM/non-ischemic cardiomyopathy (NICM) (~1.7- to ~2.0-fold) and ICM (~1.6- to ~2.1-fold) patients, while MYPN was upregulated in both DCM/NICM (~1.5- to ~1.6-fold) and ICM (~1.6- to ~1.7-fold) patients (Sweet et al., 2018; Yang et al., 2014). The absent modulation of MYPN in ICM patients in this study may be due to different patient cohorts and detection methods used. The 90 kDa PALLD isoform is the predominant isoform in the heart, while the 200 kDa PALLD isoform is expressed at much lower levels, explaining the upregulation of the 200 kDa PALLD isoform despite the overall reduced transcript level of PALLD. Notwithstanding the transcriptional modulation of both MYPN and PALLD in DCM and ICM patients, no changes in protein expression were found, so the altered transcript levels are unlikely to have any functional effects. As previously reported, CARP/Ankrd1 protein levels were significantly increased in DCM patients (Kempton et al., 2018; Nagueh et al., 2004; Zolk et al., 2002).

Altogether, our data demonstrate an important role of PALLD for normal heart function, suggesting PALLD as a possible candidate gene for cardiomyopathy. Based on the absence of a phenotype in cPKO mice, loss-of-function mutations are unlikely to affect cardiac function and would likely lead to embryonic lethality due to the essential role of PALLD in other tissues and cell types. However, as for MYPN, whose ablation does not severely affect cardiac function, it is possible that mutations with dominant negative effects may be linked to cardiac disease. The reason why a putative link between PALLD mutations and cardiac disease has not been established could be due to lack of focus on the PALLD gene.

All data generated and analysed during this study are included in the manuscript and figure supplements. Source data files have been provided for all figures.

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Decision letter after peer review:

Thank you for submitting your article "Ablation of palladin in adult heart causes dilated cardiomyopathy associated with intercalated disc abnormalities" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Didier Stainier as the Senior Editor. The following individuals involved in the review of your submission have agreed to reveal their identity: Jason R. Becker (Reviewer #2); W. Glen Pyle (Reviewer #3).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

1) Authors propose that the DCM phenotype observed in the inducible PALLD cKOi hearts is a consequence of intercalated disc (ICD) defects. Please provide data assessing the integrity of the ICD in the constitutive PALLD cKO. If similar changes occur in these hearts, then this defect is not likely the cause of the phenotype observed in the inducible KO. Similarly, to ensure that the ICD phenotype is caused by the absence of PALLD rather than being a consequence of tamoxifen administration, please assess ICD integrity in Myh6:MerCreMer mice administered tamoxifen.

2) To ensure that apoptosis is not contributing to the phenotype observed, please repeat the TUNEL experiment at an earlier timepoint (4-week post-induction). In the current version of the manuscript, apoptosis was assessed 24-week post-induction, which is a rather late timepoint and might miss early events.

3) Please determine if CARP and FHOD1 localization are altered in PALLD cKO versus cKOi. This will improve the connection between the first section of the manuscript (identification of novel interactants) and the phenotypic characterization of conditional mutants, and might shed some light on why the two models diverge phenotypically.

4) As pointed out by all Reviewers, the human mRNA data alone does not provide enough evidence that PALLD might play a role in human DCM. It is particularly difficult to reconcile the fact that deletion of PALLD in mice causes DCM, whereas in human DCM patients the transcripts encoding this protein are upregulated. It is possible that PALLD protein levels are actually reduced in human DCM and the observed mRNA upregulation is part of a compensation mechanism. To test this, please provide measurements of PALLD protein level in human DCM samples.

Reviewer #1 (Recommendations for the authors):

1. The introduction has a long list of PALLD interactions with other proteins that is not necessary for the reader to understand the work here presented. Please shorten this text and simply mention that whereas multiple interactions have been previously identified for the proline-rich domains and last 3 Ig domains (as nicely summarized in Figure 1A), nothing is known regarding putative interactors in the N-terminal domain (encompassing the first 2 Ig domains).

2. Please alter Figure 1A to include not just the long isoform, but also the other relevant variants of PALLD. Please add also information regarding expected molecular weights. This will make it easier to understand in subsequent western blots what isoforms are expressed (and ablated in cKOs) in cardiomyocytes.

3. Is the signal of all antibodies used in Figure 1 specific? In other words, do these antibodies produce any signal when applied to tissues from PALLD KO/KO mice?

4. Based on the antigen information described in the text (whole protein, C-terminus, second proline-rich region), please add to Figure1 a diagram reporting what isoforms are recognized by each of the antibodies.

5. In Figure 1D, the nuclear labeling produced by antibody PALLD 622 seems to be enriched in specific foci. Is this a consistent observation, or a particular of this image? If this is consistent, do the authors know what sort of foci are these? Nucleoli?

6. The interactants and immunofluorescence data shown in Figure 1 are somehow disconnected from the rest of the manuscript. This could be improved if, for example, authors would perform immunofluorescence analyses on their cKOs to assess if the nuclear PALLD signal disappears in their cKO and icKO animals.

7. Lines 144-145: "we generated Palld floxed (Palldfl/fl) mice containing LoxP sites flanking exon 15". Please add here what is the expected consequence of ablation of exon 15 for the different isoforms of PALLD. What domain of the protein does exon 15 correspond to? After floxing out, is there an early termination codon leading to truncated variants?

8. Lanes 166-167: "in response to (…) TAC, no significant differences were found between cPKO and control mice as shown by echocardiography (Figure 2D and Figure 2—figure supplement 4) (…) although there was a tendency towards reduced contractile function in cPKO mice from 2 weeks after TAC". This description might cause some confusion, as the graphs do show significant differences (EF and FS) between cPKO and Cre- negative controls. Please revise the text to read "no significant differences were found between cPKO and Cre+ control mice".

9. Histological data in Figure 2E and respective quantification in 2F: in addition to the genotype, please add "4-week post TAC" to the label. Also, please highlight (with a box) in the low magnification whole heart image the area shown in the high magnification panels. The high magnification images show areas of interstitial myocardium, however, fibrosis post-TAC is frequently concentrated in the vicinity of large coronaries. Please provide also images and quantification of fibrosis in perivascular areas of control and cKO TAC hearts. Please include non-TAC hearts as control.

10. For increased clarity, in Figure 4A, please move genotype labels to the top, instead of the bottom position they currently occupy. On the left side of the panel, please add a label stating what sort of staining is being shown. Please highlight in the low magnification image the area shown in higher magnifications.

11. The discussion is rather long and has elements that belong in the Results section (for example describing tamoxifen injection into the constitutive KO mice).

12. Lines 460-461: "Based on the absence of a phenotype in PKO mice". Please correct to "Based on the absence of a phenotype in cPKO mice".

Reviewer #2 (Recommendations for the authors):

1. Are there changes in the PALLD interacting proteins depending on if PALLD is deleted in the embryonic versus adult period?

2. To better support the claim that ICD ultrastructural changes are contributing to DCM in adult PALLD deletion, it would be important to determine:

a. Do these same alterations in ICD ultrastructure occur in the mice with embryonic deletion of cardiomyocyte PALLD?

b. Are these ICD ultrastructure changes a non-specific result of ventricular dilation and failure? For example, do they occur in mice with DCM secondary to other causes (MI, chronic aortic banding, etc.)?

3. Since the ICD ultrastructure defects are postulated as the mechanism regulating DCM development in the adult PALLD deletion mice, it would be important to confirm that Myh6:MerCreMer mice administered tamoxifen do not develop similar ICD ultrastructural changes.

4. Apoptosis assessment at 2 or 4 weeks after tamoxifen administration would be helpful to better determine if cardiomyocyte apoptosis contributed to the 8 and 24 week DCM phenotype in the Myh6:MerCreMer/PALLD fl/fl mice.

5. Figure 4F – There appears to be a 4-fold reduction in Myh7 gene expression in Myh6:MerCreMer mice administered tamoxifen. Do the authors have an explanation for this result?

Reviewer #3 (Recommendations for the authors):

1. Is there a sufficient sample in the human tissue to measure protein levels or MYPN and PALLD to confirm (or refute) the mRNA analysis?

2. Identification of the novel binding partners is interesting but would be enhanced with data showing whether CARP and/or FHOD1 localization or levels are affected by the knockdown models. Without these links, the connection between Figure 1 and the rest of the paper is weakened.

3. It is not clear what the evidence is for the claim that PALLD is a candidate gene for cardiomyopathy. That changes in protein expression correlate with a disease phenotype (under certain conditions) is insufficient to offer this claim.

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Ablation of palladin in adult heart causes dilated cardiomyopathy associated with intercalated disc abnormalities" for further consideration by eLife. Your revised article has been evaluated by Didier Stainier (Senior Editor) and a Reviewing Editor.

Despite considering that not all Reviewer's comments have been fully addressed, all three Reviewers recommend acceptance of the manuscript, provided that the Discussion section is altered to show awareness of study limitations. Please note that, as further detailed, additional corrections are needed in Figure 2 supplement 2.

Please submit the manuscript with the requested alterations highlighted so that we can proceed with formal acceptance.

Recommendations for the authors:

1. The new Figure 1, although helpful for the understanding of the paper, does not contain any results from original research. Having this in consideration, please make this a supplemental figure.

2. To conciliate the text with data presented and show awareness of study limitations please do the following alterations to the Discussion section:

2a. In line 385, after "unaffected", please include the following statement, or any combination of sentences conveying a similar message: "As measurements of contractility and ca²⁺ transients of isolated ventricular cardiomyocytes were performed at ca²⁺ concentrations that cannot be reached under physiological cardiac contraction, we cannot exclude that these observations might not fully recapitulate in vivo mechanisms".

2b. In line 430, after "dysfunction in cPKOi mice", please include the following statement, or any combination of sentences conveying a similar message: "Our data has shown that, in addition to the ICD, PALLD localizes to other cellular compartments, including the nucleus. Therefore, we cannot rule out the possibility that ultrastructural changes observed in cPKOi cardiomyocytes might be a consequence of LV dilation rather than the primary cause of the observed phenotype".

2c. New studies added to the revised manuscript showed absence of changes in MYPN and PALLD at the protein level in human patients. Therefore, please remove the following sentence (461-462): "Nevertheless, our results suggest the potential use of MYPN and PALLD as biomarkers".

2d. No disease-causing PALLD mutations are known, therefore correct lanes 469-470 to read "The reason why a putative link"

Shortly after submission of the revised manuscript, authors communicated to the editorial office a mistake in Figure 2 supplement 2. A corrected version of this figure has been submitted, however this needs further correction. The red channel image displayed for the middle panels does not match the merged image (it is still the same as in the original Figure 2 supplement 2). Please correct this.

Essential revisions:

1) Authors propose that the DCM phenotype observed in the inducible PALLD cKOi hearts is a consequence of intercalated disc (ICD) defects. Please provide data assessing the integrity of the ICD in the constitutive PALLD cKO. If similar changes occur in these hearts, then this defect is not likely the cause of the phenotype observed in the inducible KO. Similarly, to ensure that the ICD phenotype is caused by the absence of PALLD rather than being a consequence of tamoxifen administration, please assess ICD integrity in Myh6:MerCreMer mice administered tamoxifen.

Since cPKO mice showed no pathological phenotype, we discontinued the line to reduce mouse costs and liberate space in the mouse facility. Therefore, unfortunately we don’t have the possibility to assess the integrity of the ICD in these mice. However, since ICD abnormalities are known to be associated with DCM, we consider it highly unlikely that cPKO mice would have ICD defects as observed in cPKOi mice. In fact, we are not aware of the existence of any mouse model with increased ICD fold amplitude and normal cardiac function.

In the previous Figure 5C and D, the groups named Palld^fl/fl;TAM were in fact pooled results from MRM^+/0 TAM and Palld^fl/fl;TAM mice as we found no differences in ICD fold amplitude between the two control groups. We realize that we should have made this clear. We have now included the controls groups separately in the new Figure 6C and D. As it is clear from our measurements, TAM-injected Myh6:MerCreMer mice show no increase in ICD fold amplitude or other ICD abnormalities. It should be noted that Myh6:MerCreMer mice have been extensively used in the literature and that no adverse effects have been associated with administration of tamoxifen at a dose of 30 mg/kg/day for 5 days (Rouhi et al., J Cardiovasc Aging 2, 8, 2022).

2) To ensure that apoptosis is not contributing to the phenotype observed, please repeat the TUNEL experiment at an earlier timepoint (4-week post-induction). In the current version of the manuscript, apoptosis was assessed 24-week post-induction, which is a rather late timepoint and might miss early events.

As requested, we performed TUNEL staining on cPKOi (Palld^fl/fl;MCM^+/-) and control mice (Palld^fl/fl and MCM^+/-) 4 weeks after TAM induction. As shown in Author response image 1, no apoptosis was detected in cPKOi mice or any of the control mice 4 weeks after injection. Consequently, we have modified line 221 to: “No apoptosis was detected by TUNEL staining at 4 and 24 weeks after TAM induction”.

Author response image 1

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3) Please determine if CARP and FHOD1 localization are altered in PALLD cKO versus cKOi. This will improve the connection between the first section of the manuscript (identification of novel interactants) and the phenotypic characterization of conditional mutants, and might shed some light on why the two models diverge phenotypically.

The Reviewer may have missed that in Figure 6—figure supplement 2 (new Figure 7—figure supplement 2), we already provided immunofluorescence stainings, showing no alterations in the localization of CARP, FHOD1, and other known interaction partners of PALLD in cardiomyocytes (CMCs) from cPKOi vs. control mice. As we have discontinued the cPKO line, we don’t have the possibility to perform stainings on CMCs from cPKO mice. However, considering that we found no alterations in the localization of PALLD-interacting proteins in cPKOi mice, which have a pathological cardiac phenotype, we find it unlikely that CARP and FHOD1 would be mislocalized in CMCs of cPKO mice, which do not have any pathological cardiac phenotype.

To make it clearer that we performed immunofluorescence stainings for CARP, FHOD1 and other PALLD-interacting proteins, we modified the text in line 267-270 to:

“Immunofluorescence stainings of isolated adult CMCs showed no alterations in the localization of ICD proteins (Figure 7—figure supplement 1) and PALLD interacting proteins, including CARP, FHOD1, nebulette, SORBS2, MRTF-1, and cypher (Figure 7—figure supplement 2).”

4) As pointed out by all Reviewers, the human mRNA data alone does not provide enough evidence that PALLD might play a role in human DCM. It is particularly difficult to reconcile the fact that deletion of PALLD in mice causes DCM, whereas in human DCM patients the transcripts encoding this protein are upregulated. It is possible that PALLD protein levels are actually reduced in human DCM and the observed mRNA upregulation is part of a compensation mechanism. To test this, please provide measurements of PALLD protein level in human DCM samples.

We agree with the Reviewers that altered mRNA levels do not provide sufficient evidence for a role of PALLD in human DCM. As we didn’t have any tissue left of the original biopsies, we obtained new biopsies from 10 DCM and 7 ICM patients as well as 7 controls hearts on which we performed both qRT-PCR and Western blot analyses. qRT-PCR on the additional samples confirmed our previous results of increased transcript levels of the 200 kDa PALLD isoform, MYPN, and ANKRD1 in DCM patients. In addition, we found a significantly reduction in overall transcript levels of PALLD in both DCM and ICM patients, which is consistent with the results of two RNA-Seq studies (Sweet et al., BMC Genomics, 19, 812, 2018; Yang et al., Circulation, 129, 1009-1021, 2014). The increased transcript level of the 200 kDa PALLD isoform despite the overall reduced transcript level of PALLD can be explained by the fact that the 200 kDa PALLD isoform is expressed at much lever than the 90 kDa PALLD isoform, which is the predominant isoform in the heart. By Western blot analyses, we found no changes in the protein expression of PALLD and MYPN in DCM and ICM patients vs. controls. On the other hand, the previously reported upregulation of CARP/Ankrd1 in DCM patients was confirmed (Kempton et al., Heliyon, 4:e00514, 2018; Nagueh et al., Circulation, 110, 155-162, 2014; Zolk et al., Biochem Biophys Res Commun, 293, 1377-1382, 2002). Thus, the transcriptional changes in PALLD and MYPN expression in human DCM are unlikely to affect cardiac function. Nevertheless, our findings suggest the potential use of PALLD and MYPN as biomarkers for DCM.

Reviewer #1 (Recommendations for the authors):

1. The introduction has a long list of PALLD interactions with other proteins that is not necessary for the reader to understand the work here presented. Please shorten this text and simply mention that whereas multiple interactions have been previously identified for the proline-rich domains and last 3 Ig domains (as nicely summarized in Figure 1A), nothing is known regarding putative interactors in the N-terminal domain (encompassing the first 2 Ig domains).

To address the Reviewer’s comment, we removed the list of interactions with non-muscle proteins, which are not relevant for the role of PALLD in striated muscle. However, we prefer to include the list of PALLD-binding proteins in striated muscle as these are relevant for understanding the structural role of PALLD in the Z-line and ICD as well as its role in regulating the actin cytoskeleton. In our analysis of cPKOi mice, we performed Western blot and immunofluorescence staining for PALLD interacting proteins and we therefore find it important to mention them in the introduction.

2. Please alter Figure 1A to include not just the long isoform, but also the other relevant variants of PALLD. Please add also information regarding expected molecular weights. This will make it easier to understand in subsequent western blots what isoforms are expressed (and ablated in cKOs) in cardiomyocytes.

We agree that a figure showing the different PALLD isoforms will facilitate the understanding of the paper. As there are at least 10 PALLD isoforms it is not feasible to include all the isoforms in Figure 1A. Therefore, we have instead included a new Figure 1, illustrating all the currently known isoforms with predicted and apparent molecular weights, when known. In the new Figure 1—figure supplement 1, we have included a figure showing the exon structure of all known isoforms. This is particularly useful for understanding which isoforms are targeted by our approach.

3. Is the signal of all antibodies used in Figure 1 specific? In other words, do these antibodies produce any signal when applied to tissues from PALLD KO/KO mice?

All the used PALLD antibodies have been extensively characterized in different labs and are specific as shown by the absence of the targeted PALLD isoforms by Western blot analysis (Figure 3B and 4B).

Due to extensive isoform diversity, it wasn’t possible to target all PALLD isoforms and in cPKO mice both an N-terminal and a C-terminal isoform not targeted by our approach are expressed (see the new Figure 1—figure supplement 1). Unfortunately, a C-terminal isoform with a start codon 3’ of the exon that we targeted was discovered after the mice had been generated. For that reason, the PALLD antibodies also stain cPKO tissue and it is therefore difficult to distinguish cPKO mice from control mice based on immunofluorescence stainings. Thus, to avoid confusion, we have not included pictures of KO tissue stained with PALLD antibodies.

4. Based on the antigen information described in the text (whole protein, C-terminus, second proline-rich region), please add to Figure1 a diagram reporting what isoforms are recognized by each of the antibodies.

The epitopes against which the PALLD (Proteintech) and 4D10 antibodies have been raised are indicated in the new Figure 1. Unfortunately, although the PALLD 622 antibody (and the PALLD 621 antibody raised against the same epitopes) has been extensively used in the literature, it has not been possible to find out the exact epitopes to which it was raised and for this reason we have not included the PALLD 622 antibody in the figure. From personal communication with Prof. Carol Otey, in whose lab the PALLD 622 antibody was generated many years ago, the antibody was raised against both N- and C-terminal epitopes and should thus recognize all isoforms.

5. In Figure 1D, the nuclear labeling produced by antibody PALLD 622 seems to be enriched in specific foci. Is this a consistent observation, or a particular of this image? If this is consistent, do the authors know what sort of foci are these? Nucleoli?

We thank the Reviewer for pointing this out. Both the PALLD 622 and Proteintech antibodies showed the absence of PALLD from nuclear regions strongly stained with DAPI, suggesting its localization in the transcriptionally active regions of the nucleus. Furthermore, with the PALLD 622 antibody we often observed a punctuate staining pattern, which appears to correspond to nucleoli. To point this out we have added the following sentence:

Line 121: “PALLD was found to be absent from nuclear regions strongly stained with DAPI, corresponding to heterochromatin, suggesting the localization of PALLD in the transcriptionally active euchromatin regions of the nucleus. Additionally, stronger PALLD staining in a punctuate pattern was often observed in regions with absent DAPI staining, likely corresponding to nucleoli”.

6. The interactants and immunofluorescence data shown in Figure 1 are somehow disconnected from the rest of the manuscript. This could be improved if, for example, authors would perform immunofluorescence analyses on their cKOs to assess if the nuclear PALLD signal disappears in their cKO and icKO animals.

As mentioned above, unfortunately, due to extensive isoform diversity it is not possible to target all PALLD isoforms, so there are both an N-terminal and a C-terminal isoform not targeted by our approach (see the new Figure 1—figure supplement 1). For that reason PALLD staining is also observed in cPKO and cPKOi mice and to avoid confusion we therefore didn’t include immunofluorescence stainings on KO animals.

7. Lines 144-145: "we generated Palld floxed (Palldfl/fl) mice containing LoxP sites flanking exon 15". Please add here what is the expected consequence of ablation of exon 15 for the different isoforms of PALLD. What domain of the protein does exon 15 correspond to? After floxing out, is there an early termination codon leading to truncated variants?

To address this, we have now included Figure 1—figure supplement 1, showing the exon structure of all known PALLD isoforms. The floxed exon is indicated with yellow and corresponds to a region between the second proline-rich region and the third Ig domain. The length of the floxed exon is not evenly divisible by 3 and Cre-mediated recombination thus causes a frameshift and consequently a premature stop codon. This results in RNA instability and consequent RNA degradation by nonsense-mediated decay. Thus, the location of the floxed exon is not important as all isoforms containing the exon get degraded.

We initially referred to the targeted exon as exon 15 according to the nomenclature of Wang and Moser (Wang and Moser, 2008). However, while preparing the figure, we realized that exon 12 had been counted as two exons by Wang and Moser and for that reason we now refer to the floxed exon as exon 14. As evident from the figure, the last two isoforms shown are not targeted by our approach.

8. Lanes 166-167: "in response to (…) TAC, no significant differences were found between cPKO and control mice as shown by echocardiography (Figure 2D and Figure 2—figure supplement 4) (…) although there was a tendency towards reduced contractile function in cPKO mice from 2 weeks after TAC". This description might cause some confusion, as the graphs do show significant differences (EF and FS) between cPKO and Cre- negative controls. Please revise the text to read "no significant differences were found between cPKO and Cre+ control mice".

The text has been revised as requested in line 169 of the revised manuscript. Additionally, in the discussion “although cPKO mice showed a tendency towards reduced systolic function compared to control mice” has been removed from the sentence in line 379, so it now reads:

“Unexpectedly, cPKO mice exhibited no pathological cardiac phenotype at basal levels and showed a normal hypertrophic response to TAC”.

9. Histological data in Figure 2E and respective quantification in 2F: in addition to the genotype, please add "4-week post TAC" to the label. Also, please highlight (with a box) in the low magnification whole heart image the area shown in the high magnification panels. The high magnification images show areas of interstitial myocardium, however, fibrosis post-TAC is frequently concentrated in the vicinity of large coronaries. Please provide also images and quantification of fibrosis in perivascular areas of control and cKO TAC hearts. Please include non-TAC hearts as control.

As requested by the Reviewer, we have included hearts from mice subjected to SHAM as controls, indicated the location of high-magnification areas with a box, and modified the labels (new Figure 3E). In addition, we have included pictures of perivascular areas and performed the quantification of both interstitial and perivascular areas. No significant difference in fibrosis was found between cPKO and control hearts.

10. For increased clarity, in Figure 4A, please move genotype labels to the top, instead of the bottom position they currently occupy. On the left side of the panel, please add a label stating what sort of staining is being shown. Please highlight in the low magnification image the area shown in higher magnifications.

The figure (now Figure 5A) has been modified as requested by the Reviewer. In addition, pictures of perivascular areas have been included and quantification of both interstitial and perivascular areas has been performed. Both interstitial and perivascular fibrosis were significantly increased in cPKOi hearts.

11. The discussion is rather long and has elements that belong in the Results section (for example describing tamoxifen injection into the constitutive KO mice).

We agree that the tamoxifen injection of cPKO mice belongs to the result section. This has now been moved to line 195-200 of the result section and the results are presented in Figure 4—figure supplement 1.

Line 200-205: “To rule out the possibility that the phenotype of cPKOi mice is due to the toxic effect of TAM, we injected cPKO mice with TAM and evaluated cardiac function by echocardiography 8 weeks after TAM injection (Figure 4—figure supplement 1). No pathological cardiac phenotype was observed after TAM injection, suggesting that early ablation of PALLD allows for the activation of compensatory mechanisms.”

We are not aware of other elements of the discussion belonging to the result section.

12. Lines 460-461: "Based on the absence of a phenotype in PKO mice". Please correct to "Based on the absence of a phenotype in cPKO mice".

Thanks for noticing this. This has now been corrected.

Reviewer #2 (Recommendations for the authors):

1. Are there changes in the PALLD interacting proteins depending on if PALLD is deleted in the embryonic versus adult period?

As we didn’t find any changes in the expression or localization of PALLD interacting proteins in cPKOi mice, which have a pathological cardiac phenotype, we didn’t check for changes in cPKO mice, which have no pathological phenotype. Unfortunately, the cPKO line has been discontinued as a result of the absent pathological phenotype and it is therefore not possible to check. Considering that we found no alterations in localization of PALLD-interacting proteins in cPKOi mice, which have a pathological cardiac phenotype, we find it unlikely that PALLD interacting proteins would be mislocalized cPKO mice.

2. To better support the claim that ICD ultrastructural changes are contributing to DCM in adult PALLD deletion, it would be important to determine:

a. Do these same alterations in ICD ultrastructure occur in the mice with embryonic deletion of cardiomyocyte PALLD?

As explained above, since cPKO mice showed no pathological phenotype, we discontinued the line to reduce mouse costs and liberate space in the mouse facility. Therefore, unfortunately we don’t have the possibility to assess the integrity of the ICD in these mice. However, since ICD abnormalities are known to be associated with DCM, we consider it highly unlikely that cPKO mice would have ICD defects as observed in cPKOi mice. In fact, we are not aware of the existence of any mouse model with increased ICD fold amplitude and normal cardiac function.

b. Are these ICD ultrastructure changes a non-specific result of ventricular dilation and failure? For example, do they occur in mice with DCM secondary to other causes (MI, chronic aortic banding, etc.)?

ICD widening and been observed in various mouse models of DCM and ICD disorganization has been associated with human DCM (Wilson et al., Cell Mol Life Sci, 71, 165-181, 2014; Ito et al., Scientific reports 11, 11852, 2021; Ortega et al., PLoS One 12; e0185062, 2017). Also, chronic TAC has been associated with ICD widening (Zankov et al., Scientific Reports 7, 39335, 2017). However, it is unclear whether ICD disorganization is a cause or a result of DCM. We previously reported that MYPN KO mice subjected to 4 weeks of TAC develop severe DCM associated with increased ICD fold amplitude (Filomena et al., eLife 10:e58313, 2021). However, although MYPN subjected to TAC develop a much more severe DCM phenotype than cPKOi mice, the ICM abnormalities are more severe in cPKOi mice. Based on this, the ICD abnormalities in cPKOi mice appear to be more than just a secondary result of the DCM phenotype and likely related to the association of PALLD with the ICD.

3. Since the ICD ultrastructure defects are postulated as the mechanism regulating DCM development in the adult PALLD deletion mice, it would be important to confirm that Myh6:MerCreMer mice administered tamoxifen do not develop similar ICD ultrastructural changes.

In the previous Figure 5C and D, the groups named Palld^fl/fl;TAM were in fact pooled results from MRM^+/0 TAM and Palld^fl/fl;TAM mice as we found no differences in ICD fold amplitude between the two control groups. We realize that we should have made this clear. We have now included the controls groups separately in the new Figure 6C and D. As it is clear form our measurements, TAM-injected Myh6:MerCreMer mice show no increase in ICD fold amplitude or other ICD abnormalities. It should be noted that Myh6:MerCreMer mice have been extensively used in the literature and that no adverse effects have been associated with administration of tamoxifen at a dose of 30 mg/kg/day for 5 days (Rouhi et al., J Cardiovasc Aging 2, 8, 2022).

4. Apoptosis assessment at 2 or 4 weeks after tamoxifen administration would be helpful to better determine if cardiomyocyte apoptosis contributed to the 8 and 24 week DCM phenotype in the Myh6:MerCreMer/PALLD fl/fl mice.

Based on the literature there is no reason to think that administration of tamoxifen at a dose of 30 mg/kg/day for 5 days to Myh6:MerCreMer mice would result in apoptosis. As requested, we have performed TUNEL staining on cPKOi (Palld^fl/fl;MCM^+/-) and control mice (Palld^fl/fl and MCM^+/-) 4 weeks after TAM induction. As shown in Author response image 1, no apoptosis was detected in cPKOi or any of the control mice 4 weeks after injection. Consequently, we have modified line 221 to: “No apoptosis was detected by TUNEL staining at 4 and 24 weeks after TAM induction”.

5. Figure 4F – There appears to be a 4-fold reduction in Myh7 gene expression in Myh6:MerCreMer mice administered tamoxifen. Do the authors have an explanation for this result?

In a recent qRT-PCR study, TAM induction of Myh6:MerCreMer mice at a dose of 30 mg/kg/day for 5 days was found to cause transient changes in CMC gene expression 2 weeks after TAM injection without affecting cardiac function, myocardial fibrosis, apoptosis, or induction of double-stranded DNA breaks (Rouhi et al., J Cardiovasc Aging 2, 8, 2022). Consistent with the downregulation of Myh7 in our study, Myh7 expression was reported to be 1.9-fold reduced in CMCs 2 weeks after TAM induction. Thus, the downregulation of Myh7 is likely an effect of the TAM induction of Myh6:MerCreMer mice.

Reviewer #3 (Recommendations for the authors):

1. Is there a sufficient sample in the human tissue to measure protein levels or MYPN and PALLD to confirm (or refute) the mRNA analysis?

We didn’t have sufficient human tissue tissues left for measuring protein of MYPN and PALLD. However, we were able to obtain new biopsies from 10 DCM and 7 ICM patients as well as 7 controls hearts on which we performed both qRT-PCR and Western blot analyses. qRT-PCR on the additional samples confirmed our previous results of increased transcript levels of the 200 kDa PALLD isoform, MYPN, and ANKRD1 in DCM patients. In addition, we found a significantly reduction in overall transcript levels of PALLD in both DCM and ICM patients, which is consistent with the results of two RNA-Seq studies (Sweet et al., BMC Genomics, 19, 812, 2018; Yang et al., Circulation, 129, 1009-1021, 2014). The increased transcript level of the 200 kDa PALLD isoform despite the overall reduced transcript level of PALLD can be explained by the fact that the 200 kDa PALLD isoform is expressed at much lever than the 90 kDa PALLD isoform, which is the predominant isoform in the heart. By Western blot analyses, we found no changes in the protein expression of PALLD and MYPN in DCM and ICM patients vs. controls. On the other hand, the previously reported upregulation of CARP/Ankrd1 in DCM patients was confirmed (Kempton et al., Heliyon, 4:e00514, 2018; Nagueh et al., Circulation, 110, 155-162, 2014; Zolk et al., Biochem Biophys Res Commun, 293, 1377-1382, 2002). Thus, the transcriptional changes in PALLD and MYPN expression in human DCM are unlikely to affect cardiac function.

2. Identification of the novel binding partners is interesting but would be enhanced with data showing whether CARP and/or FHOD1 localization or levels are affected by the knockdown models. Without these links, the connection between Figure 1 and the rest of the paper is weakened.

The Reviewer may have missed that in Figure 6—figure supplement 2 (new Figure 7—figure supplement 2), we already provided immunofluorescence stainings, showing no alterations in the localization of CARP, FHOD1, and other known interaction partners of PALLD in CMCs from cPKOi vs. control mice. As we have discontinued the cPKO line, we don’t have the possibility to perform stainings on CMCs from cPKO mice. However, considering that we found no alterations in the localization of PALLD-interacting proteins in cPKOi mice, which have a pathological cardiac phenotype, we find it unlikely that CARP and FHOD1 would be mislocalized in cPKO CMCs.

To make it clearer that we performed immunofluorescence stainings for CARP, FHOD1 and other PALLD-interacting proteins, we modified the text in line 267-269 to:

“Immunofluorescence stainings of isolated adult CMCs showed no alterations in the localization of ICD proteins (Figure 7—figure supplement 1) and PALLD interacting proteins, including CARP, FHOD1, nebulette, SORBS2, MRTF-1, and cypher (Figure 7—figure supplement 2).

3. It is not clear what the evidence is for the claim that PALLD is a candidate gene for cardiomyopathy. That changes in protein expression correlate with a disease phenotype (under certain conditions) is insufficient to offer this claim.

This claim was made based on the phenotype of the cPKOi mice, not the fact that PALLD transcript levels are changed in DCM patients. In the paper we demonstrated that PALLD plays a role in the heart. Like for its homologue MYPN, it is possible that dominant PALLD mutations could be associated with cardiac disease, while PALLD ablation would likely lead to embryonic lethality due to the essential role of PALLD in other tissues and cell types. MYPN KO mice show only mild DCM, while human MYPN mutations with dominant negative effects have been shown to cause dilated, hypertrophic, and restrictive cardiomyopathy (Filomena et al., eLife 10: e58313, 2021). We discuss this in the last section of the discussion.

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

Recommendations for the authors:

1. The new Figure 1, although helpful for the understanding of the paper, does not contain any results from original research. Having this in consideration, please make this a supplemental figure.

We have now included Figure 1 and Figure 1—figure supplement 1 as new Figure 1—figure supplement 1 and Figure 1—figure supplement 2. Consequently, all figure numbers have been changed.

2. To conciliate the text with data presented and show awareness of study limitations please do the following alterations to the Discussion section:

2a. In line 385, after "unaffected", please include the following statement, or any combination of sentences conveying a similar message: "As measurements of contractility and ca²⁺ transients of isolated ventricular cardiomyocytes were performed at ca²⁺ concentrations that cannot be reached under physiological cardiac contraction, we cannot exclude that these observations might not fully recapitulate in vivo mechanisms".

The Ionoptix studies on isolated cardiomyocytes were performed in the presence of a physiological amount of CaCl₂ (1.0 mM). It was the experiments on myofibril preparations that were performed at non-physiological ca²⁺ concentrations. We have therefore modified the suggested sentence to the following:

“As biomechanical properties of myofibril preparations from the LV were measured at ca²⁺ concentrations that cannot be reached under the physiological conditions of cardiac contraction, we cannot exclude that our observations might not fully recapitulate in vivo mechanisms”.

2b. In line 430, after "dysfunction in cPKOi mice", please include the following statement, or any combination of sentences conveying a similar message: "Our data has shown that, in addition to the ICD, PALLD localizes to other cellular compartments, including the nucleus. Therefore, we cannot rule out the possibility that ultrastructural changes observed in cPKOi cardiomyocytes might be a consequence of LV dilation rather than the primary cause of the observed phenotype".

2c. New studies added to the revised manuscript showed absence of changes in MYPN and PALLD at the protein level in human patients. Therefore, please remove the following sentence (461-462): "Nevertheless, our results suggest the potential use of MYPN and PALLD as biomarkers".

2d. No disease-causing PALLD mutations are known, therefore correct lanes 469-470 to read "The reason why a putative link"

The requested changes 2b, 2c, 2d to the discussion have been made as suggested.

Shortly after submission of the revised manuscript, authors communicated to the editorial office a mistake in Figure 2 supplement 2. A corrected version of this figure has been submitted, however this needs further correction. The red channel image displayed for the middle panels does not match the merged image (it is still the same as in the original Figure 2 supplement 2). Please correct this.

We apologize for this mistake. The figure has now been corrected.

Author details

1. Giuseppina Mastrototaro

   1. University of Milan-Bicocca, Milan, Italy
   2. IRCCS Humanitas Research Hospital, Milan, Italy

   Present address

   AGC Biologics, Milan, Italy

   Contribution

   Formal analysis, Investigation, Writing – original draft

   Competing interests

   No competing interests declared

2. Pierluigi Carullo

   1. IRCCS Humanitas Research Hospital, Milan, Italy
   2. Institute of Genetic and Biomedical Research, National Research Council, Milan unit, Milan, Italy

   Contribution

   Formal analysis, Investigation

   Competing interests

   No competing interests declared

3. Jianlin Zhang

   University of California, San Diego, La Jolla, United States

   Contribution

   Formal analysis, Investigation

   Competing interests

   No competing interests declared

4. Beatrice Scellini

   Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy

   Contribution

   Formal analysis, Investigation

   Competing interests

   No competing interests declared

5. Nicoletta Piroddi

   Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy

   Contribution

   Formal analysis, Investigation

   Competing interests

   No competing interests declared

6. Simona Nemska

   IRCCS Humanitas Research Hospital, Milan, Italy

   Contribution

   Formal analysis, Investigation

   Competing interests

   No competing interests declared

7. Maria Carmela Filomena

   1. IRCCS Humanitas Research Hospital, Milan, Italy
   2. Institute of Genetic and Biomedical Research, National Research Council, Milan unit, Milan, Italy

   Contribution

   Investigation

   Competing interests

   No competing interests declared

8. Simone Serio

   IRCCS Humanitas Research Hospital, Milan, Italy

   Contribution

   Formal analysis

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-7294-2094

9. Carol A Otey

   Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, United States

   Contribution

   Conceptualization, Resources

   Competing interests

   No competing interests declared

10. Chiara Tesi

    Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy

    Contribution

    Formal analysis, Investigation

    Competing interests

    No competing interests declared

11. Fabian Emrich

    Department of Cardiac Surgery, Goethe University, Frankfurt, Germany

    Contribution

    Resources, Provided patient biopsies

    Competing interests

    No competing interests declared

12. Wolfgang A Linke

    Institute of Physiology II, University of Münster, Münster, Germany

    Contribution

    Resources, Supervision

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-0801-3773

13. Corrado Poggesi

    Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy

    Contribution

    Resources, Supervision, Funding acquisition, Writing – original draft

    Competing interests

    No competing interests declared

14. Simona Boncompagni

    Department of Neuroscience, Imaging and Clinical Sciences and Center for Advanced Studies and Technology, University G. d'Annunzio, Chieti, Italy

    Contribution

    Formal analysis, Investigation, Writing – original draft, Writing - review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-5308-5069

15. Marie-Louise Bang

    1. IRCCS Humanitas Research Hospital, Milan, Italy
    2. Institute of Genetic and Biomedical Research, National Research Council, Milan unit, Milan, Italy

    Contribution

    Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Writing – original draft, Project administration, Writing - review and editing

    For correspondence

    marie-louise.bang@cnr.it

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-8859-5034

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