Review Papers Claim by Claim

1) Core claims and evidence (what the authors show)

• ZEB1 protein levels are altered in human cardiomyopathy and mouse models: reduced in human ICM and DCM samples and reduced in Rbm20 KO, but increased in Mybpc3 KO (HCM model) — data and statistics reported in Figure 1 and quantified in the manuscript ZEB1 protein changes in cardiomyopathy models
• Gain of function: Zeb1 overexpression in NRCMs and via AAV9 in adult mice drives hypertrophic changes, upregulates Nppa/Nppb, increases cardiomyocyte size, induces fibrosis and reduces ejection fraction (EF) by week 7 (statistical p-values provided) ZEB1 overexpression phenotypes
• Loss of function: Cardiomyocyte-specific Zeb1 conditional knockout (Zeb1cKO) and inducible icKO cause progressive systolic dysfunction and chamber dilation; female mice are markedly more susceptible, showing reduced survival and very low EF in older animals (EF ~8–10%) ZEB1 knockout cardiac dysfunction
• Structural and bioenergetic defects: TEM and isolated cardiomyocyte measurements show sarcomeric disarray, increased sarcomere length and Z-disc thickness, reduced mtDNA copy number, and lower maximal OCR after Zeb1 loss (Seahorse) Sarcomere and mitochondrial defects
• Transcriptional regulation: Bulk RNA-seq (female LVs) shows strong upregulation of epithelial and ECM genes (e.g., Cdh1, Krt8) after Zeb1 deletion; de novo motif analysis (HOMER) finds Zeb1 motif enriched among upregulated genes; ChIP-seq in NRCMs maps Zeb1 binding genome-wide with promoter-proximal enrichment and identifies high-confidence promoter-bound DEGs including mitochondrial genes Acaa2 and Smim20 RNA-seq and ChIP-seq integration

2) Strengths of the study

1. Comprehensive multi-level approach: human tissue, multiple mouse genetic models (constitutive cKO plus inducible icKO), viral overexpression, NRCM experiments, TEM, Seahorse, RNA-seq and ChIP-seq — convergent evidence from structure, function and genomics strengthens causal interpretation Study multimodal design
2. Integration of ChIP-seq with RNA-seq to nominate direct targets (promoter-bound plus motif-containing DEGs) is an important mechanistic advance and supports both repressive (epithelial genes) and activating (mitochondrial genes) roles for ZEB1 in cardiomyocytes ChIP RNA integration

3) Limitations, potential confounders and blindspots

• Developmental confounder for the constitutive cKO: The αMHC-Cre used in the constitutive Zeb1cKO becomes active perinatally; therefore some phenotypes could reflect disrupted perinatal cardiomyocyte maturation or proliferation rather than pure maintenance roles in adult CMs. The authors partly address this with a MerCreMer inducible icKO, but the duration (7 weeks) and readouts could miss later consequences and sex-hormone interactions Developmental confounder note
• Sex difference unexplained: Female-biased sensitivity is robust in the data but mechanisms (estrogen signaling, X-chromosome effects, transcriptional cofactors) are not experimentally probed; sex-hormone manipulation or gonadectomy experiments would be needed to move beyond descriptive sex-differences Sex difference observation
• ChIP-seq in neonatal rat cardiomyocytes: ChIP-seq was performed in NRCMs because of higher Zeb1 expression and chromatin yield; extrapolating neonatal rat promoter binding to adult mouse cardiomyocytes and to human disease contains species and developmental-state assumptions that can introduce misannotation of targets — ideally adult CM ChIP or CUT&RUN from mouse CMs would confirm promoter occupancy in the adult context ChIP cell type caveat
• Limited human sample sizes and heterogeneity: Human ICM/DCM/NF sample numbers are modest (NF N~5, ICM/DCM N~8) and clinical heterogeneity of samples (age, medications, comorbidities) could confound protein-level comparisons; nevertheless statistical differences are reported Human sample limitations
• Functional rescue tests are limited: The paper robustly shows loss and gain phenotypes, but therapeutic or rescue experiments (restore ZEB1 in cKO, or rescue mitochondrial genes) are not presented, which would strengthen causal chain from ZEB1->target gene->phenotype Lack of rescue experiments

4) Specific methodological points and reproducibility

• Datasets reported as deposited: authors indicate raw sequencing SRP630684 (SRA), ChIP-seq E-MTAB-15712 and processed GEO accession GSMXXXXX; these deposits increase reproducibility when complete and accessible (curators should verify release and sample metadata) Data availability statement
• Statistical reporting: P-values are reported for key comparisons (e.g., EF, mtDNA, OCR), sample sizes are provided in many figures (N per group), and standard workflows (edgeR, HOMER) are used; raw count tables and ChIPpeak BED files would be needed to fully re-run analyses and confirm thresholds and filtering choices Statistical and methods summary

5) Biological interpretation and plausibility

ZEB1 is a canonical EMT transcription factor known to repress epithelial genes (e.g., CDH1/E-cadherin) and to act via co-repressors/co-factors in other tissues; the paper's finding that ZEB1 represses epithelial/ECM programs in cardiomyocytes is mechanistically plausible. The additional observation that ZEB1 binds promoter regions of mitochondrial genes and appears to support mitochondrial gene expression and function suggests a context-specific activator role (or cofactor-mediated activation) in cardiomyocytes, which is novel and biologically credible given tissue-specific cofactor repertoires and previous reports of non-canonical roles for EMT TFs ZEB1 binding to mitochondrial genes

6) How could the principal conclusions be falsified?

1. If inducible adult cardiomyocyte-specific re-expression of Zeb1 in Zeb1icKO animals (rescue) fails to restore mitochondrial gene expression, mtDNA, OCR and sarcomere structure, the claim of direct Zeb1 maintenance role would be weakened.
2. If ChIP/CUT&RUN from adult mouse cardiomyocytes (not neonatal rat cells) fails to show promoter binding at the nominated mitochondrial genes (Acaa2, Smim20) or at epithelial genes, that would question direct promoter regulation in adult CMs.
3. If sex-hormone manipulation (ovariectomy or estrogen replacement) abolishes the female-specific vulnerability independently of Zeb1 status, the sex-dimorphic interpretation may need revision.

7) Practical implications and next steps

• Follow-up experiments I recommend (concise): (A) adult CM CUT&RUN/ChIP for ZEB1 to confirm promoter occupancy in the adult mouse and human CMs; (B) rescue: AAV9-Zeb1 re-expression in adult icKO to test reversal of mitochondrial and sarcomeric phenotypes; (C) mechanistic dissection of sex differences using gonadectomy and hormone replacement; (D) interrogate ZEB1 co-factors in cardiomyocytes (e.g., CTBP1, SMARCA4/BRG1, or cardiac-specific partners) to explain promoter-activating vs repressive behavior Authors recommended next directions

8) Reproducibility checklist (what to share to help others reproduce)

• Provide processed RNA-seq count matrices, DE analysis code, edgeR parameters and gene lists (FC and FDR thresholds).
• Share ChIPpeak BED files, narrowPeak files and the exact HOMER command lines and motif outputs.
• Clarify human sample clinical metadata (age, sex, medications, cause of death) for the NF/ICM/DCM groups.
• Make raw Seahorse trace files and TEM quantification spreadsheets available.

9) Short actionable summary for researchers

ZEB1 is a bona fide regulator of cardiomyocyte identity and energetics. The balance of evidence supports that maintaining physiologic ZEB1 is necessary for adult cardiomyocyte mitochondrial and sarcomere integrity. Consider ZEB1 and its downstream mitochondrial program (Acaa2, Smim20) as candidate nodes for mechanistic and therapeutic follow-up; prioritize adult-CM-specific occupancy and rescue experiments and dissect sex-differences before translational extrapolation Paper conclusion and translational note

Key direct citation (paper)

Primary ZEB1 cardiomyocyte preprint