Early cardiac proteomic profiling in a long-latency doxorubicin cardiomyopathy model exhibits sexual dimorphism for cardioprotection

Figures

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  Fig. 1. Longer latency Doxorubicin Cardiotoxicity Model (A) Experimental design-Created in BioRender: Male and female rats received a cumulative dose of 6 mg/kg doxorubicin or an equivalent volume of saline injection. Starting one month after the final injection, cardiac function was evaluated monthly by transthoracic echocardiography for up to 7 months. (B) Fractional shortening (FS) in saline- and doxorubicin-treated male rats (left) and female rats (right). Each data point represents an individual animal. Sample sizes per group vary across timepoints due to scheduled sacrifice for tissue collection; exact n per timepoint: Male Dox (n = 11, 9, 8, 5, 4, 3, 3), Male Saline (n = 8, 8, 7, 2, 10, 2, 2), Female Dox (n = 23, 22, 23, 22, 21, 17, 18), Female Saline (n = 14, 11, 13, 13, 12, 9, 9) for months 1–7 respectively. (C) Representative echocardiograms from conscious male rats showing left ventricular M-mode recordings (1-second duration) from saline controls (FS 62%, top) and doxorubicin-treated rats (FS 33%, bottom). (D) Representative histological sections of hearts from saline-treated (top) and doxorubicin-treated (bottom) male rats.

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  Fig. 2. Global proteomic differences driven by doxorubicin treatment and sex. (A) Experimental design and proteomic analysis workflow-Created in BioRender (B) Principal component analysis (PCA) of proteins significant in the global F-test from a two-way limma model (p < 0.05) demonstrates clear separation between doxorubicin-treated and saline-treated hearts along PC1, indicating a dominant treatment-associated proteomic shift present in both sexes. Separation along PC2 reflects underlying sex-associated differences in the cardiac proteome.(C) Hierarchical clustering heatmap of the same protein set reveals distinct expression patterns across the proteome. n = 3 biologically independent animals per group (Male Saline, Male Doxorubicin, Female Saline, Female Doxorubicin). Each sample represents an individual rat heart.

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  Fig. 3. Impact of Doxorubicin Treatment on the Cardiac Proteome Independent of Sex. (A) Hierarchical clustering heatmap of proteins significantly altered by doxorubicin treatment (Doxo vs Saline, combined across females and males; two-way limma, p-value <0.05).Two major clusters (Clusters 1 and 2) capture the dominant proteomic response to doxorubicin. (B) Ingenuity Pathway Analysis (IPA) of these significantly altered proteins. Orange bars highlight pathways inferred to be activated, while blue bars represent pathways inferred to be inhibited (Fisher's exact test). (C) Predicted upstream regulators associated with the doxorubicin-induced proteomic changes. Orange indicates predicted activation, and blue indicates predicted inhibition compared to saline controls. (D) STRING network analysis of the same protein set, revealing coordinated enrichment of distinct functional modules. Blue nodes represent proteins downregulated in doxorubicin-treated hearts compared to saline controls, whereas yellow nodes represent upregulated proteins. n = 6 biologically independent animals per treatment group (Doxorubicin: 3 male +3 female; Saline: 3 male +3 female). (For interpretation of the references to…

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  Fig. 4. Baseline sexual dimorphism in the cardiac proteome, in the absence of doxorubicin: (A) Hierarchical clustering heatmap of significantly changing proteins (Male vs Female in Saline; two-way limma, p-value <0.05) highlighting four major clusters (Clusters 3, 4, 5 and 7) that distinguish male and female saline-treated hearts. (B) Heatmap of proteins exhibiting significant differences between female and male saline hearts. Out of 5184 proteins detected, 421 showed significant sex-based variation. (C) Ingenuity Pathway Analysis (IPA) reveals pathways inferred to be activated in female saline hearts (Fisher's exact test) compared to male saline hearts. (D) Upstream regulators inferred to be activated in female hearts compared to male saline hearts. (E) STRING network clustering of female-enriched proteins demonstrates coordinated enrichment in distinct biological modules. (F) IPA of male-enriched proteins identifies pathways inferred to be activated (Fisher's exact test) compared to female saline hearts. (G) Upstream regulators inferred to be activated in male hearts to female saline hearts. (H) STRING network analysis of male-enriched proteins reveals organization into distinct…

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  Fig. 5. Sex-dependent proteomic responses to doxorubicin revealed by interaction-based network analysis. (A) Heat map with hierarchical clustering of proteins exhibiting a significant sex × treatment interaction (two-way limma, p-value <0.05), indicating proteins whose response to doxorubicin differs between females and males. (B) STRING network analysis of the same interaction-significant protein set shown in (A), highlighting functionally connected modules within the sex-dependent doxorubicin response. In each node, the inner circle represents the log₂ fold change (doxorubicin vs. saline) in females, while the outer circle represents the corresponding change in males. Yellow indicates upregulation and blue indicates downregulation in doxorubicin-treated animals. n = 3 biologically independent animals per group (Male Saline, Male Doxorubicin, Female Saline, Female Doxorubicin). Each sample represents an individual rat heart. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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  Fig. 6. Box plots of mitochondrial–related proteins. Exhibiting a significant sex-by-treatment interaction (p < 0.05), identified by two-way limma analysis. Shown are normalized log₂ protein abundances across saline- and doxorubicin-treated male and female groups. Boxes represent the interquartile range (IQR) with the horizontal line indicating the median; individual data points are overlaid. P values correspond to pairwise contrasts extracted from the fitted two-way LIMMA model. n = 3 biologically independent animals per group (Male Saline, Male Doxorubicin, Female Saline, Female Doxorubicin). Each data point represents an individual rat heart.

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  Fig. 7. Box plots of excitation–contraction coupling proteins. Exhibiting a significant sex-by-treatment interaction (p < 0.05), identified by two-way limma analysis. Shown are normalized log₂ protein abundances across saline- and doxorubicin-treated male and female groups. Boxes represent the interquartile range (IQR) with the horizontal line indicating the median; individual data points are overlaid. P values correspond to pairwise contrasts extracted from the fitted two-way limma model. n = 3 biologically independent animals per group (Male Saline, Male Doxorubicin, Female Saline, Female Doxorubicin). Each data point represents an individual rat heart.