Abstract Chronic reductive stress (cRS), induced by constitutive activation of Nrf2 in transgenic (TG) mouse hearts leads to pathological cardiac remodeling and diastolic dysfunction. Transcriptomic analysis revealed that both pro-reductive (PR) and reductive stress (RS) conditions disrupt ER-associated gene expression in a dose-dependent manner, with pronounced dysregulation in high-expressing TG (TGH) mice. These shifts were associated with persistent activation of the unfolded protein response (UPR), impaired ER function, and redox imbalance marked by elevated glutathione and reduced ROS levels. Proteostasis disruption under cRS led to protein misfolding, ER dilation, and aggregation of mis/unfolded proteins. TGH mice showed increased ubiquitination and accumulation of aggregated proteins, alongside inadequate proteasome activity, indicating inadequate protein quality control (PQC) mechanisms. RNA-seq data revealed transcriptional upregulation of ubiquitin-proteasome genes and downregulation of key chaperones, suggesting a failed compensatory response. Speckle-tracking echocardiography (STE) detected myocardial dyssynchrony and progressive strain abnormalities in TGH mice, correlating with increased proteotoxic burden and impaired redox homeostasis. Elevated TEI index values confirmed systolic and diastolic dysfunction. Time- and dose-dependent upregulation of Nogo/Reticulon4 transcripts and proteins further supported maladaptive cardiac remodeling. Collectively, these findings highlight that chronic RS disrupts ER homeostasis, induces proteotoxicity, and impairs cardiac structure and function, particularly in high transgene-expressing hearts. Keywords: Reductive stress, Nrf2 signaling, Proteotoxicity, Cardiac hypertrophy, Diastolic dysfunction, ER stress Graphical abstract [49]Image 1 [50]Open in a new tab 1. Introduction Chronic reductive stress (cRS), driven by sustained Nrf2/antioxidant activation, contributes to adverse cardiac remodeling and diastolic dysfunction, though its mechanisms remain unclear [[51][1], [52][2], [53][3]]. Chronic or excessive Nrf2 activation induces reductive stress (RS), causing cellular dysfunction and disease. Constitutive Nrf2 activation in caNrf2-TG mice similarly trigger RS, leading to pathological remodeling and diastolic dysfunction [[54][1], [55][2], [56][3], [57][4], [58][5], [59][6], [60][7], [61][8]]. We previously showed that hR120GCryAB-TG mice develop proteotoxic cardiomyopathy with enhanced reductive-redox capacity [[62]9]. The endoplasmic reticulum (ER) is crucial for protein folding, trafficking, and multiple cellular processes, including protein quality control, lipid metabolism, and calcium homeostasis [[63][10], [64][11], [65][12], [66][13], [67][14]]. Maintaining a pro-oxidative ER environment is essential for oxidative protein folding and cellular proteostasis [[68]10,[69]11]. ER dysfunction, closely linked with oxidative stress (OS), drives disease progression and contributes to the pathology of neurodegenerative, diabetic, and cardiovascular conditions [[70][15], [71][16], [72][17], [73][18]]. Unfolded protein response (UPR) regulates protein-folding demand by inducing chaperones, antioxidant genes, inflammatory mediators, and autophagy [[74]19,[75]20]. Chronic ER stress impairs UPR, leading to proteotoxicity [[76][21], [77][22], [78][23]]. Each organelle has a distinct redox state essential for its function [[79]24,[80]25]. The nucleus and mitochondria are moderately reduced, while the ER maintains a pro-oxidative environment for protein folding [[81][24], [82][25], [83][26]]. Disruptions in redox balance through oxidative or reductive stress (OxS/RS) can compromise ER function and trigger pathological outcomes. Protein folding in the ER generates ∼25 % of cellular reactive oxygen species (ROS), while ROS and lipid peroxides can activate the UPR [[84]27]. Glutathione depletion induces a hyper-oxidative state, impairing ER function [[85]26]. We hypothesized that chronic reductive stress disrupts ER function, causing proteotoxicity, remodeling, and dyskinesia, with a focus on ER stress response mechanisms in cRS-induced cardiac pathology. 2. Methods 2.1. Animals The Institutional Animal Care and Use Committee (IACUC) of the University of Utah, Salt Lake City and University of Alabama at Birmingham approved all animal experimental procedures. The heart-specific, constitutively active Nrf2 transgenic mouse (caNrf2-TG) with low (TG-L) and high expression (TGH), along with the non-transgenic (NTG) littermates, were used for the study at 3 and 6 months of age [[86]1,[87]3,[88]8,[89]28]. Detailed methods for RNA isolation, NGS sequencing, immunoblotting, proteasome activity, qPCR, immunostaining and speckle tracking echocardiography are provided in the supplemental section. 2.2. Statistical analysis Statistical analyses between two groups were conducted using Student's t-test with GraphPad Prism 10.2.2. Tukey's multiple comparisons test was used for comparing three or more groups. Statistical significance was defined as ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; and ∗∗∗∗p < 0.0001. 3. Results We reported that chronic cRS in the TG mouse hearts expressing constitutively active Nrf2, a transcriptional regulator of antioxidants, induces pathological cardiac remodeling and diastolic dysfunction [[90][1], [91][2], [92][3],[93]8,[94]11]. 3.1. Pro-reductive (PR) and reductive stress (RS) induce distinct transcriptomic shifts in ER-associated genes Principal component analyses (PCA) of NGS-based myocardial transcriptome demonstrated distinct changes in the ER gene expression between TGL and TGH groups suggesting a dose-dependent impacts for pro-reductive (PR) and reductive stress (RS) conditions ([95]Fig. 1A–B). Notably, caNrf2-TGH mice exhibit a pronounced shift in gene expression compared to NTG and caNrf2-TGL, indicating greater transcriptional dysregulation in the ER under chronic RS. Pathway enrichment analysis indicated that RS in caNrf2-TGL/TGH mice drives the upregulation of genes involved in UPR suggesting that RS impairs ER function at the transcriptional level ([96]Fig. 1C and D). Quantitative PCR validation for selected gene sets involved in protein folding pathway (Atf4, Atf6, Grp78, Grp94, Ero1 alpha, Perk, Xbp1, Calr, Calnx) at 3 and 6 months in the TG hearts confirmed the dysregulation of UPR pathways ([97]Fig.S1 A&B). Fig. 1. [98]Fig. 1 [99]Open in a new tab Evidence for ER stress transcriptome dysregulation under pro-reductive and reductive stress stage. (A) Representative diagram showing the Redox stages in endoplasmic reticulum- Reductive Stress (RS), Pro-Reductive State (pRS), and Normal Redox (NR). (B) PCA plot showing distinct clustering of ER genes with unique expression profiles across NTG, caNrf2-TGL, and caNrf2-TGH groups, indicating differential ER gene regulation. (C&D) Sankey plot showing the flow of differentially expressed genes between caNrf2 TGL/caNrf2 TGH vs NTG. Genes that are upregulated or downregulated in either condition are indicated by the thickness of the connecting bands, with thicker bands representing substantial changes in gene expression. Color coding is used to distinguish upregulated (red) and downregulated (blue) genes between the two conditions. The plot highlights enriched pathways with significance represented by log10(p-value) and gene ratio. The plots were generated using SR-PLOT for data visualization. 3.2. Chronic RS induces unresolved endoplasmic reticulum (ER) stress leading to ER dysfunction NAD(P)H quinone oxidoreductase 1 (NQO1), a downstream target of Nrf2 and redox marker [[100]29], was significantly upregulated in TG mice at 3 and 6 months in a dose- and time-dependent manner indicating a reductive-redox condition ([101]Fig. 2A, Fig.S1 C & D.[102]Fig.S1 C). Dose-dependent increases of PDI, ELF2-alpha and VCP were observed in both TG strains at 3 & 6 months of age, suggesting sustained unfolding stress under cRS, which may result in persistent trafficking of misfolded proteins to the ER. Similarly, increased levels of ATF6, an ER membrane-anchored transcription factor and activator of UPR, were observed in TG hearts compared to NTG mice ([103]Fig. 2A), suggesting impaired ER/UPR function [[104]30,[105]31]. This indicates a disruption in proteostasis within the ER when exposed to physiological stresses. Fig. 2. [106]Fig. 2 [107]Open in a new tab Chronic RS induces persistent ER dysfunction in the TG mouse myocardium. (A) Representative Immunoblotting of ER-resident/associated proteins from non-transgenic (NTG) and caNrf2 transgenic low (TGL) and high (TGH) mice at 3 and 6 months of age (n = 4 mice/group). (B) gProfiler analysis for genes associated with endoplasmic reticulum (ER) stress. Bar Graph illustrating the top enriched Gene Ontology (GO) terms related to ER stress. Terms are color-coded by functional category (Molecular function, Biological Pathways and cellular component) to highlight the signatures in the caNrf2-TG mice. Bubble Plot displaying the relationships between ER stress genes and the most significantly enriched KEGG pathways. The size of each bubble corresponds to the number of genes in the pathway and the color intensity represents the significance level of the enrichment. (C) Intra ER glutathione (GSH) level in the caNrf2-TG and NTG mice was examined by fluorescence imaging using anti-GSH-NEM antibodies. Fluorescent signal from the anti-GSH antibody (green) co-localized with KDEL (red), and the merged signal (yellow) indicates elevated GSH levels specifically within the ER (n = 4 mice/group). Regions with moderate GSH intensity suggest pro-reductive stress, while areas of high GSH intensity indicate apparent reductive stress. Quantitative analysis of KDEL and GSH staining using image J demonstrated distinct variations in ER stress and redox imbalances in myocardial tissues. Transgene-dose and age-dependent increase in cardio-vascular HSP (cvHSP), a chaperone marker of protein folding [[108]32], was observed in TG mice at 3 months, which was mostly stabilized at 6 months of age, suggesting an inefficient response to cRS ([109]Fig. 2A). gProfiler analysis of differentially expressed genes identified significant enrichment of Gene Ontology (GO) pathways related to ER stress across multiple functional categories [[110]33,[111]34]. Functional annotations for ER membrane and ER lumen were identified as highly enriched, suggesting substantial involvement of ER structural components under cRS ([112]Fig. 2B). Fluorescence imaging using anti-GSH-NEM antibodies revealed a dose dependent increase in intra-ER glutathione (GSH) levels in caNrf2-TGL and TGH when compared to NTG mice ([113]Fig. 2C and D). Regions stained with low GSH intensity indicate pro-reductive stress, whereas areas with high GSH intensity reflect reductive stress. A significant decline in DMPO-protein adducts in the ER compartments of both TGL and TGH, compared to NTG myocardium, indicates reduced ROS levels due to RS ([114]Fig. S2A&B). Together, these observations indicate that the ER environment experiences a less ROS availability with a hyper reductive-redox state, which could directly impact ER function. 3.3. Chronic RS-induced aggregation of mis-/unfolded proteins causes proteotoxicity To evaluate protein aggregation in cRS mouse hearts, we employed Proteostat®, a fluorescent dye that specifically binds to cellular aggresomes [[115][35], [116][36], [117][37]]. Presence of cytoplasmic and peri-nuclear protein aggregates of mutant human R120G-αB-crystallin (hR120GCryAB) in the hearts of hR120GCryAB transgenic mice [[118]9] served as a positive control ([119]Fig. 3A). Transgene dose-dependent augmentation of misfolded/aggregated proteins was detected in the caNrf2-TG mouse hearts ([120]Fig. 3B and C). A significant accumulation of aggregated proteins in TGH hearts at ≥6 months of age, as detected by Proteostat®, indicates cRS induces misfolding of proteins. TEM analysis showed pronounced ER dilation in the subsarcolemmal regions of TG mouse hearts, indicating structural damage that could contribute to its dysfunction ([121]Fig. 3D). These observations indicate that ER structural integrity is compromised under cRS due to persistent misfolded protein accumulation and inadequate chaperone-mediated folding. Fig. 3. [122]Fig. 3 [123]Open in a new tab Persistent ER dysfunction mediated mis-/unfolding of proteins is coupled with their ubiquitination in RS Hearts (A-C) The specificity of Proteostat® staining was validated in a well-established protein aggregation mouse model (i.e., transgenic mouse heart expressing human mutant alpha-B-crystallin/hR120G-CryAB). Cardiac tissue stained with Proteostat® (red) demonstrating that Proteostat® could detect cellular protein aggregates. Heart sections of non-transgenic (NTG) and caNrf2 transgenic low (TGL) and high (TGH) mice at 6 months of age were stained with Proteostat® and imaged using a fluorescence microscope for quantification. Profile plot showing ImageJ quantification of grey values, demonstrating a progressive increase in intensity corresponding to protein aggregation levels. The highest grey values were observed in the caNrf2-TGH group, followed by caNrf2 TGL, and then NTG. Pronounced Proteostat® staining in hR120G-CryAB-TG mouse hearts indicates severe protein aggregation (served as positive control). (D) TEM mages: Substantial ER dilation (Yellow arrow) in subsarcolemmal areas is evident in TG mouse hearts, indicating sustained ER stress. Mitochondria (Red arrow) in TGH mice exhibit structural heterogeneity, varying in shape and size. The contractile apparatus remains intact in some regions but is frequently disrupted or distorted in TG hearts. (E) Co-immunostaining of Proteostat with anti-ubiquitin antibody showing the localization of more ubiquitinated proteins towards base and apex of myocardium. Immunofluorescence of ubiquitinated (green) proteins that positively stained with Proteostat® (red) and their co-localization (yellow) in the base (upper) and apex (lower) of the ventricles from TG vs. NTG heart tissues (n = 3/group). Similarly, protein aggregates co-localized to nuclei (DAPI marker, blue) yield a pink overlay, indicating protein aggregation within the nucleus. (F) proteasomal enzymes activity. Significance: ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001; ns, no significance (N = 4 mice/group). (G) Global transcriptomics analysis of myocardium showing differential expression of ER/ERAD/ubiquitin pathway genes in caNrf2TGL/caNrf2TGH vs NTG myocardium. Significantly altered genes were highlighted with asterisk. 3.4. Stagnation of ubiquitinated misfolded/unfolded or aggregated proteins is evident in TG-high mice at 6 months of age Immunohistochemistry of the apex and base regions of the ventricles in the myocardial tissue showed increased ubiquitination and lodging of several proteins in the cytoplasm of TG mice at ≥6 months of age. Co-localization of the Proteostat® (RED – mis/unfolded or aggregated proteins) and anti-ubiquitin (GREEN – ubiquitinated proteins) was evident in the TGL and TGH hearts ([124]Fig. 3E). These findings suggest that cRS results in progressive accumulation of mis-/unfolded proteins due to impaired UPR, leading to proteotoxicity. In addition, we assessed the proteasome function through kinetic assays for the (a) caspase-like, (b) chymotrypsin-like and (c) Trypsin-like activities ([125]Fig. 3F). We found trending activation of caspase-like and chymotrypsin-like functions in TGH vs. TGL or NTG mice, but the Trypsin-like activity remained unchanged, suggesting an insufficient proteasome response to cRS. NGS-RNA sequencing data sets from TGL, TGH and NTG hearts at >6 months of age revealed significant changes among the genes involved in ubiquitin-proteasome system (UPS) ([126]Fig. 3G). Several ubiquitin-proteasome genes, including asterisk-marked HSP genes, were significantly upregulated in TGH compared to TGL and NTG mice at 6 months, suggesting a compensatory transcriptional response to sustained reductive stress. Genes that were upregulated significantly include the key regulators of the ubiquitin signaling, such as UBXN1 (2.25-Fold), Mapk9 (2.2-Fold) and Serpina3c (2.2-Fold). Heat shock proteins (Hsps), including Hspa1a, Hspa1b, Hsp90aa1, Hsp90b1, Hsp8, Hsph1 and Hspb1, were found to be dose-dependently downregulated in both TGL and TGH suggesting an impaired protein-folding machinery under reductive stress condition. Trib1, Stip1 and Calr were significantly downregulated in TGH vs TGL, suggesting overwhelmed UPS response ([127]Fig. 3G). These observations possibly reveal that a compensation to decompensation responses, at the transcriptional level, leads to a permanent impairment of PQC under cRS. 3.5. Speckle-tracking echocardiography (STE) analysis of left ventricle (LV) demonstrate dyssynchrony and myocardial deformation in TGH hearts experiencing cRS-induced proteotoxicity Time-dependent progression of cardiac remodeling was assessed by STE in TG and NTG mice at 3 and 6 months of age. The RS and proteotoxicity resulted in significant left venricle (LV) dyssynchrony in TGH vs. TGL or NTG at 3 months ([128]Fig. 4). Radial strain measurements showed comparable wall motions among TGL and NTG, but the TGH mice had significant deformation at the mesocardium at 3 months, indicating an onset of the cardiac remodeling. Interestingly, at 6 months, we noticed a significant difference in the radial strain between the TGL vs. NTG and this was further exacerbated in TGH mice ([129]Fig. 4A). TGL and TGH groups showed significantly increased global radial strain vs. NTG at both 3 and 6 months, with TGH > TGL ([130]Fig. 4B). While TGL and TGH showed significantly reduced (more negative) longitudinal strain vs. NTG at both time points, with the greatest impairment in TGH ([131]Fig. 4C). Of note, we could not find distinct endocardial deformation within the TGL between the two time-points (3 & 6 months), but this was further intensified in TGH at 6 months. Elevated radial endocardial segmental strain along with global reduction of the longitudinal myocardial strain in TGL suggest the onset of a dyssynchrony in the myocardium. However, the persistent RS in TGH further elevates radial segmental strain and decreases longitudinal strain in TGH mice, suggesting the existence of diastolic dysfunction and myocardial stiffening at ≥6 months ([132]Fig. 4C and D; [133]Supplementary fig S3). Fig. 4. [134]Fig. 4 [135]Open in a new tab Strain analysis of left ventricular systolic and diastolic functions using speckle tracking - echocardiography. (A) Three-dimensional LV radial and longitudinal strain deformation shows contraction (A; orange/positive values) and relaxation (B; blue/negative values) over six LV segments during six consecutive cardiac cycles for NTG, TGL and TGH at 3 & 6 months. The Vevo 2100 software based auto-scaling of LV radial and longitudinal strains shows significant differences in TGH and TGL vs. NTG. A red line indicates the changes in myocardial motion/tension among the three groups at 3 and 6 months. (C&D) LV radial and longitudinal time-to-peak measurements and strain curves (six LV segment) are shown for NTG, TGL and TGH at 6 months (n = 8–10/group). (E) TEI index, a measure of global cardiac function, was significantly elevated in both caNrf2-TGL and caNrf2-TGH mice compared to NTG. This increase suggests impaired cardiac performance in the caNrf2-TG hearts. Higher TEI index observed in the TGH group, indicates a more pronounced dysfunction under RS induced ER stress. Data presented as mean ± SEM for n = 6 to 8 mice per group. ∗∗∗∗p < 0.0001. (F) NGS-RNA seq – FPKM counts for Nppa and Nppb in caNrf2-TGL/TGH mice at 6 months (n = 3–4/group). (G) Immunoblots for Nogo expression in caNrf2-TGL/TGH at 3 vs. 6 months (n = 5–6/group). (H) mRNA levels (qPCR) for Nogo A in caNrf2 TGL/TGH at 3 vs. 6 months (n = 3–4/group). NGS FPKM counts for Nogo in caNrf2 TGL/TGH at 6 months (n = 3–4/group). TEI index, a measure of global cardiac function [[136]38], was significantly elevated in both TGL and TGH mice compared to NTG. This elevated TEI index suggests impaired systolic and diastolic function in the caNrf2 hearts ([137]Fig. 4E). Higher TEI index observed in the TGH group, indicates a more pronounced dysfunction under RS induced ER stress. Correlation analysis revealed a positive link between proteotoxicity and myocardial redox, function, and structure, highlighting its role in impaired contraction and cardiac output ([138]Fig. S4). Increased mRNA levels for Nogo/Reticulon4 transcript levels by qPCR and NGS-RNA sequencing ([139]Fig. 4F) reflected on the time-dependent upregulation of its protein. NGS-RNA sequencing showed a dose-dependent increase in Nppa in TGL and TGH hearts at 6 months, indicating rising myocardial stress, while Nppb remained unchanged. Nogo A and B, initially low at 3 months, were significantly upregulated by 6 months—paralleling Nppa expression—suggesting a shift from an early adaptive response to maladaptive signaling. This was supported by increased Nogo mRNA by qPCR and RNA-seq gene counts in TGH at 6 months, consistent with maladaptive cardiac remodeling ([140]Fig. 4G and H). 4. Discussion Using the cardiac-specific constitutively active Nrf2 transgenic mice, we discovered that chronic reductive stress shifts the ER environment to a reductive state, sustaining unresolved ER stress. This leads to protein misfolding, proteotoxic insult, and pathological cardiac structural remodeling in a transgenic mouse model of cardiac-specific, constitutively active Nrf2 over time. We also found time/dose-dependent influences of pro-reductive vs. RS conditions on myocardial strain deformation. Shifting the redox balance towards the reductive arm drives the transition from an “active ER response” to “sustained ER stress”. Increased Elf2-alpha, VCP, PDI, and ATF6 in 3-month-old TG mice indicateUPR and ER stress. Increased VCP expression under RS conditions may serve as a compensatory mechanism to manage misfolded proteins through degradation or refolding. VCP maintains protein homeostasis by mediating ERAD, extracting misfolded proteins for proteasomal degradation, and supporting autophagy through autophagosome maturation and lysosomal fusion [[141]11,[142][39], [143][40], [144][41]]. ATF6 increase further intensifies in TGH mice at 6 months, indicating a progressive stress response. Notably, calreticulin expression decreases, suggesting excessive cytosolic calcium overload, a potential trigger for ER stress [[145]42,[146]43]. Prolonged RS in 6-month-old TGH hearts sustains ER stress, likely due to persistent glutathione levels depleting ROS signaling, which is normally triggered by oxidative insult [[147]44]. Other reports indicate that a sustained reducing environment may neutralize basal ROS, disrupting cellular homeostasis [[148]45,[149]46]. RS significantly upregulates reticulon 4 (Nogo-A/B), which stabilizes ER structure. The biphasic Nogo-A/B expression—low at 3 months, and high at 6 months, suggests a shift from early compensation to maladaptive remodeling. TEM analysis confirms ER dilation in subsarcolemmal regions, indicating structural abnormalities. This supports the idea that beyond redox imbalance, cRS drives ER remodeling, as seen in dilated/idiopathic cardiomyopathy (DCM/ICM) patients [[150]47]. We showed that cRS, a toxic gain of function for antioxidants, disrupts proteostasis, leading to proteotoxic insults, contractile dysfunction, and pathological cardiac remodeling [[151]1,[152]11,[153][48], [154][49], [155][50], [156][51], [157][52], [158][53]]. Sustained PQC impairment can drive cell- and organ-specific pathologies [[159]54,[160]55]. Excess GSH and antioxidants may interfere with native disulfide formation in nascent proteins, promoting non-native disulfides (isomerization), leading to unfolding or misfolding [[161]56,[162]57]. Such proteins may escape clearance mechanisms, accumulating as aggregates [[163][58], [164][59], [165][60]]. Caspase, chymotrypsin, and trypsin enzymes degrade ubiquitinated proteins via the UPS to maintain proteostasis [[166]61]. The lack of robust enzyme activity and persistent ubiquitinated proteins in TG mouse hearts under cRS suggest insufficient proteasomal function, with dysfunctional UPR/ER activity potentially contributing to proteotoxic cardiac diseases. We demonstrate that cRS induces unresolved ER stress and, along with inefficient UPS, results in proteotoxicity, causing structural remodeling of the heart leading to dyskinesia. The rise in TEI index reflects a combination of diastolic and systolic dysfunction [[167]62], consistent with maladaptive cardiac remodeling and reduced efficiency of myocardial relaxation and contraction under cRS-induced unresolved ER stress. The correlation between proteotoxic stress and impaired contractility suggests a mechanistic link where proteotoxic overload impedes maladaptive hypertrophic response, ultimately compromising cardiac performance. Misfolded and ubiquitinated protein accumulation disrupts cytoskeletal and EC coupling proteins, impairing myocardial contractility [[168]63]. Speckle-tracking echo analysis revealed segmental defects in cRS hearts due to ER and sarcomere disintegration. cRS-induced ER dysfunction promotes protein misfolding, unfolding, and aggregation, leading to proteotoxic cardiomyopathy. While transient ER stress is adaptive, chronic redox imbalance (OxS or RS) causes persistent stress. We propose that oxidative stress promotes non-native oxidation, whereas RS hinders native protein oxidation, driving protein unfolding, aggregation, and proteotoxicity. 5. Conclusions and clinical implications Our findings indicate that chronic reductive stress induces ER stress, disrupts protein clearance, promotes aggregation, and drives pathological remodeling—positioning cRS as a key driver of proteotoxic heart disease. These insights suggest therapeutic strategies such as restoring redox balance, modulating the unfolded protein response , and enhancing proteasomal function to alleviate ER stress and myocardial dysfunction. Moreover, identifying ER stress and proteotoxic biomarkers may facilitate early detection of pathological remodeling and guide the development of targeted therapies to prevent heart failure. CRediT authorship contribution statement Sini Sunny: Writing – original draft, Methodology, Investigation, Formal analysis, Data curation. Rajesh Kumar Radhakrishnan: Methodology, Formal analysis, Data curation. Asokan Devarajan: Writing – review & editing. Juan Xavier Masjoan Juncos: Methodology, Data curation. Mohit Bansal: Formal analysis. Xiaosen Ouyang: Formal analysis, Data curation. Jianhua Zhang: Writing – review & editing, Methodology. Min Xie: Methodology, Resources. Silvio H. Litovsky: Formal analysis, Data curation. Joel D. Trinity: Writing – review & editing. Matthew Might: Writing – review & editing, Data curation. Steven M. Pogwizd: Writing – review & editing, Formal analysis. Maria I. Kontaridis: Writing – review & editing, Formal analysis. Namakkal S. Rajasekaran: Writing – review & editing, Writing – original draft, Supervision, Project administration, Investigation, Funding acquisition, Conceptualization. Declaration of generative AI and AI-assisted technologies in the writing process During the preparation of this work, the author(s) used ChatGPT-3.5 to improve language clarity and correct syntax errors. Following its use, the author(s) thoroughly reviewed and edited the content and take full responsibility for the final version of the manuscript. Funding This study was supported by grants from the NHLBI (2HL118067, HL118067, R01HL122238, R01HL102368), NIA (AG042860), and the American Heart Association, including a Beginning Grant-in-Aid (0865015F), Transformative Project Awards (20TPA35490426, 23TPA1065811), and a Postdoctoral Fellowship to SS (PDF-909324, 24POST1194403). Additional support was provided to NSR by start-up funds from the Department of Pathology and the School of Medicine at the University of Alabama at Birmingham (UAB), as well as the UAB-AMC21 grant (2021–23). JT was supported by NHLBI grant R01HL146203. This work was also supported by the National Institutes of Health (Grants R01-HL122238, R01-HL102368), the Department of Defense Lupus Impact Award (W81XWH2110784), the American Heart Association Transformation Grant Awards (20TPA35490426, 23TPA1065811), and the Masonic Medical Research Institute to M.I.K. Authors thank Dr. Anil Kumar Challa for his technical and editorial support. Declaration of competing interest Other authors declare that they have no financial or personal relationships with any individuals or organizations that could potentially influence the work presented in this manuscript. Also confirms that there are no conflicts of interest related to the research, data analysis, interpretation, or publication of this work.M.I.K. has received grant funding from Onconova Therapeutics and is a consultant for BioMarin Pharmaceuticals Inc; both funding and consulting projects are independent of the work in this manuscript. Footnotes ^Appendix A Supplementary data to this article can be found online at [169]https://doi.org/10.1016/j.redox.2025.103713. Appendix A. Supplementary data The following are the Supplementary data to this article. Multimedia component 1 [170]mmc1.docx^ (51.8KB, docx) Multimedia component 2 [171]mmc2.pdf^ (1.3MB, pdf) Multimedia component 3 [172]mmc3.pdf^ (32.2MB, pdf) Multimedia component 4 [173]mmc4.pptx^ (389.6MB, pptx) Multimedia component 5 [174]mmc5.pdf^ (497.3KB, pdf) Data availability Data will be made available on request. References