Doxorubicin-induced Cardiotoxicity is Propagated by Paracrine Signaling through Small Extracellular Vesicles

14

Cardiovascular disease (CVD) is the leading cause of death in the United States and 15 worldwide. While most of these deaths are the result of chronic heart diseases, some CVDs are 16 induced artificially. Doxorubicin (DOX) is a chemotherapeutic that is commonly used to treat 17 breast cancer which is one of the most common types of cancer in the United States. While 18 DOX is an effective anti-cancer agent, over 10% of treated women show signs of acute 19 cardiotoxicity immediately following treatment, and approximately 2% develop severe 20 cardiotoxicity up to 10 years after the end of treatment. Despite this prevalence, the mechanism 21 by which the onset of this cardiotoxicity occurs over time is not well understood. Here, we show 22 that treatment of cardiac cells with DOX changes the cardiac function and the resulting 23 paracrine signaling profile. Subsequent exposure of healthy cells to these altered paracrine 24 agents can recapitulate the effects of direct DOX exposure in 2D and 3D in vitro models. We 25 suggest that this is the result of an altered paracrine miRNA profile and other paracrine factors 26 that propagate the initial disruption caused by direct DOX exposure. Plasma EV miRNA profiling 27 of blinded patient samples revealed distinct clustering by DOX-cardiotoxicity risk, with high-risk 28 patients exhibiting miRNA signatures similar to those from DOX-treated tissue-engineered 29 models. Pathway analysis of the most distinguishing miRNAs linked them to cardiac 30 homeostasis and cardiotoxicity-related mechanisms, supporting the potential of plasma EV 31 miRNAs as noninvasive biomarkers for early risk stratification and personalized cardioprotective 32 interventions in oncological care, and the targeting of key clusters of miRNAs to enhance both 33 understanding of and intervention strategies for preventing the onset of DOX cardiotoxicity. 34 35 36 37 38 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 13, 2026. ; https://doi.org/10.64898/2026.02.11.705398doi: bioRxiv preprint

39 Cardiovascular disease (CVD) is the leading cause of death in the United States and 40 worldwide1. While most of these deaths are the result of chronic heart diseases which lead to 41 heart failure after myocardial infarction (MI), some CVDs can be induced artificially, such as via 42 off-target effects of other therapies 2. This is particularly common in chemotherapy and remains 43 a pervasive issue with both established and novel targeted chemotherapeutics 3, often being 44 referred to as “off-target toxicity”. Off-target effects which compromise or otherwise damage 45 cardiac health and functionality are commonly referred to as “off-target cardiotoxicity”, although 46 there is little consensus on a more specific definition for this term in literature 4. For many 47 chemotherapeutics, the cause of cardiotoxicity can be linked directly to exposure to the 48 chemotherapy agent 5, spurring advances in chemotherapy delivery vehicles and targeting 49 techniques3,4. For some chemotherapeutics, however, the mechanism by which cardiotoxicity 50 occurs is less clear. 51 Doxorubicin (DOX), an anthracycline, is a chemotherapeutic that has been commonly used to 52 treat breast cancer, one of the most common types of cancer in the United States. Breast 53 cancer affects 1 in 8 women both in the United States and globally and comprises 30% of yearly 54 cancer diagnoses and 12.5% of cancer diagnoses globally 6. Since the initial formulation of DOX 55 in the 1960’s and approval for medical use in 1974, DOX has proven to be highly effective in 56 treating cancers including breast, bladder, stomach, lungs, ovarian, thyroid, soft tissue sarcoma, 57 multiple myeloma, lymphoma, and leukemia 7,8, as have the more than 2000 DOX analogues. 58 While DOX and anthracycline cocktails are very successful in mitigating or otherwise destroying 59 breast cancer and other cancers, over 10% of women showed signs of acute cardiotoxicity 60 immediately following treatment 7 and approximately 2% developed severe cardiotoxicity up to 61 10 years after the end of treatment 9, despite known clearance of DOX in less than 48 hours 10. 62 This DOX-induced cardiotoxicity has since been well-established in DOX and many of the DOX 63 analogues, both alone and in chemotherapy cocktails, and is thought to primarily operate 64 through transcriptional and mitochondrial damage 8,9. However, the precise mechanisms by 65 which DOX cardiotoxicity is initiated as well as how such effects could persist even after DOX 66 clearance are the subject of intense debate 7. We hypothesize that early DOX exposure 67 pathologically disrupts the paracrine signaling of myocardial cells, which, over time, propagate 68 this dysfunction to the surrounding tissue to eventually result in observable cardiotoxic effects. 69 This can be demonstrated by independently recapitulating the effects of DOX treatment using 70 only extracellular vesicles (EVs) isolated from DOX-treated cells. 71 Extracellular vesicles (EVs) are traditionally defined as apoptotic bodies (~1 µm – ~5 µm 72 diameter), microvesicles (~200 nm – ~1000 nm diameter), and exosomes (~30 nm – ~200 nm 73 diameter)11, with exosomes being particles of particular interest due to exosomes acting as 74 major vehicles for paracrine and endocrine transfer of proteins and nucleic acids 12. Recently, 75 however, the identification of numerous difficult to separate subgroups of exosomes and non-76 exosome EVs with sub-200 µm diameter has developed into separate classification of EVs as 77 medium or large EVs (mEVs or lEVs, respectively) with diameters typically greater than 200 µm, 78 and small EVs (sEVs) with diameters typically less than 200 µm 13. These sEVs, like exosomes, 79 are commonly vehicles for the transport of nucleic acids and proteins, notably micro RNAs 80 (miRNAs) and cytokines, which influence many diverse and pathologically relevant biological 81 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 13, 2026. ; https://doi.org/10.64898/2026.02.11.705398doi: bioRxiv preprint processes, including angiogenesis, immunomodulation, mitochondrial activity, and epithelial to 82 mesenchymal transition 11,13. Additionally, exosome-like sEVs have been demonstrated to be 83 actors in chronic CVDs 14,15, and have been suggested as actors in the onset of DOX-related 84 cardiotoxicity16. While evidence suggests that paracrine signaling plays some role in the onset 85 of DOX-cardiotoxicity, the proposed mechanisms and involvement in the propagation of 86 cardiotoxicity are often confounding 16,17. Some studies have suggested that sEVs directly 87 maintain and transport DOX to mediate cardiotoxic effects, but current literature does not wholly 88 support this and instead individual miRNAs, transported by sEVs, are under investigation as 89 major actors18,19. We suggest that DOX exposure in cardiac cells induces a global shift in the 90 miRNA population carried by sEVs, where the collective dysregulation of multiple miRNAs, not 91 any single miRNA, drives the propagation of cardiotoxic effects. By characterizing a total miRNA 92 population shift, rather than identifying a single miRNA, we are also able to identify novel 93 miRNA biomarkers for DOX-related cardiotoxicity and validate them in clinical samples. 94 In this study, we show for the first time in literature that DOX-related cardiotoxicity can be 95 propagated by paracrine factors independent of direct exposure to DOX in 2D, 3D, and heart-96 on-a-chip models. Furthermore, we identify novel biomarkers of DOX-related cardiotoxicity from 97 sEVs in conditioned media of DOX-exposed cells and validate those markers in clinical 98 samples. Finally, we perform downstream analysis of identified miRNA targets to ascertain 99 potential involved pathways for future therapeutic intervention, and cross reference with 100 miRNAs and pathways currently suspected of involvement in DOX-related cardiotoxicity. The 101 identification of sEVs as crucial elements in the onset and propagation of DOX-related 102 cardiotoxicity will help elucidat e specific mechanisms of both acute and chronic cardiotoxicity, 103 as well as assist in the development of enhanced chemotherapeutic approaches to limit off-104 target cardiotoxic effects. 105

106 DOX-EVs Induce Similar Dysfunction to Direct DOX Treatment in Cardiac Muscle and 107 Stromal Cells : To assess the effects of DOX-EVs on cardiac cells compared to direct DOX 108 treatment, hiPSC-derived cardiomyocytes (iCM) and cardiac fibroblasts (iCFs) were treated with 109 a blank control, DOX-conditioned media, or media conditioned with EVs from DOX-treated iCFs 110 (Figure 1A). EVs from iCFs were used in 2D culture, as CFs act as master regulators of the 111 myocardial microenvironment and local signaling in vivo20. 112 First, due to the known effects of DOX in inducing oxidative stress in cardiac cells 21, relative 113 ROS signal was assessed for each cohort. Interestingly, DOX-EV treatment significantly 114 increased ROS signal in iCMs (32%), though not as much as direct DOX treatment (65%) 115 (Figure 1B, left). Beating analysis of iCMs treated for 48 h with either the control, DOX media, or 116 DOX-EV media (Supplemental Video 1-2-3) revealed that both treatment groups reduced 117 beating velocity (DOX, 27%; EV, 54%) (Figure 1B, middle) and a modest decrease in average 118 beating rate (DOX, 9%; EV, 20%) (Figure 1B, right). 119 To better assess changes in beating rate and overall beating regularity, a live Ca 2+ stain was 120 used to measure the beating behavior of the iCMs temporally (Supplemental Video 4-5-6). The 121 control cells demonstrated a regular period of ~4.5 s, where the DOX-treated cells 122 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 13, 2026. ; https://doi.org/10.64898/2026.02.11.705398doi: bioRxiv preprint demonstrated an irregular period ranging from ~2 s to ~6 s, and the DOX-EV group showed a 123 similar period to DOX-treated cells, ranging from ~2 s to ~5 s (Supplemental Figure S1). When 124 overlayed, the control peaks tended to overlap, while both the DOX and DOX-EV group peaks 125 tended to appear sporadically (Figure 1C). Beating signals were transformed via fast Fourier 126 transform (FFT) and decomposed into sine wave components to allow for more direct 127 comparison of the signals. The control group showed regular peaks of decreasing amplitude, as 128 expected, whereas both the DOX and DOX-EV groups showed irregular peaks with random 129 amplitude (Supplemental Figure S2). 130 Next, the differences in the effects of DOX-EVs and direct DOX treatment on iCFs were 131 evaluated. First, relative ROS signal was assessed for each cohort. As with iCMs, both DOX 132 treatment and DOX-EV treatment significantly increased ROS signal (Figure 1D, left). 133 Additionally, both DOX and DOX-EV treatment induced a significant increase in α SMA 134 expression, with DOX increasing expression by nearly 300% compared to the control and DOX-135 EVs inducing a nearly 200% increase over the same time period (Figure 1D, middle). 136 Additionally, DOX treatment resulted in a more than 3-fold increase in daily cell death rate, from 137 ~3% to over 15%, though this was not replicated by treatment with DOX-EVs alone (Figure 1D, 138 right). 139 DOX Treatment of iECs Induces Different Dysfunction from other Cardiac Cells: In 140 addition, the effects of direct DOX treatment and DOX-EV treatment on hiPSC-derived 141 endothelial cells (iECs) were evaluated. As with iCMs and iCFs, ROS signal was evaluated 142 under all three treatment conditions. Interestingly, however, neither DOX nor DOX-EVs induced 143 a significant increase in ROS expression in iECs (Figure 1E, left), though both DOX and DOX-144 EV groups showed a non-significant increase. To evaluate overall metabolic rate of iECs under 145 each treatment, a cell metabolism assay was run on each cohort. In this case, both DOX and 146 DOX-EV-treated iECs showed significantly decreased mitochondrial activity compared to the 147 control (Figure 1E, right). Furthermore, the decrease observed in both DOX and DOX-EV 148 groups was very similar. 149 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 13, 2026. ; https://doi.org/10.64898/2026.02.11.705398doi: bioRxiv preprint 150 Figure 1: EVs from DOX-Treated Cells can Independently Recapitulate DOX Toxicity in vitro. (A)151 Experimental setup for assessing DOX-induced cardiotoxicity in 2D and 3D culture systems. (B) ROS152 level (left), beating intensity (middle), and beating rate (right) of iCMs when treated with DOX, DOX- EVs,153 or a PBS blank for 48 h. (C) Overlaid beating patterns of iCMs by cohort. (D ) ROS level (left), α SMA154 expression (middle) and cleaved caspase-3 expression (right) of iCFs by cohort. (E ) ROS level (left) and155 cell metabolic activity (right) of iECs by cohort. (E) Beating intensity (left) and cell survival (right) 48 h after156 seeding with control or DOX- treated iCFs with iCMs in a 3D model, and treated with control or DOX157 media. (F) Overlaid beating patterns of 3D models with control (left) or pre-treated (right) iCFs. n ≥ 3 for158 all groups tested, * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001 assessed by one- way ANOVA with159 Tukey’s post-hoc. 160 A) S s, A nd ter X for ith (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 13, 2026. ; https://doi.org/10.64898/2026.02.11.705398doi: bioRxiv preprint Pre-treatment of iCFs with DOX Induces DOX-like dysfunction in 3D Cardiac Models : To 161 assess the ability of local signaling to propagate DOX-cardiotoxicity, 3D bioprinted co-culture 162 models of iCMs and iCFs were constructed. Prior to seeding the gel, iCFs were cultured for 48 h 163 with DOX-conditioned media or a blank control, and iCMs were cultured under normal 164 conditions. After 3D bioprinting, constructs were allowed to adjust in a 1:1 mixture of CM(+) and 165 DMEM complete for 14 days. Constructs were then separated into 3 groups: control iCFs 166 treated with regular media (control), control iCFs treated with media supplemented with 10 nM 167 DOX (DOX), and DOX pre-treated iCFs treated with regular media (pre-treated). 168 Beating behavior was assessed after 48 h media treatment. After 48 h, a significant decrease in 169 beating velocity was observed in both the DOX (22%) and pre-treated (43%) groups, with the 170 pre-treated group reducing beating velocity substantially, but not significantly, more (Figure 1F, 171 left). Additionally, while the DOX treated group showed significantly increased cell death, the 172 pre-treated group did not (Figure 1F, right). In addition to significant disruptions to beating 173 velocity and rate, DOX pre-treatment also substantially disrupted the beating regularity of the 3D 174 models (Figure 1G), similarly to what was observed in 2D culture, while the control group 175 showed even greater regularity in beating compared to 2D culture. Subsequent FFT and sine 176 wave decomposition further support this interpretation (Supplemental Figure S2). 177 DOX-EVs Alone Induce Metabolic Dysfunction Expected in DOX-Cardiotoxicity : Following 178 regular cell culture assay, the mitochondrial respiration and associated functions of cells were 179 analyzed via Seahorse. All three cell types: iCMs, iCFs, and iECs, were seeded on Seahorse-180 compatible 96-well plates and allowed to settle. After attachment, cells were treated for 48 h 181 with DOX media, a PBS blank, or DOX EV media where the EVs originated from either DOX-182 treated CFs (CF DOX-EVs) or DOX-treated MFs (MF DOX-EVs). This was done to see if DOX-183 EVs from non-cardiac tissues could induce similar dysfunction to those from CFs. Five major 184 categories were considered: basal respiration (showed no significant difference in any group), 185 maximal respiration (MR, Figure 2A), ATP-production coupled respiration (ACR, Figure 2B), 186 proton leak (PL, Figure 2C), and spare respiratory capacity (SRC, Figure 2D). In iCMs and iCFs, 187 both the MR and SRC were significantly decreased by all treatment groups, demonstrating that, 188 independent of DOX treatment, DOX-EVs are sufficient to induce significant metabolic distress 189 in cardiac cells, and that the cells contributing to these effects do not necessarily need to be of 190 cardiac origin. This is in conjunction with a significant increase in ACR in iCFs subjected to both 191 DOX and CF DOX-EV treatment, and in iCMs subjected to CF DOX-EV treatment but not DOX 192 treatment alone. The combination of decreased MR and SRC (Figure 2A, D) with increased 193 ACR (Figure 2B) is characteristic of DOX-associated mitochondrial dysfunction in 194 cardiomyocytes (CMs) 22,23 and with larger-scale anthracycline-induced progressive 195 cardiotoxicity24. This is consistent with the results of the 2D and 3D models. MF DOX-EVs, 196 alternatively, significantly decreased PL, a measure of mitochondrial inefficiency (Figure 2C), 197 which may indicate a different, though still disruptive, influence from non-cardiac tissues. On the 198 other hand, iECs experienced significantly increased MF and SRC under all conditions. This 199 combined with the increase in ACR, suggests either increased overall mitochondrial activity or 200 an increase in the number of mitochondria. With the increase in PL as well, however, the 201 mitochondrial efficiency also likely decreased significantly. This pattern aligns with the 2D 202 model, which showed a non-significant increase in ROS alongside a significant reduction in 203 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 13, 2026. ; https://doi.org/10.64898/2026.02.11.705398doi: bioRxiv preprint overall mitochondrial activity (Figure 1E)204 205 Figure 2: DOX-EVs Induce Metabolic Dysfunction Similar to Direct DOX Treatment . Seaho rse Mit oStr es s206 assay results fo r ( A) maximal respir atio n, ( B) ATP -production coupl ed r espira ti on, ( C ) proton l eak, an d207 ( D ) spare respirat ory capacity af ter 48 h of trea tmen t. n ≥ 3 for all gro ups tes ted , * p < 0.05 , * * p < 0 .01 ,208 assessed by one-way ANOVA with Tukey’ s post-hoc 209 DOX Treatment Induces Pathology-Associated Shift in EV Characteristics : To establish210 that DOX-treatment alters the paracrine signaling behaviors of cardiac cells (Figure 3 A), EVs211 were collected from conditioned, exosome-free media from iCM s, iCFs, and iECs treated with a212 blank control or 10 nM DOX. T o demonstrate that changes in EV populations were not a result213 of direct DOX export in EVs, we first performed UV- Vis spectrophotometry to assess the214 presence of DOX in a PBS blank, DOX-conditioned media, and EVs from both control and DOX-215 treated iCMs and iCFs (Figure 3B, Supplemental Figure S3). We observed more than 2- order of216 magnitude difference between DOX- conditioned media and any other group, and that all EV217 groups were nearly identical. Furthermore, the absorbance spectra for all EVs were virtually218 indistinguishable, whereas the spectra from DOX- conditioned media was clearly distinguishable219 with peaks not present in any other spectra (Figure 3B). 220 Following this, the size profile of isolated EVs was assessed via Nanoparticle Tracking Analysis221 (NTA) (Figure 3C). This showed that EVs from DOX- treated cells tended be smaller than those222 from control cells in iCMs (Control, mode: 174 nm; DOX, mode: 141 nm; p < 0.01) and iCFs223 (Control, mode: 153 nm; DOX, mode: 118 nm; p < 0.005), but not in iECs (Control, mode: 144224 E) s d , sh Vs a ult he - of V lly le is se Fs 44 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 13, 2026. ; https://doi.org/10.64898/2026.02.11.705398doi: bioRxiv preprint nm; DOX, mode: 147 nm) (Figure 3D). All EVs measured fall within the expected range for EVs 225 (< 200 µm diameter), and all populations have calculated PDI below 0.2, indicating that the EV 226 populations are mostly monodisperse and therefore not splitting into easily separable sup-227 populations. 228 Western blot was performed to identify characteristic exosome markers CD9, CD63, and 229 TSG101 (Supplemental Figure S4). This was done to both identify whether the population 230 contained exosomes, as well as to assess the relative expression of CD63 and TSG101 to CD9 231 (Figure 3E-F), which we have previously suggested may be correlated with damage or other 232 dysfunction in the myocardium 14. These results showed that the isolated EV populations did 233 contain exosomes and showed significant changes in some surface marker expression. 234 Compared to the blank control, relative CD63 expression was increased in DOX-treated iCMs (p 235 < 0.001) and iCFs (p < 0.05), and relative TSG101 expression was increased in DOX-treated 236 iCMs alone (p < 0.005), in line with our previous findings regarding pathology-related changes in 237 EV tetraspanin expression25. 238 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 13, 2026. ; https://doi.org/10.64898/2026.02.11.705398doi: bioRxiv preprint 239 Figure 1 : DOX-EVs show disease- like morphology and protein profiles without acting as direct240 doxorubicin carriers. (A) Diagram briefly showing the process of events by which DOX cardiotoxicity241 may be propagated by local paracrine signaling. (B) The absorbance of a PBS blank, DOX- conditioned242 media, and EVs from control and DOX-treated iCMs and iCFs quantified by UV-Vis spectrophotometry for243 480 nm. (C) Nanoparticle tracking analysis (NTA) with error area for EVs from control or DOX- treated244 iCMs (red, left), iCFs (blue, middle), or iECs (green, right), with (D) subsequent quantification of mode245 shift in each population. Quantification of the western blot band intensity of CD63 (E) or TSG101 (F)246 relative to CD9 intensity for EVs obtained from control or DOX- treated iCMs, iCFs, and iECs. Data are247 presented as the mean ± standard deviation. n ≥ 3 for all groups tested, * p < 0.05, ** p < 0.01, *** p <248 0.005, **** p < 0.001 assessed by one-way ANOVA with Tukey’s post-hoc for (D), (E), and (F). 249 DOX Treatment Substantially Alters EV miRNA Profile of Cardiac Cells: miRNA profiling via250 Nanostring analysis revealed highly upregulated clusters of exosomal miRNA populations in251 EVs from DOX-treated cells relative to control cells (Figure 4A). Interestingly, despite this252 separation between control and DOX-treated cell EVs, the miRNA populations of EVs from253 control iCMs and DOX- treated iCFs clustered together, though the miRNA profiles are still254 notably distinct. From over 800 miRNAs profiled, unsupervised analysis revealed 146 miRNAs255 ct ity ed for ed de F) re < ia in is m till As (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 13, 2026. ; https://doi.org/10.64898/2026.02.11.705398doi: bioRxiv preprint (Supplemental Table S1) that were selected as being meaningfully altered as a result of DOX 256 treatment (Figure 4B). These miRNAs clearly clustered between control and DOX treated cell 257 EVs, and more miRNAs were upregulated in the DOX treated cell EVs than in those from 258 control cells. There was also substantial overlap between both DOX treated cell EV profiles and 259 the control CM profile, though many of these miRNAs mapped to normal cardiac processes. 260 miRNAs upregulated in DOX-cell EVs are associated with cardiotoxicity pathways: To 261 assess potential overlap and pathways of interest, MetaCore pathway analysis software was 262 used to build networks for the 145 identified distinctive miRNAs. This network analysis identified 263 several pathways of interest that were regulated by two or more of the identified target miRNAs, 264 often with multiple miRNAs regulating a single target or series of targets. The most involved 265 pathway identified was that of CDC42 and upstream Neuropilin-1 (Figure 4C, full pathway: 266 Supplemental Figure S5A), a cell proliferation pathway often desirably hindered by 267 chemotherapy and is vital in some cancer progression 26, and utilized 6 miRNAs (Supplemental 268 Table S2). This indicates that miRNAs delivered by sEVs from DOX treated cells continue to 269 promote anti-cancer effects. Also notable was the regulation of PPARGC1-a (Figure 4D), the 270 “master regulator” of mitochondrial biogenesis 27. PPARGC1-a is also responsible for translation 271 of mechanical stimuli to mitochondrial biogenesis 28 and the promotion of M2-phenotype 272 macrophage polarization29 in the heart, and downregulation of this gene as a result of genetic 273 mutation was found to increase risk of left ventricular diastolic dysfunction 30. This interaction 274 was regulated by 5 miRNAs (Supplemental Table S2). Both of these interactions were found in 275 the same pathway (Supplemental Figure S5A). Another pathway of interest revealed four 276 interactions downstream of c-Myc (Figure 4E, full pathway: Supplemental Figure S5B). Of 277 interest in these interactions were the upstream and downstream regulation of Cullin 4B, a 278 known effector of cardiac antioxidant pathways and sarcomere quality control and which the 279 downregulation of is associated with heart failure 31. This interaction involves 2 miRNAs 280 (Supplemental Table S2). The other interaction of major interest is the inhibition of CAS-L by 281 miR-203-5p. CAS-L is a cardiac redox agent via MICAL1 involved in the sensing and binding of 282 Ca2+, the inhibition of which is associated with ventricular tachycardia 32. Finally, two more 283 interactions downstream of c-Myc in a different pathway were identified (Figure 4F). The first 284 interaction is the downregulation of a PI3K component by 3 miRNAs (Supplemental Table S2). 285 Although PI3Ks are desirable targets for arresting breast cancer, PI3Ks are also essential 286 cardioprotective agents and the inhibition of them by pharmaceuticals is frequently met with off-287 target cardiotoxicity and arrhythmia 33. PI3Ks are an expected, though not well mapped-out, 288 target of anthracyclines and the application of DOX with PI3K supplementation through 289 pharmaceuticals or ischemic preconditioning has alleviated some of the cardiotoxic effects of 290 DOX treatment in animal models 34. The other interaction of interest was the inhibition of IL33 by 291 3 miRNAs (Supplemental Figure S5C) and subsequent inhibition of Alpha 1 antitrypsin (AAT). 292 IL33 has recently been implicated an essential component of a mechanically sensitive CF 293 cardioprotective paracrine signaling machinery, protecting against hypertrophy, fibrosis, and 294 heart failure 35 and AAT deficiency is associated with many CVDs and systemic failure of the 295 heart through unknown mechanisms36. 296 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 13, 2026. ; https://doi.org/10.64898/2026.02.11.705398doi: bioRxiv preprint 297 Figure 4: DOX- EVs demonstrate a differential miRNA profile and target disease pathway298 regulation. (A) Heatmap showing the full miRNA profiling of isolated EVs from iCMs and iCFs after 48 h299 treatment with DOX-conditioned media or a blank control. (B) miRNA profiling results for 145 identified300 targets for downstream analysis. (C- F) Results of MetaCore pathway analysis for the identified target301 miRNAs. Identified pathways were considered for analysis only for p-value < 0.05. 302 ay h ed et (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 13, 2026. ; https://doi.org/10.64898/2026.02.11.705398doi: bioRxiv preprint Distinct Plasma EV miRNA Profiles Differentiate High- and Low-Risk Cardiotoxicity 303 Patients: EVs were isolated from blinded clinical plasma samples of patient cohorts classified 304 as high or low risk for DOX-induced cardiotoxicity to profile their miRNA content and identify 305 molecular signatures associated with cardiotoxicity risk. The patient samples clustered distinctly 306 by risk category, revealing that high-risk patients exhibit plasma EV miRNA signatures similar to 307 those observed in DOX-treated cell culture models (Figure 5A). To pinpoint the miRNAs most 308 responsible for this separation, we focused on the top 10% of miRNAs (n = 80) showing the 309 highest percent coefficient of variance (%CV; red dashed line, Figure 5B). Among these, 23 310 miRNAs displayed particularly pronounced variability across five distinct cohorts (Supplemental 311 Table S3), as visualized in the heatmap and clustering analyses (Figure 5A). These cohorts 312 consisted of the identified 23 miRNA targets and adjacent miRNAs (mostly consisting of others 313 of the 80 most enriched miRNAs), and were distinguished to the right of the heatmap as follows: 314 cohort 1 – grey; cohort 2 – orange; cohort 3 – blue; cohort 4 – green; cohort 5 – purple (Figure 315 5A, Supplemental Table S3). Importantly, 35 of the miRNAs enriched in patient EVs closely 316 overlapped with miRNAs previously identified in DOX-treated cell culture EVs (Figure 5C), and 317 were largely enriched in patients with high risk of cardiotoxicity (Figure 5D). 318 Enriched miRNAs in High DCT Risk Pati ents Regulate Cardiotoxic Pathways : Pathway 319 enrichment analyses indicated that many of these miRNAs (both shared and unique) are 320 implicated in pathways governing cardiac homeostasis, chronic CVD development, and the 321 progression of cardiotoxicity (Figure 5E-H), though analysis of cohort 5 showed little specificity 322 for cardiotoxicity and was more closely related to general dysregulation of cardiac homeostasis 323 (Supplemental Figure S6), possibly indicating an overselection of targets. Specifically, miRNAs 324 from cohort 1 miR-584-3p 37,38and miR-217-5p41,42 have the most direct reported links to DCT, 325 whereas miR-497-5p 43,44 and miR-1262 45,46 have been independently associated with both 326 CVD-related pathways and anti-tumor responses (Figure 5E, Supplemental Figure S6A). 327 MetaCore analysis also identified significant co-operative regulation of DCT-related pathways, 328 including GRP75 47 and AKT 48,49,50,51,52, known to be recruited during DOX-induced stress 329 signaling, and downstream FOXO53,54,55, linked to metabolic disruption and apoptosis after DOX-330 mediated disruption of AKT, in parallel with disruption of ZBP1/IMP1 56,57, a disruption which has 331 been shown to be necessary for DCT onset in animal models (Figure 5E). Additionally, cohort 1 332 miRNAs also disrupted c-Jun related signaling, shown to cause metabolic stress and 333 pathological remodeling in the heart, via AP-1 58,59, c-Abl 60, and C/EBP 61, with c-Abl being 334 directly implicated in DCT onset (full pathway: Supplemental Figure S6A). 335 The miRNAs clustered in cohort 2 were implicated in chronic CVD and fibrotic remodeling and 336 are being investigated as targets for intervention, including miR-50478,79 miR-55376,77, miR-60481, 337 miR-59582, and miR-545-3p 83 and miR-1250-5p 80 (Figure 5F, full pathways: Supplemental 338 Figure 6B-C). While the cluster 2 miRNAs were not as directly implicated in the onset of DCT in 339 existing literature, MetaCore analysis revealed several major DCT-related proteins being co-340 operatively regulated by DOX-shifted miRNAs across two overarching pathways, both 341 downstream of or in parallel to c-Myc. Both CDK6 62,63 and TMUB1 64,65,66 have been linked to 342 chemotherapy-related cardiotoxicity, specifically via p53 regulation and metabolic stress, and in 343 this pathway are in parallel downstream of c-Myc (Figure 5F, upper). Additionally, in parallel with 344 the above pathway, NRF1 67,68, GRK2 69,70,71, and AGTR1 72 signaling were also disrupted, the 345 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 13, 2026. ; https://doi.org/10.64898/2026.02.11.705398doi: bioRxiv preprint disruption of each of which has been directly linked to DOX-induced damage, particularly in 346 suppression of mitochondrial function and p53 regulation (Figure 5F, lower, full pathways: 347 Supplemental Fig. 6B-C). 348 The miRNAs clustered in cohort 3 were largely linked to DOX-induced oxidative stress and 349 subsequent CM death, with miR-152-3p 73,74,75 being a well-known individual miRNA target in 350 DCT studies. MetaCore analysis of cluster 3 linked these miRNAs to largely fibrosis and chronic 351 CVD-related pathways. In particular, the disruption of EZH2 84,85, p66a86,87,88, FOXF189,90,91, and 352 FIP20092,93 have been mechanistically linked to the onset of excessive fibrosis in a number of 353 CVDs via both pathway regulation and gene activation, with p66a being actively explored as an 354 anti-fibrotic drug target (Figure 5G, upper). In parallel, however, cluster 3 was also highly 355 involved in pathways directly related to chemotherapy-induced cardiac fibrosis, with both 356 HDAC194,95 and p66b 96,97 being implicated (though for trastuzumab, not DOX), and both 357 PAX898,99 and ATF/CREB/FGF2 100 axis disruption exacerbating any induced fibrosis (Figure 358 5G, lower, full pathways: Supplemental Fig. 7A-B). 359 The miRNAs clustered in cohort 4 were also largely implicated in the onset of DCT, such as 360 miR-182-5p101, via regulation of major DCT nodes. MetaCore analyses linked this cluster to 361 highly interconnect nodes associated with a wide array of chemotherapy induced cardiac toxicity 362 and fibrosis, including UBB 102,103,104 FOXM1 105,106,107,108 Cyclin E kinase 109,110 363 HGFR/HIF1111,112,113,114, all of which are major targets of investigation for the onset of chemo-CT, 364 and, interestingly, all of which have been shown to be induced to pathology via irregular miRNA 365 signaling (Figure 5H, upper). More directly, YY1 115,116 AIP1117, and GRP75118 have been directly 366 implicated in DCT, poor ion handling, and DCT-related heart failure, with AIP1 being known to 367 be directly regulated via EV-chauffeured miRNAs to manipulate pathway activation (Figure 5H, 368 lower). While further biological replicates and direct, targeted clinical studies are required to 369 validate both these markers and the observed trends and interactions, these preliminary data 370 suggest that plasma EV miRNA profiling could serve as a promising biomarker tool for 371 identifying patients at risk for DOX-induced cardiotoxicity, with translational alignment between 372 in vitro and clinical settings. 373 374 375 376 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 13, 2026. ; https://doi.org/10.64898/2026.02.11.705398doi: bioRxiv preprint 377 Figure 5: Clinical samples mimic DOX-miRNA enrichment in low vs high DCT-risk patients (A)378 Heatmap showing hierarchical clustering of the top 10% most distinguishing miRNAs (n = 80 by %379 coefficient of variance (%CV) across four blinded patient samples (S783, S326, S 930, S632). Five major380 expression clusters are color-coded on the right. (B) Scatter plot of miRNA z-scores versus %CV, with the381 top 10% most distinguishing miRNAs outlined (blue box, right of red dashed line). Each dot is color-coded382 by patient sample. (C) Bar graph quantifying the overlap between high- %CV plasma EV miRNAs and383 those identified in DOX-treated tissue- engineered models: fully overlapping (blue), partially related384 (orange), or unrelated (gray) with a heatmap representing relative enrichment of these miRNAs in each385 patient sample (D). MetaCore pathway analysis was performed to identify relevant pathways for identified386 miRNA targets of each cohort, and pathways relating to chemotherapy- induced cardiotoxicity were387 identified in cohort 1 (E), cohort 2 (F), cohort 3 (G), and cohort 4 (H). 388 389

390 In this study, we evaluated the effects of DOX treatment and treatment with EVs derived from391 DOX-treated cells side-by-side in both 2D and 3D in vitro models. These models primarily392 A) % jor he ed nd ed ch ed re m ily (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 13, 2026. ; https://doi.org/10.64898/2026.02.11.705398doi: bioRxiv preprint consisted of iCMs and iCFs, cells which make up a vast majority of the heart 20, and iECs, which 393 are commonly used in stem cell-derived cardiac tissue models to emulate vasculature 25. In all 394 cell types we evaluated ROS prevalence and mitochondrial activity, and for cardiac cells 395 specifically we evaluated metrics of cardiac health including beating velocity in iCMs and pro-396 fibrotic transdifferentiation in iCFs. The healt h and survivability of iCMs was then evaluated 397 again in a 3D bioprinted cardiac tissue model containing iCFs that had been pre-treated with 398 either DOX or a PBS blank, and iCMs and allowed to grow for 14 days. T hese constructs were 399 then treated with either control media (for both the control and pre-treated groups), or DOX-400 containing media for 48 h, after which the CM beating, beating regularity, and viability of the 401 cells was analyzed. Following this, the effects of direct DOX treatment and DOX-EVs from 402 cardiac and breast fibroblasts on mitochondrial respiration in 2D cultures were assessed using 403 the Seahorse MitoStress assay, which quantifies oxygen consumption rates (OCR) to derive 404 metrics of mitochondrial function. After establishing the effects of DOX-EV treatment relative to 405 DOX, we evaluated the size, morphology, and surface protein profiles of EVs derived from 406 iCMs, iCFs, and iECs with or without DOX treatment.Once complete, the miRNA cargo of the 407 EVs and DOX-EVs from iCMs and iCFs were profiled using Nanostring miRNA profiling and 408 investigated for downstream interactions pertaining to DOX cardiotoxicity via MetaCore pathway 409 analysis. Following this, we isolated EVs and their miRNA cargo from the plasma of patients 410 receiving DOX treatment and assessed for high or low risk of cardiotoxicity, and performed 411 Nanostring miRNA profiling for each subject. We were able to successfully bifurcate these 412 populations based off of the miRNA profiles obtained and compare distinguishing miRNAs from 413 the clinical samples with those from our models. From this, we were able to identify 5 major 414 clusters of miRNAs highly related to the onset of DCT and further evaluate the activities of these 415 miRNA clusters via MetaCore pathway analysis. From these data, we identified miRNAs which 416 could be obtained from plasma which may serve as a basis to develop quantitative assays for 417 the early detection of DCT in chemotherapy patients, and identified major miRNA regulated 418 pathways which may bolster the development of future intervention strategies to inhibit or 419 prevent the onset of DCT. 420 Herein, we have shown, for the first time in literature, that EVs secreted from cardiac stromal 421 cells, in this case iCFs, can independently replicate the effects of DOX cardiotoxicity without 422 ever directly exposing the target cells to DOX. While the assays performed in this study are by 423 no means exhaustive, in iCMs it was possible to replicate increased oxidative stress, whether 424 that be through increased ROS or decreased antioxidants, and significantly dysfunctional 425 beating behaviors reminiscent of DOX cardiotoxicity but without directly using DOX. These 426

were consistent in iCFs as well, which showed distinctly pro-fibrotic and oxidatively 427 stressed behaviors which were not the result of excessive cell death. Furthermore, these effects 428 did not require deliberate EV exposure via conditioned media, which could introduce trace 429 amounts of DOX if EVs are not isolated or washed appropriately. In the 3D culture, the iCFs 430 which were exposed to DOX were washed thoroughly before seeding and allowed 14 days to 431 settle, which, evaluating the substantial increase in cell death when iCFs were exposed to DOX, 432 would be sufficient time for any residual DOX to induce significant amounts of cell death. 433 However, no significant change in cell death was observed in the pre-treated models and the 434 iCMs within those models exhibited significant dysfunction without ever having been directly 435 exposed to DOX. This strongly suggests that DOX cardiotoxicity can be induced over time via 436 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 13, 2026. ; https://doi.org/10.64898/2026.02.11.705398doi: bioRxiv preprint affected CFs in the heart, rather than directly from DOX itself. This may help explain a central 437 paradox of anthracycline cardiotoxicity, namely, how the heart can be so strongly affected given 438 the known rapid clearance time of DOX (terminal half-life of 30 h and systemic clearance within 439 48 h). 440 Additionally, in iCMs and iCFs, but not iECs, CF DOX-EVs were able to induce similar metabolic 441 dysfunction to direct DOX treatment, and this dysfunction was consistent with what is observed 442 in DOX cardiotoxicity in in vivo models22, though this effect was not consistent with MF DOX-443 EVs. This discrepancy may be due to mammary fibroblasts having a slightly different, less 444 cardiotoxic response to DOX treatment than cardiac fibroblasts, or it may be due to differences 445 between primary and iPSC-derived fibroblast behaviors and paracrine signaling. However, it 446 remains interesting that CF DOX-EVs were able to recapitulate the impact of DOX treatment on 447 both iCMs and iCFs. These data, taken together with the 2D and 3D model data, suggest that 448 DOX cardiotoxicity can, to a degree, become self-propagating by inducing DOX-like dysfunction 449 in nearby CFs. Thus, even a brief initial exposure of the heart to DOX may be sufficient to affect 450 a subset of cells and substantially elevate the long-term risk of cardiotoxicity. 451 The effects of DOX treatment on iCM and iCF paracrine signaling further implicate EVs as key 452 contributors to DOX-induced cardiotoxicity. As EVs are essential agents for maintaining tissue 453 homeostasis in the heart 119, significant alterations in their size or cargo can have a profound 454 impact on the development of chronic CVDs 120. Furthermore, a significant decrease in EV size 455 has previously been linked to chronic CVD 120, as has alterations in the surface tetraspanin 456 web25. Moreover, these changes were not a result of EVs being repurposed for DOX export 457 from cells, although EVs are sometimes deliberately loaded with DOX to generate an 458 endogenous vehicle for drug delivery 121. These data, however, support preliminary conclusions 459 from the cell culture and Seahorse MitoStress assay data. As DOX-EVs do not contain DOX 460 itself, there must be some other alteration occurring in or to the produced EVs which are 461 inducing the observed DOX cardiotoxicity-like changes. 462 Full miRNA profiling of EVs from control iCMs and iCFs compared to EVs from iCMs and iCFs 463 treated with DOX for 48 h provides interesting insight into what specifically these changes may 464 be. While the full profile does not provide much useful information, unsupervised analysis of the 465 dataset revealed 145 potentially relevant miRNA targets, and, when clustered, these 145 targets 466 clearly delineate between control and DOX-treated cell EVs. More interesting, perhaps, is the 467 downstream activities of the identified EVs which were assessed by MetaCore. The pathways 468 affected, when in the context of cancer, are useful pathways to utilize to dysregulate redox 469 balance, immunomodulation, and disruption of regular mitochondrial functioning, though the 470 mechanisms are not well understood, and in fact partially converged (with 6 miRNAs 471 “cooperating” on this pathway) on CDC42 and upstream Neuropilin-1, a cell proliferation 472 pathway often desirably hindered by chemotherapy and is vital in some cancer progression 26. 473 When these same pathways are dysregulated in the heart, however, can cause significant 474 damage to the surrounding tissue and microenvironment by way of oxidative stress 31,33, 475 disruption of ion handling and beating regulation 30,32, maladaptive remodeling and pathology-476 associated changes in paracrine signaling35 , and other heart failure-associated effects. 477 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 13, 2026. ; https://doi.org/10.64898/2026.02.11.705398doi: bioRxiv preprint Interestingly, this connection may answer more about how DOX cardiotoxicity works than just 478 how the condition manifests long-term. While it is well established that DOX induces dysfunction 479 in CMs by damaging the mitochondria and often causing mitochondrial depolarization or 480 ferroptosis, the precise mechanisms by which this occurs in CMs are essentially unknown 122. 481 Furthermore, the general understanding of the role that mitochondria play in maintaining cardiac 482 health has recently grown by leaps and bounds. In particular, mitochondrial imbalance has been 483 a recent area of interest, especially for cancer and chemotherapy-related CVDs 123. 484 Mitochondrial imbalance is when CMs, which have a relative overabundance of mitochondria 485 compared to most other cells in the body, are incapable of disposing of dysfunctional 486 mitochondria either due to dysfunctional mitochondrial disposal or increased mitochondrial 487 damage. One way that this can manifest is observed through decreased mitochondrial capacity, 488 but increased mitochondrial activity, as was observed in the cell culture and metabolism assays 489 with CMs treated with DOX and DOX-EVs. Furthermore, it has recently been suggested that 490 DOX-induced cardiomyopathy stems largely from disrupted redox circuits in cardiac cells, 491 altered metabolic activity and resulting mitochondrial stress, and dysfunctional ion handling, 492 through secondary interactions that result from DOX exposure rather than direct DOX 493 interactions124, though the precise mechanisms by which these may occur is unknown. It is, 494 however, compelling that the top common results for pathways which the identified target 495 miRNAs are involved in directly correlate to those which are now hypothesized to drive DOX-496 induced cardiotoxicity. 497 In conclusion, we have established, for the first time in literature, that DOX-induced 498 cardiotoxicity can be induced in cardiac cells in the absence of DOX by using EVs from cells 499 which have previously been exposed. These effects are consistent in 2D and 3D cell culture 500 models, and mimic the expected dysfunction of DOX cardiotoxicity. Furthermore, we have 501 demonstrated that DOX treatment significantly alters the paracrine profile of cardiac cells, both 502 in terms of size and miRNA cargo, and that these altered miRNA cargos are highly and jointly 503 involved in processes which are hypothesized to be major drivers of DOX-induced 504 cardiotoxicity. To build upon these findings, we subsequently profiled plasma EVs from breast 505 cancer patients at high or low risk for DOX-induced cardiotoxicity and observed similar miRNA 506 alterations, further supporting the translational relevance of our in vitro models and the potential 507 of EV miRNAs as predictive biomarkers. Our analysis of plasma EV miRNAs from patient 508 cohorts further underscores the translational pot ential of EV cargo profiling as a biomarker 509 strategy for DOX-induced cardiotoxicity. The distinct clustering of high- and low-risk patients, 510 coupled with the substantial overlap between patient-derived EV miRNAs and those identified in 511 DOX-treated in vitro models, provides compelling evidence that EV miRNA signatures reflect 512 both the initiation and propagation of cardiotoxic signaling. Importantly, several of the enriched 513 miRNAs were linked to pathways central to cardiac homeostasis, mitochondrial regulation, and 514 fibrotic remodeling, suggesting that these circulating EVs may not only serve as indicators of 515 cardiotoxicity risk but also provide mechanistic insight into the pathophysiology of chronic DOX 516 injury. 517 While this study provides an excellent first step into understanding the interplay between local 518 paracrine signaling and both acute and chronic DOX cardiotoxicity, this study has several 519 limitations. First, the endothelial cells utilized in th is study are not cardiac specific, and as such 520 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 13, 2026. ; https://doi.org/10.64898/2026.02.11.705398doi: bioRxiv preprint may exhibit different behaviors than cardiac specific endothelial cells may. Second, the cell 521 assays performed in this study are fairly preliminary, although they do cover a wide breadth of 522 cells. To more completely compare and contrast the impact of DOX and DOX EVs on cardiac 523 cells, future studies would be prudent to focus more completely on a single cell type or 524 otherwise a more biomimetic model, such as microtissues or heart-on-chip devices, to provide a 525 more complete picture of what interactions may be occurring. Lastly, this study did not 526 completely investigate the interactions between non-cardiac tissues exposed to DOX. While 527 mammary fibroblasts were briefly investigated and showed notably different interactions, it 528 would be beneficial in future studies to evaluate the impact of DOX EVs from other tissues on 529 cardiac health. Great efforts are being made to target anthracyclines to the desired tissues in 530 order to circumvent cardiotoxicity, and understanding how these tissues may then downstream 531 interact with the myocardium would be extraordinarily useful. In addition, while the clinical EV 532 profiling offers strong translational support for our findings, the modest sample size limits 533 broader generalization and highlights the need for validation in larger patient cohorts. Moreover, 534 future studies incorporating intermediate models, such as animal models or ex vivo platforms, 535 could help capture systemic factors and strengthen the mechanistic link between EV signaling 536 and cardiotoxicity observed in both engineered tissues and clinical samples. Despite these 537 limitations, this study has been an incredibly successful first step down a long road of 538 understanding the interactions between EVs and DOX cardiotoxicity, and this and future studies 539 will help finally elucidate the mechanisms by which DOX-induced cardiotoxicity occurs. 540

& METHODS 541 Culture of Human Induced Pluripotent Stem Cells (hiPSCs): DiPS 1016 SevA hiPSCs, 542 which were derived from human skin fibroblasts, were cultured on Geltrex (1% Invitrogen, USA)-543 coated culture flasks in mTeSR (StemCell Technologies, Canada) media supplemented with 1% 544 penicillin (VWR, USA) under standard culture conditions from passages 40-50. 545 Cells were passaged at 80% confluency. To passage the hiPSCs, cells were detached using 546 Accutase (StemCell Technologies, Canada) and seeded onto Geltrex-coated cell culture well 547 plates or split between cell culture flasks. Seeding was performed in mTeSR media 548 supplemented with Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor (5 549 μ M, StemCell Technologies, Canada). For differentiation, cells were cultured until 95% 550 confluency before starting a protocol. 551 Differentiation and Culture of hiPSC-derived Cardiomyocytes (iCMs) : Differentiation of 552 hiPSCs to iCMs was adapted from a previously established protocol. Briefly, when hiPSCs were 553 ready for differentiation the media was changed to RPMI Medium 1640 (Life Technologies, 554 USA) supplemented with B27 without insulin (2%, Invitrogen, USA), and beta-mercaptoethanol 555 (final concentration of 0.1 mM, Promega, USA) (CM (-)) with the addition of Wnt activator, 556 CHIR99021 (CHIR) (10 μ M, Stemgent, USA) (Day 1). Exactly 24 h later, media replaced with 557 CM (-) with CHIR (2 µM) (Day 2), and again another 24 h later (Day 3). Exactly 24 h later, media 558 was changed to CM (-) with Wnt inhibitor IWP-4 (5 μ M, MA, USA) (Day 4). Exactly 48 h later, 559 media was changed to CM (-) (Day 6). Exactly 72 h later, media was changed to RPMI Medium 560 1640 supplemented with B27 (2%, Invitrogen, USA), and beta-mercaptoethanol (final 561 concentration of 0.1 mM) (CM (+)) (Day 9). Following this, every 3 days media was changed 562 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 13, 2026. ; https://doi.org/10.64898/2026.02.11.705398doi: bioRxiv preprint using CM (+). Cultures typically began to beat by day 21 of this protocol, as reported 563 previously125, and continued to be cultured in CM (+) until use. 564 Differentiation and Culture hiPSC-derived Cardiac Fibroblasts (iCFs) : Differentiation of 565 hiPSCs to iCFs was adapted from a previous protocol 126. Briefly, when hiPSCs were ready for 566 differentiation the media was c hanged to CM (-) with 10 µM CHIR (Day 1). After 24 h, media 567 was changed to CM (-) without CHIR (Day 2). After 24 h, media was replaced with CFBM media 568 supplemented with fetal bovine serum (FBS) (Gibco) and 75 ng/mL fibroblast growth factor 569 (FGF) (Day 3). Media was refreshed every 48 h until day 20. After day 20, differentiated iCFs 570 were detached from the cell plate with trypsin-EDTA (0.25%, Stem Cell Technologies, Canada) 571 and seeded into fibronectin (Sigma Aldrich, USA)-coated cell culture flasks. Seeded iCFs were 572 from then on cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Thermo Fisher) 573 supplemented with 10% FBS, 1% penicillin/streptomycin (P/S) (Life Technologies), henceforth 574 called DMEM Complete, and 3 µM SD208, a TGF-b receptor I kinase inhibitor (Sigma Aldrich) 575 on fibronectin-coated cell culture flasks. Cells were cultured with SD208 supplement to inhibit 576 transdifferentiation, and then used between passage 4 and 10 without SD208. Human 577 mammary fibroblasts (hMFs) were received from a collaborator and cultured using the above 578 protocol. 579 Differentiation and Culture hiPSC-derived Endothelial Cells (iECs) : Differentiation of 580 hiPSCs to iECs was adapted from a previous protocol 125. Briefly, when hiPSCs were ready for 581 differentiation the media was changed to a 1:1 mixture of DMEM to F12 with Glutamax and 582 Neurobasal media supplemented with N2 (1%, Life Technologies, USA), B27 (2%), CHIR (8 583 μ M) and bone morphogenic protein 4 (25 ng/ml, R&D Systems, USA) (Day 1). After 72 h, media 584 was replaced with StemPro-34 SFM medium (Life Technologies, USA) supplemented with 585 vascular endothelial growth factor (200 ng/ml, PeproTech), and forskolin (2 μ M, Sigma-Aldrich, 586 USA) (Day 4), and again after 24 h (Day 5). After 24 h, cells were sorted against vascular 587 endothelial cadherin (VE-Cad) (Abcam, United Kingdom) with magnetic assisted cell sorting 588 (MACS) using a Dynamag magnet (Invitrogen, USA) (Day 6). Cells were cultured on fibronectin-589 coated cell cultures flask in endothelial growth media 2 (Lonza, Switzerland, EGM-2) until use. 590 Doxorubicin Supplemented Media (DOX-media) Preparation: Doxorubicin (100 mM) was 591 diluted in DMEM without FBS to create a stock solution of 100 µM, then diluted 1:10 to create a 592 working solution of 10 µM for easy addition of 10 nM. Final concentration necessary was 593 determined according to experimentally determined dosages in literature 127, and can be 594 compared to in vivo measurements using following formula: 595 /g1839/g4666/g1865/g1867/g1864/g1838/g3415/g4667/g3404 /g3004 /g4666 /g3034 /g3040/g3042/g3039/g3415/g4667 /g3040 /g4666 /g3034 /g3013/g3415 /g4667 (Equation 1) 596 Where m is the molar mass of DOX (543.52 g/mol), C is the blood concentration of DOX (12.54 597 ng/mL) and M is the desired molarity (10-100 nM, depending on cohort tested). 598 Clinical Plasma Sample EV Isolation: Whole blood was collected from patients via direct 599 veinous puncture into ethylenediaminetetraacetic acid (EDTA)-treated tubes to prevent 600 coagulation. Patients were selected for their risk of developing cardiotoxicity, either high-601 baseline risk or low to moderate risk (n=4) (Supplemental Table S6).To isolate the plasma, 602 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 13, 2026. ; https://doi.org/10.64898/2026.02.11.705398doi: bioRxiv preprint whole blood was centrifuged at 1000g 5for minutes at 4°C. Patient plasma was aliquoted to 603 sterile RNAse-free tubes and shipped from the Lambe Institute for Translational Research at the 604 University of Galway to the University of Notre Dame at -80°C. A temperature sensor was 605 included during transport to ensure appropriate storage conditions were maintained. Upon 606 arrival, samples were stored at -80°C until use. 607 Extracellular Vesicle (EV) Generation and Isolation : Conditioned media was generated by 608 culturing cells in exosome-free media (no change in CM (+), DMEM Complete and EGM-2 609 substitute FBS for exosome-free FBS. Conditioned media was centrifuged three times at 500g 610 for 10 min, 2500g for 20 min, and 10,000g for 30 min, and the pellet discarded after each 611 centrifugation step to remove any remaining insoluble matrix remnants. The final supernatant 612 was centrifuged at 100,000g at 4°C for 70 min using an ultracentrifuge (Optima MAX-XP 613 Tabletop Ultracentrifuge, Beckman Coulter). The pellet was either used immediately or stored 614 dry at -80°C. Plasma samples were similarly processed to isolate EVs and pellet was stored 615 accordingly. 616 Nanoparticle Tracking Analysis (NTA) : Single pellets were resuspended in 1mL of sterile, 617 particle-free PBS and measured us ing a NanoSight NS300 machi ne (Malvern Panalytical) and 618 NTA software version 3.2.16. This method obtains the hemodynamic diameter and 619 concentration of nanoparticles with diameters from 10-1000 nm in solution via Brownian motion 620 analysis. Samples were kept at 4 °C until measurement, and measurements were taken at RT. 621 Western Blot : Pellets were lysed in RIPA buffer containing 1% proteinase inhibitor cocktail 622 (Brand, Country) at 4°C for 30 minutes, then protein concentration was assessed via 623 bicinchoninic acid (BCA) assay (Pierce Chemical). Equal amounts of protein were separated by 624 12% SDS-PAGE and transferred to blotting membranes, which were incubated overnight at 4°C 625 with the rabbit polyclonal primary antibodies against CD9 (Abcam, ab223052), CD63 (Abcam, 626 ab216130), and TSG101 (Abcam, ab30871) at 1:2000 dilution, then for 1 h at RT with HRP-627 conjugated goat anti-rabbit secondary antibody (Abcam, ab205718). Membranes were then 628 exposed to a chemiluminescent substrate (Clarity ECL, Bio-Rad) and imaged using a 629 ChemiDoc-It2 imager (UVP, Analytik Jena) equipped with VisionWorks software. Images were 630 processed using ImageJ (NIH). 631 Ultraviolet-Visible Light (UV-Vis) Spectrophotometry : Pellets were resuspended in 50 µL 632 PBS immediately after ultracentrifugati on, and analyzed using a microvolume 633 spectrophotometer (Nanodrop 2000, Thermo Fisher Scientific). For controls, an empty PBS 634 sample, DOX-spiked PBS sample, and supernatant from DOX-cell EV ultracentrifugation we all 635 run as well. Absorbance was quantified at approximately 480 nm, where DOX natively exhibits a 636 broad absorption peak (specifically between 470 nm and 500 nm128) (Supplemental Figure S3). 637 Doxorubicin-treated cell EV (DOX-EV) Conditioned Media Generation : DOX media was 638 prepared for DOX as described above, with final DOX concentration of 10 nM in media. After 48 639 h of treatment, media was collected and EVs were isolated. Media conditioned with EVs isolated 640 from DOX-treated cells was created through the addition of isolated EVs to the desired media at 641 25 µg/mL EVs/media volume. EV mass was measured by the bicinchoninic acid (BCA) assay, 642 as is standard 129, before addition to media. Before treatment, cells were washed 3x with sterile 643 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 13, 2026. ; https://doi.org/10.64898/2026.02.11.705398doi: bioRxiv preprint PBS, then room temperature control or treatment media was added and left undisturbed for 48 h 644 at 37°C, after which cells were again washed 3x with PBS and assays were performed. 645 Cardiomyocyte Beating Characterization : To analyze the contractility of iCMs, a block-646 matching algorithm was performed using MATLAB as described previously130. Briefly, iCM or 3D 647 structures were recorded in brightfield in real time under a microscope (Axio Observer. Z1, 648 Zeiss, Hamamatsu C11440 digital camera) for 30 s intervals. Videos were then uploaded to the 649 analysis software, and beating velocity and frequency were calculated, and contraction heat 650 maps were plotted. 651 Ca2+ Flux Assay: Contraction kinetics were measured by analysis of calcium (Ca 2+) flux over 652 the tissue, as previously described 131. Briefly, iCMs were removed from media, which was 653 stored warm and with separated biological replicates, and was incubated with Fluo-4 AM 654 (Thermo Fischer Scientific, USA) according to the manufacturer’s protocol for 30 min at 37°C. 655 After incubation, the staining solution was removed, and the original cell media was returned to 656 the cells. Stained cells were then immediately recorded under a microscope (Axio Observer. Z1, 657 Zeiss) for 30 s intervals, with recording being performed using a green fluorescent channel at 658 200 ms exposure. Videos were analyzed utilizing an in-hous e MATLAB code as previously 659 described. 660 After analysis, resulting waveforms were converted into the frequency domain via 1-dimensional 661 Fast Fourier Transform (FFT) and were subsequently deconstructed into component sine 662 waves, the frequencies of which were plotted. 663 Immunostaining: Cells or constructs were washed 3x with PBS to remove residual media, then 664 incubated in 4% paraformaldehyde for 15 min, followed by 0.1% Triton-X for 30 min, then 5% 665 goat serum in PBS for 2 h, all with 3x PBS was hes in between. Cells were next incubated with 666 rabbit anti-cleaved caspace-3 (Abcam) and mouse anti- α -SMA (Abcam) primary antibodies 667 (1:100 in 5% goat serum) overnight at 4°C. The cells were then washed and incubated with 668 Alexa Fluor 647-labelled anti-rabbit IgG and Alexa Fluor 488-labelled anti-mouse IgG secondary 669 antibodies (dilution: 1:200 in 5% goat serum) at 4°C for 6 h. Finally, the cells were incubated 670 with DAPI (dilution: 1:1000 in PBS) for 15 minutes at RT and imaged with a fluorescent 671 microscope (Axio Observer. Z1, Zeiss). 672 Measurement of Cell Metabolic Activity : DOX media and DOX-EV media were prepared for 673 cells as described above, and cells were treated for 48 h with either DOX media, DOX-EV 674 media, or media with a PBS bl ank. After 48 h, cells were wa shed, and media was swapped out 675 for a 10% AlamrBlue (Thermo Fisher)-media mixture 132. Cells were cultured in the 10% solution 676 for 1 h, after which the solution was transferred to a 96-well plate and the absorbance of each 677 well was measured on a plate reader. 678 Reactive Oxygen Species Detection: DOX media and DOX-EV media were prepared for cells 679 as described above, and cells were treated for 48 h with either DOX media, DOX-EV media, or 680 media with a PBS blank. After 48 h, accumulation of reactive oxygen species (ROS) was 681 assessed by a Mitochondrial ROS Assay Kit (eEnzyme, USA). Briefly, the stain solution was 682 prepared as instructed and the cells were incubated at 37 ℃ for 30 min. Fluorescent images 683 were taken immediately following incubation. 684 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 13, 2026. ; https://doi.org/10.64898/2026.02.11.705398doi: bioRxiv preprint Seahorse XF ATP Real Time Assay : Seahorse respirometry, to measure oxygen consumption 685 rate (OCR) of cells, was performed with the Seahorse XF Extracellular Flux Analyzer (Agilent, 686 Germany) as reported previously 133. Briefly, differentiated iCMs, iCFs, and iECs were detached 687 and reseeded into a 96-well Seahorse-compatible plate at 4 × 10 /i3 cells/well for iCFs and iECs, 688 and 8 × 10 /i3 cells/well for iCMs. iCMs were plated 48 hours earlier to allow additional settling 689 time, which did not affect cell count since iCMs are non-proliferative. iCFs and iECs were 690 seeded and allowed to attach overnight. After attachment, cells were treated for 48 h with DOX 691 media, DOX-EV media, where the EVs came fr om DOX-treated iCFs or hMFs, or a PBS blank. 692 Following treatment, cells were stained with Hoechst 33342 (Thermo Scientific, 8 µM) for 30 693 minutes at 37°C, and cell count in each well were subsequently quantified through ImageJ 694 image analysis. Then, the media was replaced with the provided Seahorse buffer. OCR was 695 then assessed at basal level and following sequential metabolic perturbations with inhibitors 696 prepared at the following final concentrations: 2.5 μ M oligomycin, 2 μ M FCCP, and 2.5 μ M 697 rotenone/antimycin A (Rot/AA). Analysis was performed in the Agilent Seahorse online 698 software, and analyzed data was exported to excel for statistics and graphed in R. 699 3D Cardiac Model Construction: Type 1 collagen (3 mg/ml) (HumaBiologics, AZ, USA) was 700 pH-adjusted to 7 through titration and then refrigerated at 4°C till use. GelMA, synthesized and 701 dissolved in PBS (20% w/v) and maintained in a water bath at 37°C until a fully homogeneous 702 solution was obtained. Then, Irgacure2959 photoinitiator (PI, Sigma, MO, US) stock solution 703 was prepared in PBS (1% w/v) and was added to GelMA. The components were mixed to 704 achieve a bioink with final concentrations of 10% GelMA, 1 mg/ml collagen type 1, and 0.025% 705 PI as previously reported 126,133. iCMs, DOX-free conditioned media-treated iCFs (control), and 706 pre-treated iCFs were detached using trypsin-EDTA. After centrifugation at 1000 rpm for 5 707 minutes, iCMs (15 mil/mL) were combined with both types of iCFs (5 mil/mL) and centrifuged 708 again. The supernatant was removed, and each cell pellet was mixed with collagen and GelMA 709 solution, respectfully. Then, bioink was loaded into separate syringes and submerged in the ice 710 for a minute to achieve the required consistency for printing. Droplets were bioprinted using 711 CELLINK BioX6 Bioprinter at a speed of 3 mm/s using 22G nozzles on a sterilized charged 712 glass in a 60- mm dish. Lastly, 3D bioprinted constructs were photo-crosslinked for 30 seconds 713 under UV exposure (6.9 W/cm2) and placed in a well-plate with CM (+) and DMEM complete. 714 miRNA Isolation from EVs : RNA was isolated from LVVs and clinical plasma samples using 715 the Total Exosome RNA & Protein Isolation Kit (Thermo Fisher Scientific) using manufacturer’s 716 protocol. Isolated LVVs were resuspended in exosome resuspension buffer and incubated with 717 an equal volume of denaturation solution at 4 °C for 5 min. The solution was then mixed with an 718 equal volume of Acid-Phenol:Chloroform by vortexing for 30 seconds and centrifuged for 5 min 719 at 15,000g. The resulting aqueous phase was extracted and combined with 1.25x volume of 720 100% ethanol, then transferred to the provided spin column. The spin column was centrifuged at 721 10,000g for 15 seconds to bind and wash the RNA, then the RNA was eluted in the provided 722 elution solution and quantified via a microvolume spectrophotometer (Nanodrop 2000, Thermo 723 Fisher Scientific). 724 Following isolation, the eluted miRNA was concentrated using 3 kDa microcentrifuge spin filters 725 (Amicon) according to a previously established protocol 134. Briefly, the 100 µL miRNA solution 726 was worked up to 420 µL with RNAse-free water and placed into a filter, then centrifuged at 727 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 13, 2026. ; https://doi.org/10.64898/2026.02.11.705398doi: bioRxiv preprint 14,000g for 90 minutes. Next, the filter was inverted into a fresh collection tube, and centrifuged 728 at 8,000g for 2 minutes. The resulting isolate is 20-25 µL of concentrated miRNA, which was 729 quantified by the same microvolume spectrophotometer used previously. 730 Profiling of Total miRNA Content : Concentrated miRNA was prepared for miRNA profiling 731 (NanoString). The provided miRNA codeset was mixed with the provided hybridization buffer to 732 produce a master mix, and spike-in miRNA controls were prepared at 200 pm. In order, the 733 master mix, concentrated sample miRNA, spike-in miRNA, and provided probes were mixed in 734 a PCR plate and incubated at 65 °C for 16 h. The hybridized solution was then mixed with 15 µL 735 of provided hybridization buffer, for a total volume of 30-35 µL, and added to the provided 736 microfluidic cartridge. The assay was run with the provided protocol for total miRNA analysis, 737 and data was processed and analyzed using the provided software using the recommended 738 settings. Processed data was exported to a .csv spreadsheet for l og10 normalization and Z-739 scoring and subsequent plotting in R, according to the following formula: 740 /g1852/g3404 /g3051/g2879/g3051 /g1191 /g3097 (Equation 2) 741 Where /g1852 is the Z-score value, /g1876 is the log10 normalized count value, /g1876/g1191 is the average of the 742 log10 normalized count values for a given miRNA, and /g2026 is the standard deviation of the log10 743 normalized count values for a given miRNA. 744 miRNA Pathway Analysis: Raw Nanostring data was normalized via Log10 normalization, and 745 uploaded to the Clarivate MetaCore system for pathway analysis. miRNAs were identified by 746 miRBase IDs. Analysis was conducted on the identified target miRNAs to construct a custom 747 network. Automated network analysis was conducted with 50 nodes per network. Results were 748 presented as pathways obtained from the software. 749 Statistical Analysis: Results were analyzed by one-way analysis of variance (ANOVA) with 750 post-hoc Tukey’s HSD, two-way ANOVA with post-hoc Tukey’s multiple comparison test, or a 751 two-tailed Student’s t-test with Welch’s correction for unequal standard deviation. Values are 752 presented as the mean ± standard deviation (SD) unless otherwise indicated, and differences 753 were considered significant when p ≤ 0.05. 754 755

756 1. Tsao, C. W. et al. Heart Disease and Stroke Statistics-2023 Update: A Report From the 757 American Heart Association. Circulation 147, e93–e621 (2023). 758 2. Olvera Lopez, E., Ballard, B. D. & Jan, A. Cardiovascular Disease. in StatPearls (StatPearls 759 Publishing, Treasure Island (FL), 2025). 760 3. Lin, A. et al. Off-target toxicity is a common mechanism of action of cancer drugs 761 undergoing clinical trials. Sci. Transl. Med. 11, eaaw8412 (2019). 762 4. Rhea, I. B. & Oliveira, G. H. Cardiotoxicity of Novel Targeted Chemotherapeutic Agents. 763 Curr. Treat. Options Cardiovasc. Med. 20, 53 (2018). 764 5. Babiker, H. M., McBride, A., Newton, M., B oehmer, L. M., Goeller Drucker, A., Gowan, M., 765 Cassagnol, M., Camenisch, T. D., Anwer, F. & Hollands, J. M. Cardiotoxic effects of 766 chemotherapy: A review of both cytotoxic and molecular targeted oncology therapies and 767 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 13, 2026. ; https://doi.org/10.64898/2026.02.11.705398doi: bioRxiv preprint their effect on the cardiovascular system. Crit. Rev. Oncol. Hematol. 126, 186–200 (2018). 768 https://doi.org/10.1016/j.critrevonc.2018.03.014 769 6. Siegel, R. L., Miller, K. D., Wagle, N. S. & Jemal, A. Cancer statistics, 2023. CA. Cancer J. 770 Clin. 73, 17–48 (2023). 771 7. Rivankar, S. An overview of doxorubicin formulations in cancer therapy. J. Cancer Res. 772 Ther. 10, 853–858 (2014). 773 8. Carvalho, C. et al. Doxorubicin: the good, the bad and the ugly effect. Curr. Med. Chem. 16, 774 3267–3285 (2009). 775 9. Chatterjee, K., Zhang, J., Honbo, N. & Karliner, J. S. Doxorubicin Cardiomyopathy. 776 Cardiology 115, 155–162 (2010). 777 10. Gartrell, J. et al. The effects of pazopanib on doxorubicin pharmacokinetics in children and 778 adults with non-rhabdomyosarcoma soft tissue sarcoma: a report from Children’s Oncology 779 Group and NRG Oncology study ARST1321. Cancer Chemother. Pharmacol. 89, 551–557 780 (2022). 781 11. Sheta, M., Taha, E. A., Lu, Y. & Eguchi, T. Extracellular Vesicles: New Classification and 782 Tumor Immunosuppression. Biology 12, 110 (2023). 783 12. Jo, H., Shim, K. & Jeoung, D. Exosomes: Diagnostic and Therapeutic Implications in 784 Cancer. Pharmaceutics 15, 1465 (2023). 785 13. Jia, Y. et al. Small extracellular vesicles isolation and separation: Current techniques, 786 pending questions and clinical applications. Theranostics 12, 6548–6575 (2022). 787 14. Ellis, B. W. et al. Human Heart Anoxia and Reperfusion Tissue (HEART) Model for the 788 Rapid Study of Exosome Bound miRNA Expression As Biomarkers for Myocardial 789 Infarction. Small Weinh. Bergstr. Ger. 18, e2201330 (2022). 790 15. Ronan, G., Bahcecioglu, G., Yang, J. & Zorlutuna, P. Cardiac tissue-resident vesicles 791 differentially modulate anti-fibrotic phenotype by age and sex through synergistic miRNA 792 effects. Biomaterials 311, 122671 (2024). 793 16. Tian, C. et al. Potential of exosomes as diagnostic biomarkers and therapeutic carriers for 794 doxorubicin-induced cardiotoxicity. Int. J. Biol. Sci. 17, 1328–1338 (2021). 795 17. Zhang, G. et al. Understanding the Protective Role of Exosomes in Doxorubicin-Induced 796 Cardiotoxicity. Oxid. Med. Cell. Longev. 2022, 2852251 (2022). 797 18. Ni, J. et al. Human trophoblast-derived exosomes attenuate doxorubicin-induced cardiac 798 injury by regulating miR-200b and downstream Zeb1. J. Nanobiotechnology 18, 171 (2020). 799 19. Xia, W., Chen, H., Xie, C. & Hou, M. Long-noncoding RNA MALAT1 sponges microRNA-800 92a-3p to inhibit doxorubicin-induced cardiac senescence by targeting ATG4a. Aging 12, 801 8241–8260 (2020). 802 20. Ronan, G., Bahcecioglu, G., Aliyev, N. & Zorlutuna, P. Engineering the cardiac tissue 803 microenvironment. Progress in Biomedical Engineering 6, 012002 (2023). 804 21. Songbo, M. et al. Oxidative stress injury in doxorubicin-induced cardiotoxicity. Toxicol. Lett. 805 307, 41–48 (2019). 806 22. Zhao, Y. et al. Redox proteomic identification of HNE-bound mitochondrial proteins in 807 cardiac tissues reveals a systemic effect on energy metabolism after doxorubicin treatment. 808 Free Radic. Biol. Med. 72, 55–65 (2014). 809 23. Li, X. et al. Semaglutide attenuates doxorubicin-induced cardiotoxicity by ameliorating 810 BNIP3-Mediated mitochondrial dysfunction. Redox Biol. 72, 103129 (2024). 811 24. Varga, Z. V., Ferdinandy, P., Liaudet, L. & Pacher, P. Drug-induced mitochondrial 812 dysfunction and cardiotoxicity. Am. J. Physiol. Heart Circ. Physiol. 309, H1453-1467 (2015). 813 25. Ellis, B. W. et al. Human Heart Anoxia and Reperfusion Tissue (HEART) Model for the 814 Rapid Study of Exosome Bound miRNA Expression As Biomarkers for Myocardial 815 Infarction. Small Weinh. Bergstr. Ger. 18, e2201330 (2022). 816 26. Du, D.-S. et al. Effects of CDC42 on the proliferation and invasion of gastric cancer cells. 817 Mol. Med. Rep. 13, 550–554 (2016). 818 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 13, 2026. ; https://doi.org/10.64898/2026.02.11.705398doi: bioRxiv preprint 27. Halling, J. F. & Pilegaard, H. PGC-1α -mediated regulation of mitochondrial function and 819 physiological implications. Appl. Physiol. Nutr. Metab. Physiol. Appl. Nutr. Metab. 45, 927–820 936 (2020). 821 28. Lin, J. et al. Transcriptional co-activator PGC-1 alpha drives the formation of slow-twitch 822 muscle fibres. Nature 418, 797–801 (2002). 823 29. Yakeu, G. et al. Low-intensity exercise enhances expression of markers of alternative 824 activation in circulating leukocytes: roles of PPARγ and Th2 cytokines. Atherosclerosis 212, 825 668–673 (2010). 826 30. Ingelsson, E. et al. The PPARGC1A Gly482Ser polymorphism is associated with left 827 ventricular diastolic dysfunction in men. BMC Cardiovasc. Disord. 8, 37 (2008). 828 31. Diaz, S., Wang, K., Sjögren, B. & Liu, X. Roles of Cullin-RING Ubiquitin Ligases in 829 Cardiovascular Diseases. Biomolecules 12, 416 (2022). 830 32. Konstantinidis, K. et al. MICAL1 constrains cardiac stress responses and protects against 831 disease by oxidizing CaMKII. J. Clin. Invest. 130, 4663–4678 (2020). 832 33. Sadasivan, C. et al. Cardiovascular toxicity of PI3Kα inhibitors. Clin. Sci. Lond. Engl. 1979 833 134, 2595–2622 (2020). 834 34. Maulik, A., Davidson, S. M., Piotrowska, I., Walker, M. & Yellon, D. M. Ischaemic 835 preconditioning protects cardiomyocytes from anthracycline-induced toxicity via the PI3K 836 pathway. Cardiovasc. Drugs Ther. 32, 245–253 (2018). https://doi.org/10.1007/s10557-018-837 6793-y 838 35. Sanada, S. et al. IL-33 and ST2 comprise a critical biomechanically induced and 839 cardioprotective signaling system. Journal of Clinical Investigation 117, 1538 (2007). 840 36. Pini, L. et al. Alpha1-antitrypsin deficiency and cardiovascular disease: questions and issues 841 of a debated relation. J. Cardiovasc. Med. Hagerstown Md 23, 637–645 (2022). 842 37. Xue, H. et al. MicroRNA-584-3p, a novel tumor suppressor and prognostic marker, reduces 843 the migration and invasion of human glioma cells by targeting hypoxia-induced ROCK1. 844 Oncotarget 7, 4785–4805 (2015). 845 38. Totoń -Ż urań ska, J. et al. MicroRNA composition of plasma extracellular vesicles: a 846 harbinger of late cardiotoxicity of doxorubicin. Mol. Med. 28, 156 (2022). 847 39. Sucharov, C. C., Miyamoto, S. D. & Garcia, A. M. Circulating microRNAs as biomarkers in 848 pediatric heart diseases. Prog. Pediatr. Cardiol. 49, 50–52 (2018). 849 https://doi.org/10.1016/j.ppedcard.2018.02.008 850 40. Wu, H., Wang, Y., Wang, X., Li, R. & Yin, D. MicroRNA-365 accelerates cardiac hypertrophy 851 by inhibiting autophagy via the modulation of Skp2 expression. Biochem. Biophys. Res. 852 Commun. 484, 304–310 (2017). https://doi.org/10.1016/j.bbrc.2017.01.108 853 41. Xiao, Y., Deng, T., Su, C. & Shang, Z. MicroRNA 217 inhibits cell proliferation and enhances 854 chemosensitivity to doxorubicin in acute myeloid leukemia by targeting KRAS. Oncol. Lett. 855 13, 4986–4994 (2017). 856 42. Zheng, Y. et al. MiR-24-3p modulates cardiac function in doxorubicin -induced heart failure 857 via the Sp1/PI3K signaling pathway. Cell. Signal. 124, 111407 (2024). 858 43. Chen, M. et al. Improvement of cardiac function by mesenchymal stem cells derived 859 extracellular vesicles through targeting miR-497/Smad7 axis. Aging 13, 22276–22285 860 (2021). 861 44. Li, Y., Hua, K., Jin, J. & Fang, L. miR-497 inhibits proliferation and invasion in triple-negative 862 breast cancer cells via YAP1. Oncol. Lett. 22, 580 (2021). 863 45. Sun, F. et al. Exosomes derived from the blood of patients with sepsis regulate apoptosis 864 and aerobic glycolysis in human myocardial cells via the hsa/i2 miR/i2 1262/SLC2A1 signaling 865 pathway. Mol. Med. Rep. 25, 1–9 (2022). 866 46. Zhang, W. et al. NF-κ B downstream miR-1262 disturbs colon cancer cell malignant 867 behaviors by targeting FGFR1. Acta Biochim. Biophys. Sin. 55, 1819–1832 (2023). 868 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 13, 2026. ; https://doi.org/10.64898/2026.02.11.705398doi: bioRxiv preprint 47. Xu, H. et al. IP3R-Grp75-VDAC1-MCU calcium regulation axis antagonists protect 869 podocytes from apoptosis and decrease proteinuria in an Adriamycin nephropathy rat 870 model. BMC Nephrol. 19, 140 (2018). 871 48. Singla, D. K. Akt-mTOR Pathway Inhibits Apoptosis and Fibrosis in Doxorubicin-Induced 872 Cardiotoxicity Following Embryonic Stem Cell Transplantation. Cell Transplant. 24, 1031–873 1042 (2015). 874 49. Russo, M., Della Sala, A., Tocchetti, C. G., Porporato, P. E. & Ghigo, A. Metabolic Aspects 875 of Anthracycline Cardiotoxicity. Curr. Treat. Options Oncol. 22, 18 (2021). 876 50. Fang, Q. et al. LncRNA TUG1 alleviates cardiac hypertrophy by targeting miR-877 34a/DKK1/Wnt-β -catenin signalling. J. Cell. Mol. Med. 24, 3678–3691 (2020). 878 51. Rawat, P. S., Jaiswal, A., Khurana, A., Bhatti, J. S. & Navik, U. Doxorubicin-induced 879 cardiotoxicity: An update on the molecular mechanism and novel therapeutic strategies for 880 effective management. Biomed. Pharmacother. 139, 111708 (2021). 881 52. Bezler, M., Hengstler, J. G. & Ullrich, A. Inhibition of doxorubicin ‐ induced HER3‐ PI3K‐ AKT 882 signalling enhances apoptosis of ovarian cancer cells. Mol. Oncol. 6, 516–529 (2012). 883 53. Cheng, F. et al. FOXO1-NMNAT3 axis dysregulation promotes doxorubicin cardiotoxicity: 884 NAD+ replenishment as a redox-targeted antioxidant therapy. Redox Rep. 30, 2565033 885 (2025). 886 54. Cheng, F. et al. FOXO1-NMNAT3 axis dysregulation promotes doxorubicin cardiotoxicity: 887 NAD+ replenishment as a redox-targeted antioxidant therapy. Redox Rep. 30, 2565033 888 (2025). 889 55. Fan, C. et al. Rosmarinic acid alleviates doxorubicin-induced cellular senescence and 890 cardiotoxicity by targeting the 14-3-3/Foxo1 signaling axis. Phytomedicine 148, 157482 891 (2025). 892 56. Lei, Y. et al. Cooperative sensing of mitochondrial DNA by ZBP1 and cGAS promotes 893 cardiotoxicity. Cell 186, 3013-3032.e22 (2023). 894 57. Papatriantafyllou, M. ZBP1 links mtDNA instability to sustained IFN-I responses and 895 cardiomyopathy. Nat. Cardiovasc. Res. 2, 711–711 (2023). 896 58. Wang(a), J. et al. Molecular mechanisms of doxorubicin-induced cardiotoxicity: novel roles 897 of sirtuin 1-mediated signaling pathways. Cell. Mol. Life Sci. CMLS 78, 3105–3125 (2021). 898 59. Ito-Hagiwara, K., Hagiwara, J., Endo, Y., Becker, L. B. & Hayashida, K. Cardioprotective 899 strategies against doxorubicin-induced cardiotoxicity: A review from standard therapies to 900 emerging mitochondrial transplantation. Biomed. Pharmacother. 189, 118315 (2025). 901 60. Borlak, J. et al. The Abl1 tyrosine kinase is a key player in doxorubicin-induced 902 cardiomyopathy and its p53/p73 cell death mediated signaling differs in atrial and ventricular 903 cardiomyocytes. J. Transl. Med. 22, 845 (2024). 904 61. Yarmohammadi, F., Rezaee, R., Haye, A. W. & Karimi, G. Endoplasmic reticulum stress in 905 doxorubicin-induced cardiotoxicity may be therapeutically targeted by natural and chemical 906 compounds: A review. Pharmacol. Res. 164, 105383 (2021). 907 62. Fradley, M. G. et al. Adverse Cardiovascular Events Associated With Cyclin‐ Dependent 908 Kinase 4/6 Inhibitors in Patients With Metastatic Breast Cancer. J. Am. Heart Assoc. 12, 909 e029361 (2023). 910 63. Pavlovic, D., Niciforovic, D., Papic, D., Milojevic, K. & Markovic, M. CDK4/6 inhibitors: 911 basics, pros, and major cons in breast cancer treatment with specific regard to cardiotoxicity 912 – a narrative review. Ther. Adv. Med. Oncol. 15, 17588359231205848 (2023). 913 64. Castelli, M. et al. HOPS/TMUB1 retains p53 in the cytoplasm and sustains p53‐ dependent 914 mitochondrial apoptosis. EMBO Rep. 21, EMBR201948073 (2019). 915 65. Bao, Y.-N. et al. Targeting tumor suppressor p53 for organ fibrosis therapy. Cell Death Dis. 916 15, 336 (2024). 917 66. Li, C. et al. P55PIK Regulates P53-Dependent Apoptosis in Cancer Cells by Interacting with 918 P53 DNA-Specific Domain. OncoTargets Ther. 13, 5177–5190 (2020). 919 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 13, 2026. ; https://doi.org/10.64898/2026.02.11.705398doi: bioRxiv preprint 67. Rawat, P. S., Jaiswal, A., Khurana, A., Bhatti, J. S. & Navik, U. Doxorubicin-induced 920 cardiotoxicity: An update on the molecular mechanism and novel therapeutic strategies for 921 effective management. Biomed. Pharmacother. Biomedecine Pharmacother. 139, 111708 922 (2021). 923 68. Qi, X.-M. et al. PGC-1α /NRF1-dependent cardiac mitochondrial biogenesis: A druggable 924 pathway of calycosin against triptolide cardiotoxicity. Food Chem. Toxicol. Int. J. Publ. Br. 925 Ind. Biol. Res. Assoc. 171, 113513 (2023). 926 69. Fiordelisi, A. et al. P490GRK2 is a novel early marker of cardiotoxicity in response to 927 doxorubicin. Cardiovasc. Res. 114, S119 (2018). 928 70. Kosić , M. et al. Paroxetine mitigates cardiac remodelling by doxorubicin and increases 929 survival. Biomed. Pharmacother. 145, 112411 (2022). 930 71. Ferrero, K. M. & Koch, W. J. GRK2 in cardiovascular disease and its potential as a 931 therapeutic target. J. Mol. Cell. Cardiol. 172, 14–23 (2022). 932 72. Kosić , M. et al. Paroxetine mitigates cardiac remodelling by doxorubicin and increases 933 survival. Biomed. Pharmacother. 145, 112411 (2022). 934 73. Zhang, W.-B., Lai, X. & Guo, X.-F. Activation of Nrf2 by miR-152 Inhibits Doxorubicin-935 Induced Cardiotoxicity via Attenuation of Oxidative Stress, Inflammation, and Apoptosis. 936 Oxid. Med. Cell. Longev. 2021, 8860883 (2021). 937 74. MicroRNA in the Diagnosis and Treatment of Doxorubicin-Induced Cardiotoxicity | MDPI. 938 https://www.mdpi.com/2218-273X/13/3/568. 939 75. Zhao, X., Tian, Z., Sun, M. & Dong, D. Nrf2: a dark horse in doxorubicin-induced 940 cardiotoxicity. Cell Death Discov. 9, 261 (2023). 941 76. Liang, Y. et al. Targeting the circBMPR2/miR-553/USP4 Axis as a Potent Therapeutic 942 Approach for Breast Cancer. Mol. Ther. Nucleic Acids 17, 347–361 (2019). 943 77. Ghazimoradi, M. H. & Babashah, S. The role of CircRNA/miRNA/mRNA axis in breast 944 cancer drug resistance. Front. Oncol. 12, 966083 (2022). 945 78. p53 at the Crossroads between Doxorubicin-Induced Cardiotoxicity and Resistance: A 946 Nutritional Balancing Act | MDPI. https://www.mdpi.com/2072-6643/15/10/2259. 947 79. Adamcova, M. et al. Cardiac miRNA expression during the development of chronic 948 anthracycline-induced cardiomyopathy using an experimental rabbit model. Front. 949 Pharmacol. 14, 1298172 (2023). 950 80. Feng, T. et al. Expression of miRNA-338-3p/miRNA-1250-5p/miRNA-3065-5p clusters in 951 peripheral blood mononuclear cells of ischemic stroke. Sci. Rep. 15, 11194 (2025). 952 81. Mitura-Lesiuk, M. M., Dubaj, M., Bigosi ń ski, K. & Raniewicz, M. The Role of miRNAs as 953 Predictors of Acute Lymphoblastic Leukemia Chemotherapy Toxicity in Children: A 954 Systematic Review. J. Clin. Med. 14, 5869 (2025). 955 82. Ikitimur, B., Cakmak, H. A., Coskunpinar, E., Barman, H. A. & Vural, V. A. The relationship 956 between circulating microRNAs and left ventricular mass in symptomatic heart failure 957 patients with systolic dysfunction. Kardiol. Pol. 73, 740–746 (2015). 958 83. Wang, Q. et al. The biomarkers of key miRNAs and target genes associated with acute 959 myocardial infarction. PeerJ 8, e9129 (2020). 960 84. Zhu, W.-S. et al. Targeting EZH1 and EZH2 contributes to the suppression of fibrosis-961 associated genes by miR-214-3p in cardiac myofibroblasts. Oncotarget 7, 78331–78342 962 (2016). 963 85. Lee, J. & Moon, J. H. Targeting Cardiac Fibrosis in Diabetic Heart Failure: The Role of the 964 EZH2, AMPK, and PPAR-γ Pathways (Diabetes Metab J 2024;48:716-29). Diabetes Metab. 965 J. 48, 1176–1178 (2024). 966 86. Brackertz, M., Gong, Z., Leers, J. & Renkawitz, R. p66alpha and p66beta of the Mi-2/NuRD 967 complex mediate MBD2 and histone interaction. Nucleic Acids Res. 34, 397–406 (2006). 968 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 13, 2026. ; https://doi.org/10.64898/2026.02.11.705398doi: bioRxiv preprint 87. Yamada, Y. et al. Cardiac Reprogramming and Gata4 Overexpression Reduce Fibrosis and 969 Improve Diastolic Dysfunction in Heart Failure With Preserved Ejection Fraction. Circulation 970 151, 379–395 (2025). 971 88. Dai, W. et al. Epigenetics-targeted drugs: current paradigms and future challenges. Signal 972 Transduct. Target. Ther. 9, 332 (2024). 973 89. Jin, D. & Han, F. FOXF1 ameliorates angiotensin II-induced cardiac fibrosis in cardiac 974 fibroblasts through inhibiting the TGF-β 1/Smad3 signaling pathway. J. Recept. Signal 975 Transduct. Res. 40, 493–500 (2020). 976 90. Jin, D. & Han, F. FOXF1 ameliorates angiotensin II-induced cardiac fibrosis in cardiac 977 fibroblasts through inhibiting the TGF-β 1/Smad3 signaling pathway. J. Recept. Signal 978 Transduct. Res. 40, 493–500 (2020). 979 91. Black, M. et al. FOXF1 Inhibits Pulmonary Fibrosis by Preventing CDH2-CDH11 Cadherin 980 Switch in Myofibroblasts. Cell Rep. 23, 442–458 (2018). 981 92. Lu, C., Yang, Y., Zhu, Y., Lv, S. & Zhang, J. An Intervention Target for Myocardial Fibrosis: 982 Autophagy. BioMed Res. Int. 2018, 6215916 (2018). 983 93. Chen, Y., Wang, Z., Ma, Q. & Sun, C. The role of autophagy in fibrosis: Mechanisms, 984 progression and therapeutic potential (Review). Int. J. Mol. Med. 55, 1–16 (2025). 985 94. Tao, H. et al. Histone deacetylases in cardiac fibrosis: Current perspectives for therapy. 986 Cell. Signal. 26, 521–527 (2014). 987 95. Gillette, T. G. HDAC Inhibition in the Heart: Erasing Hidden Fibrosis. Circulation 143, 1891–988 1893 (2021). 989 96. Zhou, H. et al. ZNF281 enhances cardiac reprogramming by modulating cardiac and 990 inflammatory gene expression. Genes Dev. 31, 1770–1783 (2017). 991 97. Xie, S. et al. Noncoding RNA-associated competing endogenous RNA networks in 992 trastuzumab-induced cardiotoxicity. Non-Coding RNA Res. 9, 744–758 (2024). 993 98. Wu, Y. et al. Pax8 plays a pivotal role in regulation of cardiomyocyte growth and 994 senescence. J. Cell. Mol. Med. 20, 644–654 (2016). 995 99. O’Sullivan, E. D. et al. Indian Hedgehog release from TNF-activated renal epithelia drives 996 local and remote organ fibrosis. Sci. Transl. Med. 15, eabn0736 (2023). 997 100. Wang, X.-M., Liu, X.-M., Wang, Y. & Chen, Z.-Y. Activating transcription factor 3 (ATF3) 998 regulates cell growth, apoptosis, invasion and collagen synthesis in keloid fibroblast through 999 transforming growth factor beta (TGF-beta)/SMAD signaling pathway. Bioengineered 12, 1000 117–126 (2021). 1001 101. Niu, N., Miao, H. & Ren, H. Effect of miR-182-5p on apoptosis in myocardial infarction. 1002 Heliyon 9, e21524 (2023). 1003 102. Pagan, J., Seto, T., Pagano, M. & Cittadini, A. Role of the Ubiquitin Proteasome System 1004 in the Heart. Circ. Res. 112, 1046–1058 (2013). 1005 103. Yan, K. et al. The role of K63‐ linked polyubiquitination in cardiac hypertrophy. J. Cell. 1006 Mol. Med. 22, 4558–4567 (2018). 1007 104. Willis, M. S., Schisler, J. C. & Patterson, C. Appetite for destruction: E3 ubiquitin-ligase 1008 protection in cardiac disease. Future Cardiol. 4, 65–75 (2008). 1009 105. Park, Y.-Y. et al. FOXM1 mediates Dox resistance in breast cancer by enhancing DNA 1010 repair. Carcinogenesis 33, 1843–1853 (2012). 1011 106. Bolte, C. et al. Expression of Foxm1 transcription factor in cardiomyocytes is required for 1012 myocardial development. PloS One 6, e22217 (2011). 1013 107. Zhang, Z., Li, M., Sun, T., Zhang, Z. & Liu, C. FOXM1: Functional Roles of FOXM1 in 1014 Non-Malignant Diseases. Biomolecules 13, (2023). 1015 108. Kalozoumi, G., Yacoub, M. & Sanoudou, D. MicroRNAs in heart failure: Small molecules 1016 with major impact. Glob. Cardiol. Sci. Pract. 2014, 79–102 (2014). 1017 109. Chen, J. et al. Inhibition of cyclin-dependent kinase 7 mitigates doxorubicin cardiotoxicity 1018 and enhances anticancer efficacy. Cardiovasc. Res. 120, 1024–1036 (2024). 1019 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 13, 2026. ; https://doi.org/10.64898/2026.02.11.705398doi: bioRxiv preprint 110. Fradley, M. G. et al. Adverse Cardiovascular Events Associated With Cyclin ‐ Dependent 1020 Kinase 4/6 Inhibitors in Patients With Metastatic Breast Cancer. J. Am. Heart Assoc. 12, 1021 e029361 (2023). 1022 111. Gallo, S., Sala, V., Gatti, S. & Crepaldi, T. HGF/Met Axis in Heart Function and 1023 Cardioprotection. Biomedicines 2, 247–262 (2014). 1024 112. Gallo, S. et al. Activation of the MET receptor attenuates doxorubicin-induced 1025 cardiotoxicity in vivo and in vitro. Br. J. Pharmacol. 177, 3107–3122 (2020). 1026 113. Gallo, S., Sala, V., Gatti, S. & Crepaldi, T. HGF/Met Axis in Heart Function and 1027 Cardioprotection. Biomedicines 2, 247–262 (2014). 1028 114. Sato, T. & Takeda, N. The roles of HIF-1 α signaling in cardiovascular diseases. J. 1029 Cardiol. 81, 202–208 (2023). 1030 115. Lax, A. et al. Silencing of microRNA-106b-5p prevents doxorubicin-mediated 1031 cardiotoxicity through modulation of the PR55α /YY1/sST2 signaling axis. Mol. Ther. Nucleic 1032 Acids 32, 704–720 (2023). 1033 116. Ma, Y., Grootaert, M. O. J. & Sewduth, R. N. Cardiotoxicity of Chemotherapy: A Multi-1034 OMIC Perspective. J. Xenobiotics 15, 9 (2025). 1035 117. Hao, C. et al. Overexpression of GATA4 enhances the antiapoptotic effect of exosomes 1036 secreted from cardiac colony-forming unit fibroblasts via miRNA221-mediated targeting of 1037 the PTEN/PI3K/AKT signaling pathway. Stem Cell Res. Ther. 11, 251 (2020). 1038 118. Gallo, S., Sala, V., Gatti, S. & Crepaldi, T. HGF/Met Axis in Heart Function and 1039 Cardioprotection. Biomedicines 2, 247–262 (2014). 1040 119. Thakur, A. et al. The mini player with diverse functions: extracellular vesicles in cell 1041 biology, disease, and therapeutics. Protein Cell 13, 631–654 (2022). 1042 120. Ronan, G. et al. “Comprehensive multi-omics of age-respective plasma and matrix-1043 bound extracellular vesicles identifies anti-fibrotic miRNAs validated on a heart-on-a-chip”. 1044 Biomaterials 330, 124031 (2026). 1045 121. Lennaárd, A. J., Mamand, D. R., Wiklander, R. J., El Andaloussi, S. & Wiklander, O. P. 1046 B. Optimised Electroporation for Loading of Extracellular Vesicles with Doxorubicin. 1047 Pharmaceutics 14, 38 (2021). 1048 122. Murabito, A., Hirsch, E. & Ghigo, A. Mechanisms of Anthracycline-Induced 1049 Cardiotoxicity: Is Mitochondrial Dysfunction the Answer? Front. Cardiovasc. Med. 7, 35 1050 (2020). 1051 123. Rocca, C. et al. Mitochondrial dysfunction at the crossroad of cardiovascular diseases 1052 and cancer. J. Transl. Med. 21, 635 (2023). 1053 124. Wallace, K. B., Sardão, V. A. & Oliveira, P. J. Mitochondrial determinants of doxorubicin-1054 induced cardiomyopathy. Circ. Res. 126, 926–941 (2020). 1055 https://doi.org/10.1161/CIRCRESAHA.119.314681 1056 125. Ellis, B. W., Acun, A., Can, U. I. & Zorlutuna, P. Human iPSC-derived myocardium-on-1057 chip with capillary-like flow for personalized medicine. Biomicrofluidics 11, 024105 (2017). 1058 126. Basara, G., Celebi, L. E., Ronan, G., Discua Santos, V. & Zorlutuna, P. 3D bioprinted 1059 aged human post-infarct myocardium tissue model. Health Sci. Rep. 7, e1945 (2024). 1060 127. Harahap, Y., Ardiningsih, P., Corintias Winarti, A. & Purwanto, D. J. Analysis of the 1061 Doxorubicin and Doxorubicinol in the Plasma of Breast Cancer Patients for Monitoring the 1062 Toxicity of Doxorubicin. Drug Des. Devel. Ther. 14, 3469–3475 (2020). 1063 128.Sikora, T., Morawska, K., Lisowski, W., Rytel, P. & Dylong, A. Application of Optical 1064

for Determination of Concentration of Doxorubicin in Blood and Plasma. 1065 Pharmaceuticals 15, (2022). 1066 129. Théry, C. et al. Minimal information for studies of extracellular vesicles 2018 1067 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and 1068 update of the MISEV2014 guidelines. J. Extracell. Vesicles 7, 1535750 (2018). 1069 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 13, 2026. ; https://doi.org/10.64898/2026.02.11.705398doi: bioRxiv preprint 130. Basara, G., Ozcebe, S. G., Ellis, B. W. & Zorlutuna, P. Tunable Human Myocardium 1070 Derived Decellularized Extracellular Matrix for 3D Bioprinting and Cardiac Tissue 1071 Engineering. Gels Basel Switz. 7, 70 (2021). 1072 131. Basara, G. et al. Electrically conductive 3D printed Ti3C2Tx MXene-PEG composite 1073 constructs for cardiac tissue engineering. Acta Biomater. 139, 179–189 (2022). 1074 132. Rampersad, S. N. Multiple applications of Alamar Blue as an indicator of metabolic 1075 function and cellular health in cell viability bioassays. Sensors 12, 12347–12360 (2012). 1076 133. Celebi, L. E. & Zorlutuna, P. 3D bioprinted fat-myocardium model unravels the role of 1077 adipocyte hypertrophy in atrial dysfunction. Adv. Sci. 13, 202516114 (2026). 1078 https://doi.org/10.1002/advs.202516114 1079 134. Ronan, G., Bahcecioglu, G., Yang, J. & Zorlutuna, P. Cardiac tissue-resident vesicles 1080 differentially modulate anti-fibrotic phenotype by age and sex through synergistic miRNA 1081 effects. Biomaterials 311, 122671 (2024). 1082 1083 1084

1085 The lyophilization of decellularized ECM was conducted at the Center for Environmental 1086 Science and Technology (CEST) at the University of Notre Dame. We thank the Biophysics 1087 Instrumentation (BIC) Core Facility for the use of Optima MAX-XP Tabletop Ultracentrifuge. The 1088 Nanoparticle Tracking Analysis was conducted using the NanoSight NS300 at the Harper 1089 Cancer Research Institute (HCRI) Tissue Core Facility. We thank the Flores-Mireles Lab at the 1090 University of Notre Dame for providing access to the NanoString equipment used in this study. 1091 The schematics in some figures were created using BioRender.com 1092 Funding: Research reported in this publication was supported by NSF-CAREER Award # 1093 1651385, NSF CBET Award # 1805157 and NIH Award # 1 R01 HL141909-01A1 1094 Competing Interests Statement: The authors have no competing interest to disclose. 1095 Data Availability Statement: All data required for production of the manuscript is included in 1096 this submission. Additional raw data can be provided upon request. 1097 Ethics Statement: This study was conducted in accordance with the principles of the 1098 Declaration of Helsinki. Institutional Review Board approval was not required, as the University 1099 of Notre Dame Research Compliance office determined that the work does not constitute human 1100 subjects research because the Indiana Donor Network supplies samples without any identifying 1101 information. 1102 1103 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 13, 2026. ; https://doi.org/10.64898/2026.02.11.705398doi: bioRxiv preprint