Timing of Changes in Heart Tissue Structure in Chemotherapy-Related Heart Damage

Acute AIC typically occurs within hours to two weeks following drug administration and it is relatively rare (<1%) and generally reversible [9,11,17]. Clinically, it may manifest as transient arrhythmias including supraventricular tachycardia, premature ventricular contractions, or atrioventricular conduction delays as well as acute pericarditis or reversible left ventricular dysfunction. In most cases, these effects are self-limiting, resolve upon discontinuation of the drug, and are not dose dependent [29,54,55,56,57]. However, their presence may serve as an early indicator of future cardiotoxicity, emphasizing the importance of thorough cardiovascular monitoring. Acute myocarditis, which can imitate or co-occur with these conditions, is more likely to occur in patients with pre-existing cardiovascular disease or those receiving multi-agent chemotherapy [8,58,59].

Acute cardiotoxicity is characterized by transient reductions in LVEF and a spectrum of arrhythmias, including atrial fibrillation, atrial tachycardia, atrioventricular nodal tachycardia, and premature ventricular contractions [29,54,55,56,57]. Common electrophysiologic abnormalities frequently include sinus tachycardia, repolarization changes, prolonged QT intervals, and reduced QRS voltages [60]. A prospective cohort study of breast cancer patients treated with anthracycline reported that approximately one-third of participants developed acute therapy-related cardiac dysfunction, highlighting the cardiotoxic potential of this regimen [61]. In this study, transthoracic echocardiography was utilized during treatment to detect early functional changes, particularly reductions in LVEF and GLS [61]. CMR demonstrated that early declines in LVEF did not correlate with an increased incidence of symptomatic heart failure during long-term follow-up [61]. These changes usually are associated with transient impairments in myocardial mechanics, including reductions in GLS, indicating diffuse myocardial injury and alterations in left ventricular mass index, suggesting early myocardial remodeling [54,62].

Subacute AIC refers to an intermediate rare phase of myocardial injury that typically develops within weeks to a few months after treatment [15,50,63]. It serves as a transitional stage between the transient acute effects and the possible progression to early onset chronic cardiomyopathy. Clinically, this phase is often asymptomatic or represented as indistinct chest pain [50], characterized by subclinical reductions in LVEF detectable only through sensitive imaging modalities such as global longitudinal strain (GLS) or via biomarker surveillance [11].

In a reported case, a 25-year-old male developed subacute cardiotoxicity 17 days after receiving anthracycline therapy and presented with transient left ventricular (LV) wall thickening on echocardiography that resolved within one month [50]. Despite preserved systolic function, the patient exhibited significantly reduced early diastolic velocities, suggesting isolated diastolic dysfunction as a subclinical manifestation of myocardial injury. Although the cardiac abnormalities were resolved without progressing to chronic dysfunction, this case emphasizes the potential utility of diastolic functional changes as an early indicator of anthracycline-related myocardial stress [50].

If myocardial dysfunction in this phase left unrecognized or unmanaged, persistent myocardial injury can progress into early onset chronic cardiotoxicity most commonly presenting as dilated cardiomyopathy within the first-year post–anthracycline exposure [17].

Early onset chronic anthracycline-induced cardiotoxicity is the most observed clinical presentation, typically manifesting within the first year after treatment completion [9,17]. Cardinale et al. have suggested that the distinction between early and late-onset chronic cardiotoxicity may be misleading, as many cases historically labeled as chronic could have been detected earlier with modern tools and possibly reversed with timely cardioprotective therapy [11,51]. While this perspective emphasizes the value of early detection and intervention, it is equally important not to overlook the distinct subacute phase and early chronic phase. Recognition of these subclinical windows is critical, as they represent a pathophysiologically active yet clinically silent phases during which timely interventions may prevent permanent cardiac damage and improve long-term outcomes.

This phase is clinically characterized by asymptomatic reductions in LVEF within the first-year post-treatment, and accounts for most cases in adult cohorts [59]. Despite its utility, LVEF lacks sensitivity for detecting early myocardial injury, prompting greater reliance on more sensitive imaging biomarkers. Speckle-tracking echocardiography (STE), particularly GLS, has emerged as a powerful tool for detecting subclinical myocardial dysfunction prior to LVEF decline [64,65]. A GLS value below 16% or a relative reduction >15% from baseline has been shown to signal elevated cardiotoxicity risk and may justify initiation of cardioprotective medications [11,47,51]. Diastolic dysfunction is increasingly recognized as an early marker of AIC, often preceding detectable systolic impairment [55,66,67,68].

Chronic AIC is defined by its delayed onset, often developing years to decades after completion of chemotherapy [9,11]. This prolonged latency is usually accompanied by irreversible structural and functional cardiac alterations, such as myocardial fibrosis and ventricular remodeling, and is associated with poor long-term outcomes, including increased cardiovascular mortality [51]. The risk of chronic cardiotoxicity displays a strong dose-dependent relationship, with heart failure incidence rising from a maximum of ~5% at cumulative anthracycline doses below 400 mg/m² to over 18% at doses of 700 mg/m² [8,12,69]. A sign of chronic toxicity is the progressive decline in left ventricular systolic function, which may end in dilated cardiomyopathy and symptomatic heart failure [51]. In long-term cancer survivors, subclinical indicators including impaired diastolic relaxation on echocardiography, reduced myocardial strain, and modest elevations in biomarkers are frequently detectable before overt symptoms arise [11,51].

Reductions in CMR-derived LVEF by >10%, particularly when falling below 53%, are considered indicative of therapy-related cardiac dysfunction [70]. Parametric techniques such as native T1 mapping, T2 mapping, and the extracellular volume (ECV) quantification offer unique insights into myocardial edema, diffuse fibrosis, and interstitial expansion thereby enhancing diagnostic precision and prognostic stratification [9,71]. In chronic AIC, CMR imaging has demonstrated that increased ECV fractions, indicative of diffuse interstitial fibrosis, correlate with impaired systolic and diastolic function, reduced exercise capacity, and increased long-term mortality even up to twenty years after anthracycline exposure in long-term survivors of childhood cancer [51,61,72]. These findings emphasize the need for cardiovascular surveillance, even in asymptomatic individuals, to enable early detection and intervention.

In animal models of acute anthracycline injury, myocardial histopathology is marked by dense interstitial inflammatory infiltrate composed predominantly of neutrophils, lymphocytes, and macrophages [72,79,80]. Clinical evidence aligns with these findings.

In vitro, primary CFs exposed to DOX demonstrated increased α-smooth muscle actin (α-SMA) expression and enhanced collagen synthesis, signifying early myofibroblast differentiation and enhanced ECM production [67]. Animal models have provided valuable insights into the temporal evolution of DOX-induced myocardial injury. In murine studies, the acute phase is characterized by prominent intracytoplasmic vacuolation of cardiomyocytes, particularly within the ventricles and interventricular septum, along with occasional myofiber atrophy indicating early structural disruption [72]. Myocardial cells after acute exposure showed swelling, sarcoplasmic core dilation, myofibril loss, cytoplasmic vacuolization, and enlarged nuclei with prominent nucleoli [81].

Circulating biomarkers capture the extent of this injury. High-sensitivity troponin is the earliest and most reliable indicator of acute myocyte necrosis; even modest troponin elevations identify patients at heightened risk for subsequent chronic cardiotoxicity [82]. Cardiac troponin I (cTnI) and troponin T (cTnT) are the most sensitive circulating markers of acute cardiomyocyte injury in patients receiving anthracycline chemotherapy. cTnI levels begin to rise within 24 h of each anthracycline dose and demonstrate a cumulative increase with successive cycles, reflecting ongoing subclinical myocardial injury [72,82,83]. Persistent elevation of cTnI or cTnT during or immediately after completion of therapy identifies individuals at significantly higher risk for subsequent declines in LVEF and overt cardiotoxicity [56,83,84]. These troponin elevations are often transient and may normalize if therapy is held, but their presence should prompt consideration of cardioprotective interventions (e.g., ACE inhibitors, β-blockers) [85].

Owing to the markedly accelerated course of anthracycline cardiotoxicity in animals, especially mice, histological reports of subacute injury are limited and often indistinguishable from features of acute or early onset chronic injury, a notable case report [50] detailed subacute anthracycline cardiotoxicity demonstrated that an endomyocardial biopsy performed 17 days after consolidation chemotherapy revealed marked interstitial myocardial edema without evidence of inflammatory cell infiltrates or myocyte necrosis and only minimal interstitial fibrosis suggestive of a potentially reversible stage of injury. Cellular infiltration may be a secondary response, occurring only after overt myocyte injury has taken place [50]. In a rabbit model, Bristow et al. found that subacute injury histology resembled acute phase features with predominant neutrophilic infiltration, occasional eosinophils, focal areas of myocyte degeneration, and interstitial edema [15]. On the biomarker level, case reports describe elevations in troponin and C-reactive protein (CRP) during this interval, indicating ongoing myocardial injury without full progression to chronic damage [50,86].

In contrast to the acute phase, the early onset chronic stage of DOX exposure (≥12 weeks) is characterized by more extensive myocardial remodeling. Zeiss et al. reported features including myofiber disorganization, marked variability in myofiber diameter, persistent vacuolation in some animals, and the development of fine interstitial "chicken-wire" fibrosis indicating progressive structural damage [72]. Typically, this phase is clinically silent but detectable by rising natriuretic peptides (BNP/NT-proBNP) and inflammatory markers (e.g., high-sensitivity C-reactive protein, GDF-15, sST2, MPO).

B-type natriuretic peptide (BNP) and its N-terminal fragment, NT-proBNP, are released in response to increased ventricular wall stress and can rise within days to weeks after initiation of anthracycline therapy, often before echocardiographic evidence of dysfunction appears [87]. Serial measurements of NT-proBNP during chemotherapy have demonstrated that persistently elevated or progressively increasing levels predict both early and late left ventricular dysfunction [87,88]; for example, a rise of ≥36% from baseline in NT-proBNP during treatment correlates with a significantly greater likelihood of LVEF decline at 3, 6, and 12 months post-therapy [88]. In anthracycline-treated cohorts, elevated NT-proBNP immediately post-cycle has been associated with subclinical left ventricular impairment, and patients with persistently high NT-proBNP levels exhibit worse ventricular remodeling and a higher incidence of heart failure on follow-up [87,88]. Early recognition during this window allows timely initiation of cardioprotective therapy to prevent irreversible injury [75,88].

Inflammatory and oxidative stress biomarkers have so far been characterized primarily in preclinical models and few cohort studies [75,86,89]. They also have been previously discussed in the literature [9,90,91,92,93]. However, their clinical relevance remains to be confirmed through well-designed cohort and clinical studies.

Prolonged anthracycline exposure leads to cumulative left ventricular dysfunction characterized by maladaptive remodeling. Over months to years, cardiomyocyte hypertrophy, mitochondrial impairment, and disorganization of the myocardial ECM give rise to chamber dilation and heart failure symptoms with reduced ejection fraction [12,35,47,53,94]. Histologically, this chronic stage is defined by diffuse interstitial fibrosis alongside an imbalance in matrix metalloproteinase (MMP-2/MMP-9) activity relative to their tissue inhibitors (TIMP-1, TIMP-2), promoting excessive collagen accumulation [4,20,78]. Upstream mediators such as TNF-α, IL-6, and ROS further amplify MMP expression and activation, integrating inflammatory and redox signals into the fibrotic remodeling characteristic of AIC [20,78].

The late-onset cardiotoxicity which emerges years after treatment completion is characterized by elevated novel fibrosis-related biomarkers such as PICP and galectin-3 [9,75,95]. Circulating procollagen peptides specifically the carboxy-terminal propeptide of procollagen type I (PICP) and the amino-terminal propeptide of procollagen type III (PIIINP) serve as indicators of myocardial collagen synthesis and interstitial fibrosis [96,97]. In women undergoing anthracycline-based breast cancer chemotherapy, early increases in PICP at three months post-treatment were significantly associated with subclinical reductions in LVEF and predicted development of overt cardiotoxicity at one year, whereas NT-proBNP alone showed less prognostic value [95]. PIIINP rise typically occurs after established ventricular dysfunction, limiting its utility for early detection [96].

In the healthy myocardium, CFs represent the predominant non-myocyte cell type alongside endothelial cells, vascular smooth muscle cells, and transient leukocytes and are indispensable for maintaining ECM integrity and providing mechanical support [21,98,99]. Although early investigations of DOX cardiotoxicity concentrated on direct cardiomyocyte injury as the main pathogenesis [6,37], emerging evidence has expanded the view by implicating the direct involvement of CFs and non-myocytes cellular constituents in AIC affecting fibroblast function, disrupting ECM homeostasis, provoking inflammatory signaling, driving maladaptive remodeling and in the initiation and propagation of chronic myocardial damage [21,23,100]. Collectively, these findings support the notion that fibroblast-driven mechanisms may be central to both the development and progression of long-term anthracycline cardiotoxicity.

The mechanism by which anthracyclines, particularly DOX, adversely affect CFs closely parallels their antitumor activity. Cardiac fibroblasts are highly susceptible to doxorubicin-induced oxidative stress, primarily mediated by ROS generated through mitochondrial complex I [101]. The produced lipid peroxidation, protein oxidation, and activation of stress-responsive kinases such as p38 MAPK drive apoptosis, cellular senescence, and phenotypic reprogramming toward a pro-inflammatory and profibrotic state [21,67,94].

Alteration in mitochondrial metabolism including cardiolipin remodeling have been proposed not only as a potential mechanism of AIC but also underlying sex-related differences [102]. The influence of sex and age on AIC remains incompletely characterized. Nevertheless, accumulating evidence from experimental and retrospective cohort studies indicates that these variables are major modifiers of susceptibility and should be explicitly integrated into preclinical study design, patient risk stratification, and the development of cardioprotective strategies [102,103,104,105,106]. Most experimental data in pediatric oncology suggest a higher incidence of AIC among females compared with males, whereas limited adult data raise the possibility of a sex-related protective effect in women [105].

Sex-related disparities extend to metabolic and structural outcomes under cumulative anthracycline exposure. Male rodents exhibit greater weight loss, reduced survival, pronounced myocardial dysfunction, impaired energy signaling, decreased mitochondrial biogenesis, lower cardiolipin content, diminished mitochondrial DNA, and altered mitochondrial respiration, accompanied by a stronger upregulation of myocardial stress biomarkers compared with females [104].

ECM remodeling also appears to contribute to these differences; although fibrosis surrounding cardiac muscle cells is comparable between sexes, total and reactive fibrosis are significantly greater in males, suggesting a sex-dependent propensity for adverse fibrotic remodeling [104]. These findings underscore the need for standardized diagnostic criteria and prospective studies specifically designed to study sex-based differences in AIC.

Age further modulates anthracycline toxicity. Preclinical studies consistently demonstrate a dose-dependent escalation of cardiotoxicity with advancing age. In an animal model, older rats (24 months) displayed markedly greater vulnerability to anthracycline-induced myocardial injury than younger animals (6 months) exposed to comparable doses [103].

Recent sequential studies by Ishikawa group [22] have demonstrated that sublethal concentrations of DOX induce trans-differentiation of human cardiac fibroblasts (HCFs) into myofibroblasts, characterized by increased expression of α-SMA, matrix metalloproteinase-1 (MMP-1), interleukin-6 (IL-6), and collagen. These changes are mediated via activation of the PI3K/AKT and TGF-β/Smad signaling pathways [22]. In that experiment comparing HCFs and normal human dermal fibroblasts (NHDFs), the α-SMA-mediated trans-differentiation into myofibroblasts is specific to HCFs and is absent in NHDFs except after direct TGF-β stimulation, indicating a greater susceptibility of HCFs to doxorubicin-induced fibrotic remodeling [22]. Further supporting the role of CFs in DOX-induced cardiotoxicity, these studies show that systemic administration of DOX leads to activation of fibrotic signaling pathways in CFs, including upregulation of Toll-like receptor 9, interleukin-1 (IL-1), and fibrotic markers such as galectin-3, α-SMA, and tissue inhibitor of metalloproteinases-1 (TIMP1) [21,22,94]. Skaggs et al. used proteomic profiling to show that DOX-treated primary CFs exhibit impaired ECM maintenance, marked by downregulation of ECM-regulatory genes including Col4a1, Col4a2, Col5a1, Mmp11, Mmp14, and TGFβ, indicating disruption of basement membrane integrity, matrix organization and augmented myofibroblast differentiation [23].

Transforming growth factor-beta 1 (TGF-β1) is a central mediator in the conversion of fibroblasts to myofibroblasts and drives collagen synthesis within the myocardium. In models of anthracycline-induced cardiomyopathy, TGF-β1 and its downstream effector, SMAD3, are upregulated two- to four-fold at both early and late time points in the course of the disease [20]. In a meta-analysis of AIC in animal models, Leerink et al. reported that connective tissue growth factor (CTGF), a canonical TGF-β1 target, peaks shortly after anthracycline exposure and remains elevated during long-term follow-up [20]. Additionally, transcripts encoding fibroblast activation markers such as osteonectin and tenascin-C are upregulated in large-animal studies [20,98,107]. These findings, along with persistent upregulation of collagen types I and III and progressive interstitial fibrosis, underscore the enduring nature of pro-fibrotic signaling in anthracycline-induced cardiomyopathy [108]. DOX elicits an early inflammatory cytokine response in CFs, followed by late-phase collagen production, thereby driving the sequential stages of anthracycline-induced myocardial remodeling [67]. DOX prompts HCFs to secrete pro-inflammatory cytokines and pro-fibrotic growth factors. Specifically, IL-6 and TGF-β mRNA rises in a time-dependent manner over the first 3–6 h of DOX stimulation [22]. DOX stimulates inflammatory and fibrotic pathways in HCFs via both PI3K/AKT and TGF-β/Smad signaling cascades, as evidenced by time-dependent phosphorylation of Smad2 and AKT following DOX exposure. Moreover, HCFs demonstrate a progressive increase in Col1a1 transcript levels across 72 h of DOX treatment [22]. Inhibition of TGF-β/Smad signaling attenuates DOX-induced IL-6 mRNA expression and α-SMA protein levels, while blockade of PI3K selectively suppresses MMP-1 protein upregulation in HCFs [22].

AKT, a serine/threonine kinase, plays a pivotal role in regulating cardiomyocyte survival, growth and function. Activation of the PI3K/AKT pathway is essential for normal cardiac growth, as well as for the heart's adaptive responses to stress [109,110,111]. Transient activation of AKT has been shown to confer cardioprotection by inhibiting apoptosis and inducing adaptive functional hypertrophy characterized by preserved contractile function [112,113], While prolonged AKT signaling results in extensive cardiac hypertrophy, interstitial fibrosis, and impaired contractile function [112]. These pathological changes are associated with a mismatch between cardiomyocyte growth and angiogenesis, leading to reduced capillary density and subsequent cardiac dysfunction. In experimental models of anthracycline-induced cardiomyopathy, a decrease in phosphorylated AKT levels has been observed, indicating compromised pro-survival signaling. This reduction in AKT activity undermines the heart's endogenous protective mechanisms, exacerbating ECM perturbations and contributing to the progression of cardiac injury [20,114].

In addition to TGFβ and AKT signaling changes, DOX also induces the generation of ROS, leading to oxidative stress and mitochondrial dysfunction [38]. This oxidative stress activates p53, a tumor suppressor protein that regulates cell cycle arrest and apoptosis [115,116]. In cardiac fibroblasts, p53 activation contributes to cellular senescence and the senescence-associated secretory phenotype (SASP), which includes the secretion of pro-inflammatory cytokines and pro-fibrotic factors [37,117]. These processes further exacerbate myocardial remodeling and fibrosis [37,118]. Using primary cardiac fibroblasts isolated from murine hearts, Mancilla and colleagues demonstrated that doxorubicin markedly suppresses fibroblasts expansion, reducing cell confluence by nearly 50% relative to untreated controls within 72 h in both wild type and P53 -/- cells [118]. Time-course analysis indicated that by 36 h the loss of confluence reflected predominantly cell death rather than reduced proliferation. In addition, doxorubicin reduced migration in wild-type fibroblasts, whereas genetic deletion of p53 restored migratory capacity. Wild-type cells exhibited a significant shift in cell-cycle distribution, consistent with arrest at the p53-dependent G1/S checkpoint, while p53 -/- fibroblasts did not display the same arrest, indicating that their decreased proliferation arises via alternative mechanisms. Beyond these functional effects, the study revealed a profound disruption of mitochondrial quality control. Doxorubicin markedly increased p53 protein levels and promoted its cytosolic accumulation increasing P53/parkin interaction. This prevented Parkin from localizing to damaged mitochondria, effectively blocking mitophagy, leading to mitochondrial dysfunction and accumulation of reactive oxygen species [118]. In addition, previous in vitro studies have demonstrated that doxorubicin markedly reduces cardiac fibroblast numbers predominantly by inhibiting proliferation rather than inducing cell death. Cell counts at 24, 48 and 96 h post-exposure show a pronounced reduction in proliferation, accompanied by significant downregulation of cyclin D1, consistent with doxorubicin-induced G₁/S cell-cycle arrest in fibroblasts [119].

The cardiac ECM is a dynamic and intricate network composed primarily of fibrillar collagens (types I and III), along with proteoglycans, glycoproteins, and glycosaminoglycans, which together provide structural and biochemical support to the myocardium [120]. In addition to acting as a fibrous scaffold, the ECM serves essential mechanobiological roles, facilitating force transmission, limiting myocardial overstretch, and preserving the structural integrity of cardiomyocytes and the intramyocardial vasculature [98,99,120]. Cardiac fibroblasts are central regulators of ECM homeostasis, capable of sensing biomechanical and biochemical cues and modulating the synthesis and degradation of ECM components accordingly [98,99]. However, this regulatory system becomes profoundly disrupted upon exposure to anthracyclines such as DOX, which induces dose-dependent cardiotoxicity characterized by cardiomyocyte loss, interstitial fibrosis, and exaggerated inflammatory signaling, ultimately leading to maladaptive cardiac remodeling [6,22,67,94,121].

DOX-induced oxidative stress and inflammatory signaling activate matrix metalloproteinases, enzymes that degrade ECM components, promote myofibroblast differentiation, and drive excessive collagen deposition (Figure 1) [22,67,94,118]. Disruption of ECM integrity, whether through excessive proteolysis or unrestrained matrix accumulation, destabilizes the myocardial architecture [122]. Excess collagen leads to interstitial fibrosis and vascular encasement, while matrix degradation weakens the collagen scaffold, impairing systolic and diastolic function and disrupting electrical synchrony [123]. Notably, anthracycline-induced ECM remodeling is temporally heterogeneous. Acute DOX exposure is associated with transient fibroblast activation and matrix degradation, whereas chronic exposure leads to persistent myofibroblast differentiation, pathological collagen accumulation, and irreversible fibrotic remodeling [22,23,53,94,95]. In vitro, DOX elicits a temporal response in HCFs; within 3–6 h, there is an increase in IL-6, IL-1β, and TGF-β mRNA expression, followed by upregulation of α-SMA mRNA at 24 h and α-SMA protein accumulation by 48 h culminating in collagen deposition [22,94].

Matrix metalloproteinases (MMPs) constitute a family of zinc-dependent endopeptidases that play pivotal roles in the degradation and remodeling of the ECM. These enzymes are integral to both physiological processes such as embryonic development, angiogenesis, and wound healing and pathological conditions, including tumor invasion, atherosclerosis, myocardial infarction, and heart failure [77]. In the context of cardiac tissue, several MMPs have been identified, including MMP-1, MMP-2, MMP-7, MMP-9, and MMP-14 [77,124]. Each of these enzymes exhibits distinct substrate specificities and regulatory mechanisms, contributing variably to ECM turnover and cardiac remodeling. While some MMPs are non-specific to the heart, MMP-1, 2 and 9 are directly involved with AIC [20,22,23,77,78].

MMP-1, also known as interstitial collagenase, is primarily responsible for initiating the degradation of fibrillar collagens, particularly types I and III; the principal structural proteins of the cardiac ECM [124]. Its overexpression has been implicated in pathological cardiac remodeling as it contributes to excessive ECM degradation, undermining myocardial structural integrity and promoting ventricular dilation [76]. Experimental models have shown that upregulation of MMP-1 is associated with the development of cardiac hypertrophy and progression to decompensated heart failure [122,125,126]. DOX exposure increased MMP-1 expression through the activation of the PI3K/Akt signaling pathway [22]. This abnormal upregulation of MMP-1 in HCFs contributes to ECM destabilization, fibroblast activation, and ultimately the fibrotic and functional decline observed in AIC [22].

MMP-2 and 9, also known as gelatinases, are fundamental to cardiac ECM turnover and remodeling and play dual roles (protective or pathogenic) depending on their activation timing and cellular localization [77,124]. MMP-2 is constitutively expressed in cardiomyocytes and localizes to subcellular compartments including sarcomeres, mitochondria, nuclei, and cytoskeleton, where it contributes to both normal cell signaling and structural integrity [77,78]. Anthracycline exposure, particularly to DOX, induces oxidative stress that activates latent MMP-2 and MMP-9, increasing their enzymatic activity and driving pathologic remodeling [77,94]. DOX also triggers de novo synthesis of MMP-2 and upregulates a pathological splice variant known as N-terminal truncated MMP-2 (NTT-MMP-2), which lacks the mitochondrial targeting sequence [127,128]. This variant promotes mitochondrial dysfunction, activates nuclear factor κB (NF-κB), and increases the expression of interleukin-6 (IL-6), exacerbating myocardial inflammation and injury [67]. Once activated, MMP-2 and MMP-9 degrade key contractile proteins such as troponin I and myosin light chain-1, leading to impaired sarcomeric integrity and reduced cardiomyocyte contractility [129]. Animal models have demonstrated that even a single dose of DOX significantly reduces LVEF and fractional shortening, with early diastolic dysfunction evident alongside increased myocardial MMP activity [67].

Acute DOX exposure rapidly disrupts ECM homeostasis via a combination of transcriptional regulation, oxidative stress, and inflammatory signaling in CFs. A key driver of this phenotype is DOX-mediated redox cycling, which generates excessive reactive oxygen species (ROS), leading to mitochondrial dysfunction, lipid peroxidation, and DNA damage. These ROS also activate p38 MAPK and JNK/NAD(P)H oxidase pathways, resulting in the early upregulation of MMP-2 and MMP-9 and subsequent ECM breakdown [42,126,130,131] (Table 2). Within four weeks after a single therapeutic dose of DOX, myocardial cytoskeletal proteins including tubulin, fibronectin, and myosin light-chain kinase are markedly reduced, although total interstitial collagen volume remains unchanged. This suggests that early subcellular remodeling proceeds overt fibrosis [132]. Concurrently, DOX elicits ROCK1-dependent actin cytoskeletal reorganization in CFs, leading to cell shrinkage, detachment, and apoptosis [133].

Experimental studies showed that DOX downregulates cellular communication network factor 2 (CCN2) in both NIH3T3 embryonic fibroblasts and primary CFs indicating a potential for early antifibrotic activity [100]. Consistent with this observation, proteomic and transcriptomic profiling of primary murine CFs treated with 1 μM DOX for 24 h demonstrated acute downregulation of extracellular matrix (ECM)-related genes, indicating compromised basement membrane integrity and matrix cohesion [23]. These transcriptional changes coincide with reduced collagen deposition and gross ECM destabilization in vivo. In parallel, increased activities of MMP-1, -2, and -9 contribute to myocardial fibrosis, collagen disarray, and cytoplasmic vacuolization [4,22,76,131].

In an in vitro study, CFs exhibited a shift toward a myofibroblast phenotype within 24 h of DOX exposure, characterized by early induction of α-SMA and subsequent collagen synthesis, driven by autocrine and paracrine TGF-β signaling and SMAD2 phosphorylation [67]. This myofibroblast activation spreads a pro-fibrotic loop, while ECM stiffening alters mechano-electrical coupling, potentially promoting arrhythmogenesis [134,135,136]. Finally, NF-κB signaling is triggered early, raising TNF-α and IL-6 levels a response that can be attenuated by FGF21 by reducing NF-κB p65 nuclear translocation [137].

Although infrequently characterized, the subacute phase marks a transitional period where CFs begin to exhibit a pro-inflammatory and proteolytic phenotype in response to anthracycline exposure. During this phase, increased vascular permeability and interstitial edema arise, driven by a Toll-like receptor 2 (TLR2)-mediated inflammatory response. DOX activates TLR2 signaling in endothelial cells, upregulating pro-inflammatory cytokines such as TNF-α and IL-6. [50,138]. This inflammatory amplification, while less extensively studied than in the acute or chronic phases, likely contributes to ongoing ECM degradation and fibroblast activation, setting the stage for persistent tissue remodeling and fibrotic progression. The interplay between endothelial cells and CFs during this phase reinforces cytokine production, which may exacerbate myocardial inflammation, promote myofibroblast differentiation, and prime the heart for long-term structural remodeling.

This transitional phase represents the evolution of acute injury into chronic myocardial damage [12]. It marks a range from the initial injury to the sustained structural remodeling. During this period, sustained cardiac stressors prompt CFs to undergo differentiation into α-SMA-positive myofibroblasts, reinforcing a self-amplifying fibrotic loop [139]. DOX-exposed CFs exhibit increased secretion of TGF-β, driving SMAD2 phosphorylation and sustained myofibroblast activation [22,67,139] (Table 2). Two weeks following DOX administration, isolated CFs retain this activated phenotype, with upregulated CTGF, galectin-3, MMP-2/9, and α-SMA in tissue lysates, histology and immunoblotting, confirming ongoing matrix deposition [67]. Concomitant increases in fibronectin and focal adhesion proteins further strengthen cell–matrix anchoring typical of the activated state [132]. DOX also elevates MMP-1 expression and activity in HCFs via PI3K/Akt signaling—an effect not seen in dermal fibroblasts suggesting simultaneous degradative remodeling that may weaken the ECM scaffold [22,94]. Together, these converging molecular and cellular mechanisms, including suppressed ECM homeostasis, enhanced proteolytic activity, persistent fibroblast activation, and amplified inflammation, define the early chronic ECM injury landscape. This stage lays the groundwork for irreversible myocardial remodeling if left unchecked, highlighting the need for timely intervention to mitigate long-term AIC.

Chronic anthracycline cardiotoxicity manifesting weeks in animals [108,114,140] to years in humans [141] after treatment is characterized by sustained ECM remodeling, progressive interstitial fibrosis, and declining ventricular function. Over time, CFs adopt a stable myofibroblast phenotype, continuously secreting type I and III collagens, fibronectin and periostin leading to matrix accumulation, increased myocardial stiffness, and impaired diastolic relaxation [67,98,107,134]. This persistent fibrogenic state is maintained by chronically upregulated intracellular pathways in CFs such as PI3K/Akt, MAPK, and TGF-β/Smad signaling remain activated, promoting myofibroblast survival and ECM gene transcription. CFs isolated from chronically DOX-treated hearts exhibit elevated α-SMA and collagen expression, sustained by autocrine TGF-β release and SMAD2/3 phosphorylation [22,100,142]. Oxidative stress via NADPH oxidases is central to chronic remodeling [39,143,144]. NOX2 expression stays high in DOX-treated CFs, driving persistent ROS production, pathological cross-linking of collagen fibers, and heightened fibrosis [144]. Genetic deletion or pharmacologic inhibition of NOX2 or NOX4 in animal models alleviates ECM deposition and preserves cardiac function, underscoring their key role in chronic anthracycline injury [143,144].

MMP dynamics shift during chronic injury; although initial MMP upregulation facilitates acute ECM turnover, chronic DOX exposure leads to sustained MMP-9 overexpression and activity, driving maladaptive remodeling and interstitial fibrosis [114]. In rodent models, plasma MMP-2/9 activities rise by four weeks post-DOX, with left ventricular MMP-2 activation by eight weeks changes that correlate with Akt activation, superoxide dismutase inhibition, and caspase-3 cleavage [114]. Chronic DOX also impairs cellular quality control; autophagy and mitophagy are suppressed, leading to mitochondrial dysfunction, ATP deficits, and further ROS generation that exacerbate ECM deposition [48,118] (Table 2). In a chronic rabbit model of daunorubicin-induced cardiomyopathy, proteomic analyses have revealed significant alterations in basement membrane proteins, including collagen types I and III, and extracellular matrix constituents. These molecular changes align with increased interstitial fibrosis and MMP-2 upregulation, reinforcing the progression of adverse cardiac remodeling [108]. Ultimately, these chronic molecular and structural insults result in an irreversible ECM signature, typified by excessive collagen cross-linking, loss of myocardial compliance, and functional deterioration. This maladaptive remodeling process drives the transition from subclinical myocardial dysfunction to overt heart failure in long-term anthracycline recipients.