Revolutionizing cardiac fibrosis treatment: the potential of personalized CAR T-cell therapy

Abstract

Cardiac fibrosis, a condition characterized by the deposition of excess collagen in the cardiac tissue, is a major complication of various cardiovascular diseases, including myocardial infarction, hypertension, and different types of cardiomyopathies. CAR T-cell therapy, a form of immunotherapy that involves the genetic modification of T cells to recognize and target specific antigens, has shown promise in the treatment of various cancers and autoimmune diseases. The rationale behind using CAR T-cell therapy to treat cardiac fibrosis lies in the fact that fibrosis is often driven by the activation of pro-fibrotic immune cells, such as myofibroblasts. By targeting these pro-fibrotic cells with CAR T-cells, it may be possible to reduce the severity of cardiac fibrosis. Enhancing CAR T-cell therapy through innovative nanoparticle delivery systems provides a comprehensive approach to treating cardiac fibrosis, with experimental evidence indicating potential in reducing fibrosis and improving cardiac function. Despite these benefits, significant challenges such as cardiotoxicity and cardiovascular complications remain. Therefore, this review explores the molecular mechanisms underlying cardiac fibrosis and the effects of CAR T-cell therapy on the heart, elucidating both its antifibrotic properties and associated cardiotoxic effects based on findings from recent studies.

Introduction

Collagen is essential for maintaining the structural integrity and function of the heart under healthy conditions. However, in the context of heart disease, the dysregulation of collagen synthesis and deposition can lead to adverse remodeling, impaired cardiac function, and heart failure [1, 2]. Cardiac fibrosis is considered a type of heart remodeling process. The excessive synthesis and secretion of ECM components, along with ECM-associated modulatory glycoproteins, are hallmark features of fibrosis. This condition is at least partly driven by immune system activation and inflammation that occur following cardiac stress, hypertension, myocardial infarction, chronic inflammation or exposure to various toxic substances [3]. While quiescent fibroblasts help preserve the normal architecture of heart muscle, fibroblasts that become pathologically activated due to injury or disease impair the heart’s compliance and increase its stiffness [4]. These activated fibroblasts also send signals to cardiac myocytes that further impair myocardial function. Although the fibrotic response serves as a critical barrier against myocardial rupture during infarction, it can lead to adverse remodeling that accelerates the progression of heart failure [5, 6].

This process involves post-translational modifications and cross-linking of ECM proteins, as well as the recruitment of fibroblasts that differentiate into myofibroblasts [7]. Additionally, there is often a dysregulation of ECM degradation, which is mediated by matrix metalloproteinases (MMPs) and their endogenous inhibitors [8]. These changes contribute to the pathological remodeling of tissues, leading to increased stiffness and impaired function. As a result, there is a complex interplay between cellular signaling pathways and ECM dynamics in the progression of fibrosis [9, 10].

The extent of myocardial fibrosis holds prognostic value, as it leads to contractile dysfunction and arrhythmias in structural heart diseases with diverse etiologies. Unfortunately, there are currently limited effective treatments available for established cardiac fibrosis [11]. Myocardial fibrosis is classified into three types: (1) Reactive interstitial fibrosis, linked to hypertension and diabetes, marked by collagen accumulation without injury (2) Infiltrative interstitial fibrosis, associated with storage disorders like amyloidosis; and (3) Reparative fibrosis, which occurs after myocardial infarction and in non-ischemic cardiomyopathies by collagen deposition [12–14]. During the early stages or upon the removal of underlying causes, cardiac fibrosis may regress, allowing for the restoration of normal tissue structure and function. To facilitate the regression of cardiac fibrosis, various strategies can be employed, including the regulation of myofibroblast activation and proliferation and/ or elimination of fibrogenic myofibroblasts [15].

Despite disappointing outcomes from clinical trials and extensive research on traditional methods aimed at disrupting the inflammation-fibrosis axis, antifibrotic immunotherapy remains a valuable area for future investigation, even after fibrosis and heart failure have developed [16]. However, the progress of immunotherapy presents challenges, especially concerning the use of allogeneic T cells, which have shown potential in targeting myofibroblasts and effectively treating cardiac fibrosis [17].

Therapeutic agents prescribed, such as inhibitors of the renin-angiotensin-aldosterone system (RAAS) or Peroxisome proliferator-activated receptor (PPAR-α) agonists, have shown benefits in decreasing fibrosis [18]. Despite extensive research, no antifibrotic medication has convincingly demonstrated the ability to reverse fibrosis or improve health outcomes in clinical studies. These findings underscore the need for innovative strategies that focus on targeting myofibroblasts rather than solely addressing individual molecular pathways [19, 20]. In this regard, chimeric antigen receptor T-cell (CAR T) therapy presents a promising approach to potentially halt or reverse cardiac fibrosis. CAR T therapy is an advanced form of immunotherapy that involves genetically modifying autologous T cells to specifically target and eliminate surface antigens on affected cells [21]. While CAR T-cells have shown significant success in cancer treatment, this technology can be adapted for other medical fields, positioning CAR T immunotherapy as a viable strategy for combating cardiovascular diseases [22]. Recent studies have highlighted the effectiveness of CAR T-cell therapy in treating cardiac fibrosis in murine models [23, 24]. This breakthrough suggests the potential for addressing interstitial fibrosis related to various conditions, including diabetes [25, 26].

CAR T-cell therapy involves engineering T cells to express chimeric antigen receptors (CARs) that recognize specific surface proteins on target cells. Upon encountering these target cells, the modified T cells become activated and initiate a cytotoxic response, resulting in the elimination of the targeted cells [27]. In the context of cardiac fibrosis, CAR T-cells could be specifically designed to target surface antigens found on fibrogenic myofibroblasts. This targeted approach would allow for the selective removal of these cells, potentially promoting the regression of fibrotic lesions and improving cardiac function [28]. However, while this innovative treatment holds promise, it is important to acknowledge the significant risks associated with CAR T-cell therapy. These risks include potential toxicity and adverse effects, which may encompass cardiovascular complications. Therefore, careful consideration and monitoring are essential when implementing this therapy in patients with cardiac fibrosis [29].

There is an ongoing debate regarding the prevalence of cardiotoxicity following CAR T-cell therapy.

In this context, we will explore the advantages and challenges of utilizing CAR T-cell therapy for the treatment of cardiac fibrosis.

Molecular mechanism of cardiac fibrosis

Signaling pathways

Cardiac fibroblasts are primarily responsible for establishing and maintaining the structural framework that upholds the integrity of the heart. They play a vital role in facilitating effective muscle contraction and provide stable anchorage for other cardiac cells, which modulate the activity of cardiomyocytes. Additionally, cardiac fibroblasts support the heart’s architecture by synthesizing collagen, a key component of the extracellular matrix [30].

After heart injury and during tissue remodeling, MMPs enzymes degrade collagen (types I and III) and other extracellular matrix proteins. The activity of MMPs is finely regulated by tissue inhibitors of metalloproteinases (TIMPs), which help maintain the balance between matrix synthesis and degradation. This balance is essential for normal cardiac function, as any disruption can lead to pathological conditions such as fibrosis and heart failure [31, 32]. Understanding the interactions between cardiac fibroblasts, MMPs, and TIMPs is critical for developing therapeutic strategies aimed at preserving cardiac function and preventing adverse remodeling in various heart diseases. Cardiac fibrosis occurs when the synthesis of ECM proteins exceeds their degradation. This imbalance between ECM production and breakdown can lead to structural changes in cardiac tissue, contributing to impaired heart function [33].

The nature of the initial myocardial injury significantly influences the mechanisms by which fibrogenic signals are activated. Fibroblasts are stimulated through neurohumoral pathways, both directly and indirectly, by their effects on immune cell populations [34]. Activation of cardiac fibroblasts occurs in response to various myocardial injuries via well-established mechanisms involving signaling molecules such as Transforming Growth Factor Beta (TGF-β) (Fig. 1), small mother against decapentaplegic (SMAD) proteins 2/3, interleukins (IL-1, IL-11, IL-14), platelet-derived growth factors (PDGFs), and tumor necrosis factor-alpha (TNF-α), along with other immune system interactions [35].

Fig. 1.

The molecular mechanisms underlying cardiac fibrosis are complex and involve various cell types and signaling pathways

As another involved cascade, the Wnt/Fzd signaling pathway is crucial for regulating fibroblast activation [10]. Wnt ligands serve as extracellular signaling molecules that transmit signals through Frizzled (Fzd) receptors or co-receptors LRP5 and LRP6. TGF-β induces pro-fibrotic signaling partially via the Frizzled-8 receptor [36]. Additionally, elevated levels of Wnt2 and Wnt4 activate β-catenin/NF-κB signaling pathways, facilitating cardiac fibrosis through the collaborative action of Fzd4/2 and LRP6 in fibroblasts.

Moreover, matricellular proteins and secreted fibrogenic mediators, including integrins, cytokine receptors, syndecans, and CD44, bind to various fibroblast cell surface receptors. This binding initiates intracellular signaling cascades that regulate genes involved in the synthesis, processing, and metabolism of the ECM [37]. Fibrosis not only compromises cardiomyocyte health and function but also leads to increased stiffness of the myocardium. In response to different myocardial injuries, TGF-β-SMAD2/3 pathways activate cardiac fibroblasts, prompting them to produce ECM proteins while simultaneously suppressing immune cell proliferation. Moreover, elevated levels of mediators that stimulate cardiac fibroblast activity, such as angiotensin II (Ang II), aldosterone, and catecholamines, significantly contribute to the development of reactive fibrosis in heart diseases [7, 32, 38, 39].

Genetic of myocardial fibrosis

The genetic basis of cardiomyopathies includes various variants and mutations in genes related to cell contractility, sarcomere proteins, calcium regulation, cytoskeletal components, and metabolic pathways (Table 1). These genetic alterations lead to changes in ventricular structure, function, and remodeling [40]. In genetic cardiomyopathies, these alterations typically affect the architecture and functionality of cardiomyocytes, often resulting in the development of fibrosis [41]. Previous genome-wide association studies (GWAS) have determined variants associated with cardiac structure and dysfunction. Notably, common genetic variations in the SMARCB1 gene have been linked to increased left ventricular wall thickness and enhanced TGF-β1-mediated myocardial fibrosis [42]. Other studies have associated genes such as MYH7, MYH6, CDKN1A, SH2B3, TTN, LMNA, and BAG3 with myocardial fibrosis [43–45].

Table 1.

Genes associated with cardiac fibrosis

Gene	Function/Role	Implication in Fibrosis
MYH7	Sarcomere protein involved in muscle contraction	Mutations linked to cardiomyopathies and fibrosis
MYBPC3	Cardiac muscle protein	Alterations associated with fibrotic response
COL1A1	Collagen type I	Increased expression contributes to ECM accumulation
COL3A1	Collagen type III	Similar role in ECM deposition
TGF-β1	Cytokine regulating fibroblast activity	Promotes fibroblast activation and collagen synthesis
CTGF	Connective tissue growth factor	Enhances TGF-β1 effects, promoting fibrosis
MMPs	Matrix metalloproteinases	Involved in ECM remodeling; dysregulation leads to fibrosis
TIMPs	Tissue inhibitors of metalloproteinases	Regulate MMP activity; balance is crucial for ECM homeostasis
SOD2	Antioxidant enzyme	Dysregulation linked to oxidative stress and fibrosis
NLRP3	Inflammasome component	Associated with inflammation and fibrotic response
GSK3β	Protein kinase involved in cell signaling	Regulates pathways that influence fibroblast activity
PRKCA	Protein kinase C alpha	Promotes fibroblast activation and ECM production

Additionally, researchers have indicated that alterations in genes encoding sarcomere protein components, such as MYH7, MYBPC3, and MYL2, are common among a significant proportion of patients exhibiting an inflammatory phenotype accompanied by subsequent fibrosis [46]. Phospholamban (PLN), a sarcoplasmic reticulum membrane protein expressed in cardiomyocytes, plays a crucial role in calcium regulation. Skarp et al. demonstrated that hearts with the PLN-R14del variant exhibited the highest levels of myocardial fibrosis. Whole exome sequencing of patients with myocardial fibrosis identified 21 missense variants and one nonsense variant located in genes such as cartilage acidic protein 1 (CRATC1), calpain 1 (CAPN1), unc-45 myosin chaperone A (UNC45A), and unc-45 myosin chaperone B (UNC45B). These variants contribute to the functionality of the extracellular matrix and cardiomyocytes [47]. Furthermore, 11 independent loci have been associated with T1 time, a marker of myocardial fibrosis. The identified loci implicate genes involved in various biological processes, including glucose transport (SLC2A12), iron homeostasis (HFE, TMPRSS6), tissue repair (ADAMTSL1, VEGFC), oxidative stress response (SOD2 SNP rs9457699_G), cardiac hypertrophy (MYH7B), and calcium signaling (CAMK2D, rs55754224_T), all of which are linked to myocardial fibrosis [48].

Studies have suggested that genetic predispositions can interact with lifestyle factors such as diet and exercise, further influencing the risk of developing cardiac fibrosis. Identifying specific genetic markers associated with increased susceptibility to fibrosis could pave the way for personalized treatment approaches and targeted therapies aimed at mitigating the progression of cardiac diseases [49, 50].

Epigenetic modulation of cardiac fibrosis

Environmental factors such as hypertension, diabetes, and oxidative stress can induce epigenetic changes that contribute to cardiac fibrosis. These stressors lead to persistent alterations in gene expression profiles, promoting fibrotic processes [51]. Epigenetic modulation significantly influences the pathogenesis of cardiac fibrosis by affecting gene expression patterns related to fibroblast activation and ECM accumulation. This modulation involves changes in histones and DNA, as well as the role of non-coding RNAs. Cardiac fibrosis is characterized by excessive ECM accumulation in the heart, resulting in stiffness and impaired function, highlighting the importance of understanding these epigenetic mechanisms [52].

Fibrosis is linked to elevated levels of DNA methylation. Specifically, cardiac fibroblasts (CFs) show increased DNA methylation when exposed to ischemic conditions that drive cardiac fibrosis. This phenomenon is associated with the upregulation of the DNA methyltransferases DNMT1, DNMT3a, and DNMT3b [53].

DNMT3a plays a vital role in facilitating fibroblast activation through the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway, promoting cardiac fibrosis by inducing abnormal methylation of the Ras protein activator-like-1 (Rasal-1) and Ras-association domain family 1 (Rassf-1) (Fig. 2). This process results in increased methylation and the production of fibrosis-related markers, such as α-SMA. For instance, the H3K4me3 epigenetic mark, indicative of histone modifications, has been observed to be upregulated in the promoter region of the pro-fibrotic gene Smad3 following TGF-β stimulation in cardiac fibroblasts [54, 55]. The involvement of histone acetyltransferases, particularly CBP/P300, in cardiac fibrosis is well documented, as they act as coactivators within the TGF-β signaling pathway and with the Smad3 transcription factor, thereby epigenetically modulating the expression of collagen-related pro-fibrotic genes [56].

Fig. 2.

Epigenetic modifiers can either activate or repress gene expression by targeting DNA and/or RNA, thereby influencing cellular fate

Additionally, Ang II has been shown to enhance the expression of the endothelin-1 (ET-1) gene in endothelial cells by depositing the H3K4me3 mark on the ET-1 promoter, which subsequently contributes to the formation of fibrotic tissue in the murine heart [57]. Furthermore, there is increasing evidence linking the expression of specific microRNAs, such as miR-133a, miR-208, miR-1, and miR-451, with the onset and progression of cardiac fibrosis. Emerging therapies targeting epigenetic factors to modulate antifibrotic mechanisms in the remodeling of fibrotic heart tissue may represent the future of treatment approaches for cardiac fibrosis [58–60].

CAR T-cell therapy for cardiac fibrosis

CAR T-cells are typically generated by introducing a genetically modified CAR fusion protein into a patient’s T cells. Following this modification, the T cells are expanded and subsequently infused into the patient after a lymphodepletion process [61]. Post-infusion, these T cells undergo proliferation and identify their target antigen, leading to the activation of endogenous T cells and the release of cytokines that facilitate the destruction of target cells. Selecting the appropriate cell-surface target for CAR T-cells is essential and can differ across various organs and species. To reduce ‘off-target’ impacts on healthy tissues, the optimal target should be limited to the affected tissue, where fibrogenesis is predominantly observed [62, 63].

Fibroblast activation protein (FAP), identified as an endogenous target for cardiac fibrosis, was revealed through the expression analysis of gene profiles from cardiac fibroblasts in both healthy and diseased human hearts. This gene exhibited the greatest variation between control and diseased samples [64]. FAP is a cell surface glycoprotein that is typically expressed at low levels or is absent in most normal adult mice or human tissues. It is expressed during embryonic development at sites undergoing active tissue remodeling, such as wound repair, tissue fibrosis, and various tumors [65]. This glycoprotein plays a significant role in the degradation of extracellular matrix components, including collagen. By cleaving collagen and other ECM proteins, FAP facilitates tissue remodeling, which is essential during normal healing processes but can contribute to pathological fibrosis when dysregulated [66]. In diseases characterized by excessive fibrosis (e.g., cardiac fibrosis, liver cirrhosis), this may contribute to the accumulation of collagen, leading to tissue stiffness and dysfunction [67]. Previous studies have noted that human hearts with acute myocardial infarction injury express FAP at elevated levels, while healthy human hearts exhibit minimal FAP expression [68]. Therefore, targeting FAP could potentially modulate collagen metabolism and serve as a therapeutic strategy in fibrotic diseases [69–71].

FAP CAR T-cells are generated by isolating and engineering CD8 + T lymphocytes to express a segment of a single-chain variable fragment (scFv) that specifically recognizes FAP. This scFv is fused to the cytoplasmic domains of human CD3ζ and CD28, which are essential for T cell activation and proliferation [72]. Once infused into the patient, these CAR T-cells migrate to the heart, where they selectively target myofibroblasts or activated fibroblasts that express FAP. Upon recognising it in these cells, the CAR T-cells utilize cytotoxic mechanisms to induce apoptosis in the myofibroblasts, thereby inhibiting the fibrotic process [73]. This targeted approach, which aims at reparative fibroblasts, raises significant concerns regarding potential off-target effects during tissue healing, particularly after an acute myocardial infarction (AMI). The sustained presence of CAR T-cells may lead to the depletion of reparative fibroblasts long after their beneficial functions have ceased, resulting in detrimental long-term cardiac remodeling. Targeting FAP-expressing cells in the aftermath of AMI could obstruct the essential fibrotic response, potentially causing severe structural failure of the infarcted myocardium. Research conducted in murine models indicates that the depletion of activated fibroblasts can hinder scar formation and elevate mortality rates following cardiac injury [15, 74].

This highlights the dual role of fibrosis as both a pathological and protective mechanism. Temporary CAR T approaches, including RNA-based CAR-Ts or switchable CAR-Ts, present a viable solution by restricting the duration of fibroblast depletion. RNA CAR-Ts transiently express the receptor and are degraded within days, creating a limited period of activity [75]. Alternatively, synthetic “on/off” switches or suicide gene systems can allow clinicians to regulate the lifespan of CAR T-cells [76].

Previous studies have demonstrated the effectiveness of using CAR T-cells targeting FAP to ablate tumor-promoting cancer-associated stromal cells and treatment of mesothelioma and pancreatic cancer. The efficacy of ex-vivo CAR T-cells in selectively targeting and eliminating myofibroblasts within a mouse model [77, 78]. In the AngII/PE mouse model of hypertensive cardiac injury and fibrosis, adoptive transfer of T lymphocytes expressing a CAR against FAP significantly reduced cardiac fibrosis and partially restored both diastolic and systolic function after cardiac injury in mice. These findings provide proof-of-concept for a novel immunotherapeutic approach to treating heart disease [79].

Cardio safety and scalability

Given the significant improvements that CAR T-cells have demonstrated in treating myocardial fibrosis, minimizing toxic effects will be crucial for their successful application in humans. Currently, the primary methods for generating CAR-engineered T cells involve viral gene transduction using retroviral or lentiviral vectors. These methods risk insertional mutagenesis and genotoxic effects in the effector cells [80]. Additionally, the prolonged protein expression enabled by these viral vectors could result in uncontrolled adverse outcomes if significant toxicities arise due to unintended cross-reactivity [81]. Therefore, developing safer gene delivery methods and refining CAR T-cell design will be essential to enhance their therapeutic potential while mitigating risks [82]. Furthermore, in contrast to cancer, where the advantages of immune surveillance and the sustained presence of targeted T cells are crucial for completely eradicating cancer cells, fibrotic disorders require merely a general decrease in disease burden to realize clinical improvements [83].

Another challenge associated with the prolonged activation of CAR T-cells is the treatment of human diseases linked to fibrosis, as activated fibroblasts play a crucial role in the wound healing process [84]. When chimeric antigen receptor T cells are modified through conventional methods to target fibroblasts, they may persist in the body for several months or even years. This extended presence could potentially hinder the wound-healing process over time [85]. In this context, patients with heart failure who have persistent CAR T-cells used to manage chronic fibrosis may face an increased risk of complications, such as cardiac ventricular wall rupture, particularly in the event of a subsequent myocardial infarction [86]. Therefore, it is essential to develop strategies that allow for the controlled activation and eventual deactivation of CAR T-cells, ensuring that their therapeutic effects do not compromise critical healing processes in the heart and other tissues. Additionally, alternative approaches that target fibrosis without long-term T cell persistence may be necessary to mitigate these risks [87].

mRNA- lipid nanoparticles technology

To reduce the disadvantage of permanent activation CAR T-cells, nucleoside-modified mRNA technology was developed as a temporary CAR T-cell therapy. The transient nature of the RNA by methylation to reduce immunogenic responses technique, which offers a safety advantage over prolonged CAR T-cell therapy, is an important advancement of the CAR T generation method [88]. Using this method, it is possible to generate a short-lived CAR T-cell without the need for a viral vector or the risk of genomic integration by modifying the cells to express messenger RNA rather than virally encoded DNA [89]. After cellular uptake, the mRNA escapes the endosome and is quickly translated before being degraded in the cytoplasm. Additionally, the expression of the CAR ceases once the mRNA degrades or becomes diluted due to cell division. This transient nature of CAR expression is crucial for minimizing potential adverse effects while still allowing for effective cellular responses during the therapeutic process [90, 91]. To overcome the mentioned challenges as well as to eliminate the necessity for T cell extraction and reinfusion, the latest advancement in antifibrosis CAR T-cell production is the use of lipid nanoparticles (LNPs) carrying FAP mRNA sequences, while they were coated with an anti-CD5 antibody to facilitate their targeting to T cells, when injected intravenously [92]. The in-vivo administration of the LNP-mRNA system facilitates the endocytosis of the lipid nanoparticles, leading to their degradation and subsequent release of mRNA into the cytoplasm of T cells. This mRNA is unable to integrate into the genome and has a short half-life [93]. According to Rurik et al., the delivery of LNPs to T cells led to a temporary expression of FAPCAR, which enabled the CAR T-cells to specifically eliminate FAP⁺ fibroblasts, thereby reducing fibrosis in a mouse model subjected to cardiac stress through Ang II and PE infusion and improving cardiac function. Similar to virally engineered FAP CAR T-cells [94], these LNP-generated CAR T-cells were able to efficiently kill FAP-expressing target cells in vitro in a dose-dependent manner (Fig. 3).

Fig. 3.

The generation of CAR T-cells in vivo using CD5/LNP-FAPCAR involves administering LNP coated with anti-CD5 antibodies along with mRNA that encodes the FAPCAR membrane receptor. This approach enables the production of transient CAR T-cells that specifically target and eliminate pro-fibrotic cells in the damaged myocardium. The FAPCAR membrane receptor is designed to recognize the FAP protein expressed by cardiac myofibroblasts

The use of this technology in mRNA vaccines against COVID-19 (BNT162b2 and mRNA-1273) has demonstrated its safety and scalability [95]. Significant efforts are needed to establish the appropriate dosage strategy for effectively eliminating cardiac fibrosis, given the distinct characteristics of COVID-19 infection compared to other cardiovascular diseases [96]. However, the application of transient CAR T reprogramming offers the potential for controlled “dosing” of cells. This approach requires a comprehensive analysis of the proliferation and expansion kinetics of fibroblasts in various fibrotic disease contexts where this technology may be utilized [97]. For example, fibroblast proliferation rates in response to cardiac stress can vary significantly and are closely associated with other tissue responses, such as inflammation and vascular remodeling [98]. Furthermore, the plasticity and differentiation states of activated fibroblasts can be affected by the nature and duration of the injury [99].

Epigenetic modification of CAR T-cells

The application of epigenetic mechanisms and modifications presents another promising approach to address the challenges associated with CAR T-cell therapy improvement. Epigenetic modifications serve as a mechanism to influence gene expression through chromatin remodeling, which is facilitated by various histone modifiers, including post-translational modifications such as acetylation, methylation, phosphorylation, ubiquitination, SUMOylation, and lactylation [100]. Additionally, DNA methyltransferases (DNMTs) and ten-eleven translocation (TET) enzymes, which are involved in demethylation, along with non-coding RNAs like microRNAs, play crucial roles in this process. These epigenetic modifiers can either activate or repress gene expression by targeting DNA and/or RNA, thereby influencing cellular fate [53].

In the context of CAR T-cells, strategic epigenetic modifications can mitigate the obstacles that hinder their effectiveness. Such modifications can enhance the persistence and survival of T cells, reduce T cell exhaustion, improve their infiltration into tumor sites, and promote the development of a memory phenotype [101]. Selective epigenetic reprogramming can generate less differentiated CAR T-cells, potentially reducing CAR T-cell exhaustion. “Less differentiated CAR T-cells” are in an earlier stage of development, resembling naïve or memory T cells rather than fully matured effector T cells. These cells are often preferred for therapy as they persist longer, proliferate more effectively upon encountering tumor antigens, show less exhaustion, and are linked to better long-term outcomes and durable responses [102]. The ongoing evolution of CAR T technology, alongside the technical challenges faced, aims to enhance the therapeutic efficacy of CAR T-cell therapy, particularly in the field of cardio-oncology [103].

CRS and cardiotoxicity; the main CAR T-cell therapy issue

The activation and proliferation of CAR T-cells often lead to a significant release of inflammatory cytokines, which can have a profound impact on cardiac function. This process is known as cytokine release syndrome (CRS) [104]. When CAR T-cells become activated, they secrete various inflammatory cytokines and chemokines, including IL-1, IL-6, IL-1Ra, and CC-chemokine ligands CCL2 and CCL3, as well as nitric oxide (NO). Additionally, bystander immune cells contribute to this inflammatory response by releasing IL-6, soluble IL-6 receptor (sIL-6R), IL-2, interferon-gamma, and granulocyte-macrophage colony-stimulating factor (GM-CSF) [105, 106]. Among these, IL-6 plays a critical role in mediating toxicity during CRS by the recruitment of additional T cells by increasing the expression of chemokine’s that attract them and facilitates the differentiation of lymphocytes [107]. Moreover, IL-6 activates the gp130/STAT3 signaling pathway, which exacerbates oxidative stress and contributes to mitochondrial dysfunction and cardiac hypertrophy [108]. Elevated levels of reactive oxygen species contribute to increased apoptosis in cardiomyocytes. Additionally, IL-6 alters calcium handling and reduces myocardial contractility, potentially leading to diastolic dysfunction and arrhythmias [109].

Patients undergoing CAR T-cell therapy experience a range of symptoms associated with CRS, including fever, myalgia, fatigue, hypotension, tachycardia, and capillary leakage. Cardiotoxicity related to CRS can range from arrhythmias and hemodynamic instability due to hypotension, to left ventricular (LV) dysfunction and even sudden cardiac death [110]. The severity of CRS, particularly grade 3 CRS, which includes temperature abnormalities, hypotension requiring vasopressors, and hypoxia that demands the use of high-flow nasal cannula or non-rebreather masks, has been significantly correlated with the development of adverse cardiovascular events in patients undergoing CAR T treatment [111].

Retrospective cohort studies have shown that major adverse cardiovascular events (MACE) occurred in 10–20% of patients [90]. In contrast, a meta-analysis found a higher prevalence of cardiotoxic effects: 54% of patients experienced arrhythmias, 30% developed heart failure, 20% showed signs of cardiomyopathy, 10% suffered from acute coronary syndrome (ACS), and 7% experienced cardiac arrest. These discrepancies underscore the need for more extensive, long-term cohort studies to fully evaluate the cardiotoxicity associated with CAR T-cell therapy [112].

Patients experience a range of symptoms of CRS with different severity, such as fever, myalgia, fatigue, hypotension, tachycardia, and capillary leakage. Cardiotoxicity secondary to CRS ranges from arrhythmias and hemodynamic instability due to hypotension, left ventricular (LV) dysfunction, to sudden cardiac death [110]. The degree of CRS, particularly grade 3 CRS (presence of temperature abnormalities and hypotension that necessitate the administration of vasopressors, as well as hypoxia that demands the use of high-flow nasal cannula, nonrebreather masks, or Venturi masks), has been found to be significantly correlated with the development of adverse cardiovascular events in retrospective evaluations of individuals undergoing CAR T treatment [111].

In a small number of retrospective analyses, severe cardiac dysfunction has been observed in approximately 5–12% of CAR T-cell treated cancer patients, most of whom develop CRS grade 3 [113]. The underlying pathophysiology is not fully understood, but it has been suggested that the myocardium and conduction system are dysfunctional due to systemic cytokines, similar to what occurs in septic environments. Pharmacovigilance data recently showed that the relative mortality of patients with CV and pulmonary adverse events (CPAEs) in CAR T-cell therapy was 30.9%, while the relative mortality of patients with CRS was 17.4%. This highlights the urgent need to address these issues [114].

Cardiotoxicity is a serious complication that occurs early after CAR T-cell administration, typically within the first two weeks. The incidence of death attributed to cardiotoxicity ranged from 1.4 to 4.3%. The mechanisms of cardiotoxicity of CAR T-cell therapies are thought to be primarily caused by CRS-associated systemic inflammation [115].

Cardiovascular side effects, including cardiac arrhythmias (such as atrial fibrillation, supraventricular tachycardia (SVT), atrioventricular nodal block, prolonged QT interval, and sinus and ventricular tachycardia), left ventricular dysfunction (LVD), HF, pericardial effusion, and, in rare cases, cardiac arrest, have been reported following CAR T-cell therapy [116].

Clinical observations, including those from Phase I-III trials, show that CV side effects are common in patients receiving CAR T-cell therapy, with a decline in left ventricular ejection fraction (LVEF) observed in 10–30% of cases. These side effects are also associated with significant morbidity and mortality [117].

Arrhythmias (incidence ranging from 5 to 12%), cardiomyopathy/heart failure, cerebrovascular accidents (CVAs), and myocardial infarction (MI) are examples of serious adverse cardiovascular events [118]. Within thirty days of receiving CAR T-cell therapy, 16% of patients experienced at least one MACE. However, the range of clinical symptoms is broad. It can include decompensated heart failure, new-onset arrhythmias, left ventricular systolic function (LVSD), elevated serum troponin levels, sinus tachycardia (often associated with CRS fever), and cardiovascular mortality. To date, LVSD associated with CAR T-cell therapy has been shown to be reversible at 6 months in the setting of physiologic stress resulting from CRS [119, 120].

The treatment of cardiovascular problems after CAR T-cell therapy depends on the specific issue and the patient’s status, as most cardiovascular complications arise from CRS [111]. BP monitoring is crucial to recognize hypotension and to prescribe IV fluids or vasopressors when needed. Additionally, using drugs that suppress CRS toxicity after CAR T-cell therapy, such as Tocilizumab, Siltuximab, or corticosteroids, is important [121, 122]. A study found that vasopressors were used for 13.6% of patients with hypotension following CD19 CAR T-cell therapy for lymphoma [123]. If a patient develops AF, which is common due to CRS, its management follows current guidelines and includes the need for anticoagulation, monitoring of electrolyte levels, antiarrhythmic therapy, or cardioversion [124]. Studies have shown that having preexisting cardiac disease increases the risk of cardiovascular disorders after CAR T-cell therapy [125]. Evaluating patients prior to CAR T is a crucial step, and this assessment should be conducted by a cardio-oncologist, including cardiac biomarkers, echocardiography, ECG, or examination, depending on the patient’s baseline disease and medical history. A study showed that a global longitudinal strain (GLS) measured by echocardiography six months prior to CAR T-cell therapy was associated with a higher risk of cardiovascular events after CAR T [126].

Cardiotoxicity biomarkers pre CAR T-cell therapy

Retrospective observational studies have reported a significant cardiovascular event rate ranging from 12 to 28% among patients undergoing CAR T-cell therapy, with most of these events occurring shortly after treatment, typically within 6 to 21 days post-transfusion [127]. Early detection of cardiac injury and inflammatory responses is crucial, as it allows for timely interventions that can include dose adjustments and the initiation of cardio protective therapies. By implementing proactive monitoring strategies, healthcare providers can better manage potential complications and improve patient outcomes following CAR T-cell therapy [124]. Research on biomarkers that predict cardiotoxicity associated with CAR T-cell therapy remains limited, leaving the roles of cardiac and inflammatory biomarkers in managing this cardiotoxicity insufficiently understood. However, cytokines have emerged as some of the most potent and accessible laboratory biomarkers for predicting CRS [128]. Several predictive biomarkers, including interferon-gamma, IL-6, soluble gp130 (sgp130), soluble IL-6 receptor (sIL-6R), and IL-15, have been studied in patients [129]. Research indicates that peak levels of interferon-gamma, IL-6, and IL-15 are correlated with the onset of atrial fibrillation following CAR T-cell infusion [111]. Additionally, elevated levels of high-sensitivity troponin T (hsTropT) and N-terminal pro-B-type natriuretic peptide (NT-proBNP) can help identify patients at high risk for cardiac events after CAR T-cell infusion. These findings underscore the importance of monitoring these biomarkers to better manage potential cardiotoxicity in affected patients. Elevation in troponin levels may indicate myocardial damage, similar to their role in ischemic cardiomyopathy, while increases in N-terminal pro-B-type natriuretic peptide (NT-proBNP) may serve as a marker for cardiac stress, akin to its use in heart failure [130].

Published data have shown a correlation between troponin elevation and the occurrence of CRS of grade 2 or higher, which is associated with a decrease in LVEF and an increased risk of serious cardiovascular adverse events in patients with elevated troponin levels [120]. Furthermore, these biomarkers can provide critical insights into patient management following CAR T-cell therapy. Monitoring troponin and NT-proBNP levels can facilitate early identification of cardiac complications, allowing for timely interventions to mitigate risks and improve patient outcomes. Elevated troponin levels indicate myocardial injury, while high NT-proBNP levels reflect increased cardiac stress, both of which are essential for assessing cardiovascular health in this patient population [131, 132].

In contrast, Hu JR et al. found that troponin-T levels did not change during CAR T-cell therapy, while NT-proBNP levels were elevated. However, the authors did not establish a correlation between these findings and cardiotoxicity [133]. Neither cardiac nor pro-inflammatory cytokines were associated with cardiac events following CAR T-cell therapy; rather, clinical factors such as age, baseline creatinine levels, and left atrial volume were identified as significant predictors of these events. Hence, more research is needed to clarify the role of pro-inflammatory cytokines and cardiac biomarker monitoring in patients receiving CAR T-cell therapy [107].

Pre CAR T-cell cardiovascular management

The latest guidelines from the European Society of Cardiology (ESC) on cardio-oncology recommend that all cancer patients undergo a baseline electrocardiogram (ECG), along with measurements of natriuretic peptides (NP) and cardiac troponin (cTn), prior to receiving CAR T-cell therapy [134]. For patients with pre-existing cardiovascular conditions, an echocardiogram is advised. Furthermore, the guidelines suggest that NP and cTn levels should be monitored, along with echocardiography, in patients experiencing CRS of grade 2 or lower. Grade 2 CRS is characterized by fever (≥ 38.0 °C) accompanied by hypotension that does not require vasopressor support and/or hypoxia necessitating supplemental oxygen via low-flow nasal cannula (≤ 6 L/min) or equivalent. [116].

For the best possible management of the cardiovascular consequences of CAR T-cell therapy, interdisciplinary collaboration between oncologists, neurologists, pharmacists, specialized nurses, and cardio-oncologists with competence in critical care is required [135]. Cardio-oncology evaluation will be required prior to initiation of CAR T-cell therapy if the patient has abnormalities in their biomarkers or cardiac diagnostic tests. [136]. A suggested evaluation for patients with a history of cardiovascular symptoms, abnormal echocardiogram or ECG, pre-existing CVD, or reduced exercise tolerance includes consultation with a cardio-oncologist and diagnostic testing based on the patient’s risk factors [137].

Patients with pre-existing arrhythmias or coronary artery disease, a history of chemotherapy-induced cardiomyopathy or other cardiomyopathies, or a high risk of coronary artery disease based on their calculated QRISK3-2018 score are all included in high-risk category [138]. All patients suggested undergo a 12-lead ECG, cardiac imaging with TTE (including 2-, 3- and 4-dimensional assessment of left ventricular ejection fraction, diastolic and global longitudinal strain), CMR with a fast scan protocol, and blood sampling for troponin and NT-proBNP levels to inform clinical decisions in cardio-oncology and help identify the occurrence of a CV adverse event during cancer therapy. GLS should be assessed by echocardiography as it has been shown to decrease after CAR T-cell therapy [139].

To address the very sensitive early phase, a standardized cardio-oncology follow-up with biomarkers, echocardiography and ECG performed on day 7 after CAR T-cell transfusion may be helpful. For high-risk patients, a 3-month follow-up with cardiac biomarkers, ECG, and echocardiography is recommended to evaluate any recovery of overt CV symptoms or delayed CV damage [140].

Conclusion

CAR T-cell therapy presents significant potential benefits for treating cardiac fibrosis. This innovative approach allows for targeted therapy, selectively attacking pro-fibrotic immune cells while preserving non-fibrotic cells, which enhances both safety and efficacy. Additionally, CAR T-cell therapy can be personalized to meet the specific needs of individual patients, tailoring the treatment to optimize outcomes. It also offers the possibility of being used as an adjunct therapy, combining with pharmacological agents or stem cell treatments for a more comprehensive strategy against cardiac fibrosis. However, challenges such as the high cost of CAR T-cell therapy may limit its widespread application. There is increasing interest in exploring CAR T-cell therapy’s potential for other cardiovascular diseases, like atherosclerosis and cardiac remodeling, which could lead to novel therapies targeting various aspects of cardiovascular health. Despite these advantages, understanding the complex adverse effects associated with CAR T-cell therapy is still in its early stages. The efficacy and safety of this treatment modality require validation through specialized clinical trials. A critical future focus for cardio-oncology will be managing cardiovascular events effectively while maintaining anti-cancer efficacy. Improved clinical management strategies will emerge from a deeper understanding of the molecular mechanisms underlying cardiotoxicity, potentially validating CAR T-cell therapy as a viable option and paving the way for new therapeutic paradigms in cardiovascular disease. Additionally, addressing the epigenetic dysregulation observed in patient-derived T cells is vital for developing fully functional CAR T-cells with enhanced activity. Mapping the epigenome of these cells may help reprogram them into effective therapeutic agents. This can be achieved through DNA methylation and hydroxymethylation, histone modifications such as methylation, acetylation, and phosphorylation, as well as non-coding RNA-mediated mechanisms including miRNAs, lncRNAs, and siRNAs [141]. Using allogeneic CAR T-cells from healthy donors could also be a promising strategy to overcome existing limitations.

Author contributions

S.T: literature search, writing and figures design; N.D; literature search, writing; A. J: literature search, writing and manuscript reviewing; M.H: Had the idea, writing and manuscript reviewing.

Funding

Non to report.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethical approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.