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# Uncovering the role of prokineticin pathway on Epicardial Adipose Tissue (EAT) development and EAT-associated cardiomyopathy

Martina Vincenzi, Canan Nebigil

► To cite this version:

Martina Vincenzi, Canan Nebigil. Uncovering the role of prokineticin pathway on Epicardial Adipose Tissue (EAT) development and EAT-associated cardiomyopathy. Trends in Cardiovascular Medicine, 2025, 35 (5), pp.328-338. 10.1016/j.tcm.2025.02.006. inserm-05235684

HAL Id: inserm-05235684

https://inserm.hal.science/inserm-05235684v1

Submitted on 2 Sep 2025

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<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Localization of adipose tissue in the heart. Epicardial adipose tissue (EAT) is located between the myocardium, and visceral pericardium, originates from the splanchnopleuric mesoderm, and shares its blood supply with the myocardium via coronary arteries. The EAT surrounding the left atrium differs from the EAT infiltrating the coronary arteries. Paracardial adipose tissue (PAT) does not directly contact the myocardium or receive vascularization from the coronary arteries. The combined fat surrounding the heart, including both epicardial and paracardial adipose tissues, is often referred to as pericardial fat.</figcaption></figure>

ply with the myocardium via coronary arteries. Unlike EAT, PAT does not directly contact the myocardium or receive vascularization from the coronary arteries, meaning its influence on the heart is likely less significant (Fig. 1). The combined fat surrounding the heart, including both epicardial and paracardial adipose tissues, is often referred to as pericardial fat. EAT primarily consists of adipocytes, but also includes nerve cells, inflammatory cells such as macrophages and mast cells, stromal cells, vascular cells, and immune cells. The EAT surrounding the left atrium differs from the EAT infiltrating the coronary arteries. Each localized EAT depot has a unique transcriptome and proteome, resulting in varied impacts on nearby heart structures [5].

In fact, EAT in adults retains some features of brown and beige fat, which plays a role in energy supply, mechanic cushioning, and thermogenesis, utilizing glucose and lipids to produce heat, and protecting the heart from hypothermia [6]. This thermogenic function is driven by uncoupling protein-1 (UCP-1), a protein found abundantly in EAT but nearly absent in other adipose tissues such as subcutaneous fat [7]. Over time, particularly with aging and chronic diseases, EAT undergoes a shift, losing its brown fat-like characteristics and accumulating more unilocular white adipocytes, which are primarily associated with energy storage rather than thermogenesis. Further research is needed to determine whether EAT can adapt to different metabolic conditions and function as a brown or beige fat depot when required.

In addition to its thermogenic role, EAT is metabolically active, releasing and taking up free fatty acids (FFAs) to support the heart's energy needs, since FFA oxidation accounts for about 50-70% of the myocardial energy production. Fatty acid-binding protein 4 (FABP4), which is highly expressed in EAT, plays a key role in FFA handling. Moreover, EAT can act as a buffer between the myocardium and the local vasculature, protecting the heart against lipotoxicity by sequestering excess FFAs [8]. This dual capacity for releasing and absorbing FFAs makes EAT a critical component in maintaining myocardial health, particularly under metabolic stress (Fig. 2).

## Pathophysiologic mechanism of eat mediated CVDs

EAT can be a transducer of the toxic effects of chronic inflammatory diseases and metabolic disorders on the heart [9]. EAT has a role in the progression and development of CVD such as coronary artery diseases (CAD), atrial fibrillation (AF) and heart failure (HF) (Fig. 3) [10]. Through its secretion of adipokines, inflammatory cytokines, and metabolic factors, EAT also contributes to the pathophysiology of HF and other CVDs, especially in conditions such as obesity and diabetes, rheumatoid arthritis and virus infection. Excessive and dysfunctional EAT can negatively impact the heart in two major ways: 1) by serving as a source of pro-inflammatory mediators, 2) through direct cardio-mechanical interactions, leading to CVDs (Fig. 4).

### Source of pro-inflammatory cytokines and inflammation related CVDs

#### Pro-Coronary artery diseases (CAD)

Myocardium and coronary arteries can be modulated by EAT through the vasocrine or paracrine secretion of bioactive molecules. EAT tends to accumulate at specific sites, signaling underlying metabolic disturbances and systemic inflammation. Increased EAT thickness is linked to obstructive CAD and coronary vasospasm, while greater EAT volume is associated with more vulnerable plaque types. Additionally, EAT contributes to coronary microvascular dysfunction, which can lead to HF even in the absence of obstructive CAD [11]. Thus, EAT is implicated in both epicardial CAD and dysfunction of the coronary microcirculation, making it a significant factor in the pathophysiology of heart disease (Fig. 4A).

Chronic and prolonged ischemic conditions, such as advanced stages of CAD, can suppress brown fat-like activity in EAT. Excess epicardial fat can shift the physiologic balance toward a proinflammatory, pro-fibrotic and lipotoxic milieu, which can contribute to myocardial dysfunction and plays a significant role in the development of HF [9]. Additionally, In patients with advanced CAD, genes associated with adipocyte browning and thermogenic activation are downregulated in EAT, while the expression of pro-inflammatory cytokine genes are upregulated [12]. These shifts in gene expression may result from fibrosis and apoptosis occurring in EAT during end-stage organ disease (Fig. 4A).

EAT secretes various factors that causes endothelial dysfunction. For example, resistin released from EAT increases endothelial cell permeability [13]. It also releases angiogenic factors like thrombospondin-2, vascular endothelial growth factor (VEGF), and prokineticins (PKs), which contribute to angiogenesis and myocardial remodeling. Additionally, inflammatory mediators secreted by EAT, including activin A, visfatin, and monocyte chemoattractant protein-1 (MCP-1), have been linked to fibroblast proliferation and collagen synthesis, connecting EAT to both atrial and ventricular fibrosis.

EAT is also involved in oxidative stress. Elevated levels of reactive oxygen species (ROS) and reduced expression of antioxidant enzymes promote inflammation and atherogenesis in epicardial adipocytes. In patients with CAD, the EAT transcriptome is enriched with genes related to hemostasis and coagulation, such as tissue plasminogen activator, highlighting the connection between fibrinolysis and inflammation in adipose tissue. Additionally, epicardial adipocytes in individuals with CAD overexpress markers of cellular stress, such as the kinases MAP2K3 and MAP3K5, which are linked to coronary inflammation, along with multiple proteases involved in lysosomal degradation and apoptosis [5].

#### Pro-Coronary atherosclerosis

Beyond its mechanical impact, EAT plays a critical role in the development of coronary atherosclerosis (Fig. 4A). Surrounding
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Role of EAT in health and diseases after birth, aging and in pathological condition. EAT from neonatal to adult stages, maintains some features of brown and beige fat, which plays role in energy supply, mechanic cushioning, and thermogenesis, utilizing glucose and lipids to produce heat, and protecting the heart from hypothermia. However, with aging EAT undergoes a shift, losing its brown fat-like characteristics and accumulating more unilocular white adipocytes. Additionally, the thermogenic function of EAT is decreased, and expression of pro-fibrotic and pro-apoptotic factors are increased. In pathological conditions, such as coronary artery disease, diabetes mellitus, heart failure and atrial fibrillation, EAT serves as a source of pro-atherogenic, pro-arrhythmogenic and pro-inflammatory mediators such as adipokine, leptin, tumor necrosis factor (TNF), interleukins, creatine kinases, matrix metallopeptidase (MMP), tumor protein 53, and leucine-rich repeat (LRR)-containing proteins (NLR) family members (NLRP3) inflammasome and through direct cardio-mechanical interactions.</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Role of EAT in development of cardiovascular diseases. EAT accumulation can be a transducer of the toxic effects of chronic diseases such as coronary atherosclerosis, atrial dysfunction and arrhythmias and heart failure (HF), specifically HF with preserved ejection fraction (HFpEF) by increasing inflammation and fibrosis, and direct cardio-mechanical interactions.</figcaption></figure>

the epicardial coronary arteries and sharing their microcirculation, EAT in patients with CAD exhibits a dense inflammatory infiltrate, characterized by a high prevalence of pro-inflammatory M1 macrophages [14]. Additionally, EAT shows increased expression of p53 mRNA, which is associated with inflammation, tissue damage, and elevated levels of high-sensitivity C-reactive protein (hs-CRP) [15]. EAT secretes pro-inflammatory cytokines, such as CCL2, interleukin-6 (IL-6), and tumour necrosis factor (TNF), as well as adipokines like chemerin, omentin-1, resistin, and serglycin, into the coronary lumen, thereby promoting systemic inflammation [16].

Locally, EAT inflammation contributes to the inflammation of coronary atherosclerotic plaques. Upregulation of innate immune

response pathways in EAT, including JUN N-terminal kinase (JNK), nuclear factor-κB (NF-κB), and Toll-like receptor (TLR) signalling, further stimulates the release of inflammatory mediators [17].

Additionally, the excessive influx of FFAs from EAT is mediated by group II secretory phospholipase A2 (sPLA2-II) [18] and FABP4, can infiltrate the adventitia and contribute to lipid accumulation in the perivascular adipose tissue (PVAT) that secretes pro-inflammatory adipocytokines to induce atherosclerotic plaques [19]. FABP4 enhances neutrophil recruitment and oxidative stress in macrophages, leading to structural heart disease and contractile dysfunction. In patients with CAD and chronic hyperglycaemia, increased signalling through advanced glycation end products (AGE) and their receptor (RAGE) [20], along with reduced glucose trans-
<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Mechanism of EAT-mediated cardiac diseases.</figcaption></figure>

**A. Development of coronary artery diseases:** In coronary artery disease, coronary EAT has dense inflammation, with many pro-inflammatory M1 macrophages. EAT releases pro-inflammatory cytokines and adipokines into coronary arteries, promoting systemic and local inflammation of atherosclerotic plaques. Excess free fatty acids (FFA) from epicardial fat may increase lipid infiltration. Chronic hyperglycemia exacerbates inflammation and increases oxidative stress and damaging endothelial cells (ECs).

**B. Arrythmia:** Left atrial EAT is located next to the left atrium, and releases profibrotic factors which can lead to atrial fibrosis. EAT can also contribute to atrial fibrillation by releasing pro-inflammatory factors. Additionally, excess free fatty acids (FFAs) from EAT can disrupt cardiomyocyte alignment affecting gap junctional structure, leading to irregular conduction and promoting re-entrant circuits. The increased activity of ion channel in ganglionated plexi enhances autonomic effects on atrial cardiomyocytes, prolonging action potential duration.

**C. HFpEF:** EAT can also promote myocardial inflammation through the secretion of cytokines, which may play a role in systemic inflammation in HFpEF. As obesity progresses, in EAT, there is a significant alteration of the population of T helper cell type 2 (Th2) and T regulatory cell (Treg), macrophage polarization, affecting in maintaining adipose tissue immune homeostasis and insulin sensitivity. Additionally elevated levels of monocyte differentiation, adaptive immune cells CD8-positive T lymphocytes cells as well as innate cells such as macrophages (M1 pro-inflammatory population) can be observed in EAT in obese patients. EAT accumulation can lead to local inflammation in heart, which contributes to oxidative stress, ROS accumulation in endothelial cells, microvascular damage and refraction, hypertrophic cardiomyocytes, and myocardial fibrosis, ultimately resulting in heart failure with preserved ejection fraction (HFpEF).

porter type 4 (GLUT4) levels, exacerbates oxidative stress and en-
dothelial damage.

The mechanisms by which EAT contributes to atherosclerosis are complex and involve factors such as inflammation, height-ened innate immune response, oxidative stress, endothelial injury, adipocyte stress, lipid buildup, and glucotoxicity. Patients with CAD tend to have greater EAT volume and thickness compared to in-dividuals without atherosclerosis. A coronary artery calcium (CAC) score above 10 is associated with increased EAT volume, which can predict 70 % atherosclerosis risk [21].

All together studies imply EAT involvement in both epicardial
CAD and coronary microcirculation dysfunction, highlighting its
significance in heart disease pathophysiology. Assessing EAT can
help predict the risk of major coronary events before calcium ac-
cumulation in atherosclerotic plaques, particularly in asymptomatic
individuals who are not obese.

Pro-arrhythmic effect

EAT-derived adipokines play a significant role in driving ox-
idative stress and myocardial fibrosis (Fig. 4B). Pro-inflammatory

and pro-fibrotic cytokines, such as activin A, visfatin, transform-
ing growth factor-β1 (TGF-β1), and MCP-1, promote fibroblast pro-
liferation and myofibroblast activation, contributing to both atrial
and ventricular arrhythmias [22]. Additionally, cytokines like lep-
tin, TNF-α, IL-1β, and IL-6 further exacerbate fibrotic processes.
Leptin, in particular, has been shown to affect calcium handling,
increase collagen I production in cardiac myofibroblasts, and en-
hance sodium reabsorption in the kidneys, all of which contribute
to cardiac fibrosis.

EAT can also affect local electrophysiological properties, such as the refractory period of the atria and pulmonary veins, sustaining atrial fibrillation. Research using cultured human induced pluripo-tent stem cell-derived cardiomyocytes suggest that local peri-atrial EAT accumulation, rather than overall cardiac fat, plays a key role in conduction abnormalities that underlie atrial fibrillation [23].

Recent advances in imaging, mapping, and ablation techniques have highlighted the epicardial region of the heart as a crucial factor in the development of ventricular arrhythmias associated with various cardiac diseases, including Brugada syndrome [24], arrhythmogenic cardiomyopathy (ACM), [25] and dilated cardiomy-
opathy. EAT is particularly significant in this context due to its production of pro-inflammatory and profibrotic factors. The accumulation of EAT, especially around the pulmonary veins and atrial structures, leads to both structural and electrical changes in the heart, increasing the risk of arrhythmias such as atrial fibrillation (AF). Notably, interventions targeting EAT, like epicardial left atrial appendage occlusion, seem to offer greater benefits compared to endocardial methods. These interventions can reduce levels of adrenaline, aldosterone, and sympathetic activity while increasing adiponectin.

The role of EAT in AF might be linked to its embryonic development. The embryonic epicardium can either generate coronary smooth muscle cells and cardiac fibroblasts or undergo adipogenic differentiation [2]. Atrial EAT develop from progenitor cells in the epicardium and from the secretions of atrial myocytes. Interestingly, the atrial natriuretic factor released by atrial myocytes in response to mechanical stress has adipogenic effects that may contribute to EAT formation [4]. The deregulated EAT may play adverse paracrine roles in cardiac arrhythmias and lipotoxic cardiomyopathies.

### Cardiomechanical interaction together with inflammation and neurohormonal mechanisms, leading to HFpEF

#### EAT in development of HF

Excessive accumulation of EAT within the pericardium exerts direct mechanical stress on the myocardium, leading to a constric-tive pericarditis-like situation, contributing to left ventricular hy-pertrophy, diastolic dysfunction and dilatation of the atria, which are considered typical hallmarks of heart failure with preserved ejection fraction (HFpEF) [26]. EAT-driven myocardial remodeling seems to play a significant role in the development of HFpEF espe-cially in woman where the increased peri-atrial adiposity is linked to an impaired atrial function [27].

The Multi-Ethnic Study of Atherosclerosis (MESA) provided valuable insights, revealing that higher pericardial fat volume (which includes both epicardial and pericardial fat) is associated with an increased risk of HF [28]. In this large study of 6785 par-ticipants, each standard deviation increased in pericardial fat vol-ume correlated with a 22 % higher risk of developing HF, even af-ter adjusting for baseline factors. Similarly, the Jackson Heart Study found that higher volumes of both EAT and VAT were linked to an increased risk of HFpEF [29]. The St. Francis Heart Study reveals that epicardial fat modifies the relationship between coronary cal-cium score and all-cause mortality [30].

EAT may also play a role in the development of HF through neurohormonal mechanisms involving both intrinsic adrenergic and cholinergic nerves, which interact with the external cardiac sympathetic and parasympathetic nervous systems. The increased catecholamine production by EAT might contribute to the higher incidence of atrial fibrillation in patients with HFpEF [31]. Addi-tionally, isoprenaline-stimulated expression of a macrophage apop-tosis inhibitor, CD5L, in EAT samples was higher in patients with HF [32].

EAT's unique characteristics and proximity to the heart make it an important player in the development and progression of HF-pEF, where metabolic dysfunction and inflammation are prominent (Fig. 4C). Advancing our understanding of these aspects will be crucial for developing effective, tailored therapies for HFpEF and related cardiovascular conditions. EAT is also associated with im-paired cardiorespiratory fitness and higher mortality in HFpEF.

As obesity progresses, there is also a shift in the balance of T helper type 2 (Th2) and regulatory T cells (Treg), alongside enhanced macrophage polarization and monocyte differentiation. The observed rise in pro-inflammatory macrophages (M1) and CD8+ T cells, coupled with a decline in anti-inflammatory M2

macrophages within EAT, suggests an enhanced inflammatory state linked to CAD and HF. This highlights the critical role of these immune cell populations in cardiovascular health. Furthermore, in the EAT of HF patients, the inflammatory milieu is characterized by an increased presence of innate immune cells like dendritic cells, which facilitate macrophage infiltration, and a decreased population of natural killer cells. These changes collectively amplify inflammation, increase the secretion of inflammatory cytokines, and contribute to development of HF [33].

Inflammation in EAT is also connected to the activation of the leucine-rich repeat (LRR)-containing proteins (NLR) family mem-bers (NLRP3) inflammasome, a protein complex that regulates in-flammation and pyroptosis, a form of programmed cell death. Markers of inflammation, such as α1-antichymotrypsin (serpin A3) creatine kinase B-type and matrix metallopeptidase (MMP) MMP14 and tumor protein (TP), TP53, are increased in EAT in HF, further emphasizing its inflammatory nature [34].

Among the 1554 participants (53.0 % females) without HF, EAT thickness was correlated with inflammation and fibrosis-related circulating biomarkers and negatively correlated with N-terminal pro-B-type natriuretic peptide (NT-proBNP), smaller left ventricu-lar end-diastolic dimension, myocardial longitudinal strain impair-ment, incident HF risk, and overall CVD risk [35].

In heart failure with reduced ejection fraction (HFrEF), EAT vol-ume is typically reduced compared to controls, likely due to poor nutrition and muscle wasting associated with intestinal congestion [36]. These shifts in gene expression may result from fibrosis and apoptosis occurring in EAT during end-stage organ disease. How-ever, in two other studies EAT was increased in HFrEF compared to controls and HFpEF [10,37,38]. Nevertheless, reduced EAT has been linked to worse left ventricular dysfunction and poorer outcomes [36,39]. The EAT in HFrEF may exhibit a more anti-inflammatory profile, while in HFpEF it tends to have a more pro-inflammatory profile, resulting in a direct harmful impact on the myocardium in HFpEF and a beneficial effect in HFrEF. A recent study using a deep learning model found that intrathoracic pericardial adipose tissue shares genetic links with abdominal visceral adiposity and inde-pendently predicts T2D and hypertension. However, these associ-ations lost significance after adjusting for visceral adipose tissue. The limitations of this study include the inability to distinguish between EAT and PAT, as well as reduced applicability to broader populations due to the healthier, primarily European composition of the UK Biobank cohort [40].

#### EAT in metabolic disease-mediated CVD

Diabetes, obesity, rheumatoid arthritis, human immunodeficiency virus infection can result in coronary atherosclerosis and an increased risk of myocardial infarction [9].

### Type 2 diabetes mellitus (T2DM)-
EAT is a critical player in the nexus of obesity and T2DM-mediated CVDs. Dysregulated glucose metabolism is a key clinical feature of T2DM and is closely linked to the abnormal accumulation of EAT [41]. The patients with im-paired fasting glucose, insulin resistance, or T2DM exhibit signifi-cantly greater EAT thickness over the right ventricular free wall, as well as increased EAT volume and area, compared to control sub-jects [42].

#### Obesity-
In obesity, the epicardium becomes a site of de-ranged adipogenesis, leading to the secretion of proinflammatory adipokines and causing atrial and ventricular fibrosis. EAT releases excessive FFAs, leading to lipotoxic effects on the heart. Lipotoxicity contributes to excessive oxidative stress, endoplasmic reticulum stress, and mitochondrial dysfunction, ultimately causing left ventricular (LV) diastolic dysfunction [43]. Accumulation of EAT over the ventricles is associated with myocardial fibrosis, ventricular hypertrophy, and elevated cardiac filling pressures—hallmark features of HFpEF. This relationship between EAT and myocardial dys-
<figure><img src="image_7.png" /><figcaption>**Fig. 5.** Clinically tested drugs targeting EAT. T2Diabetes and obesity and chronic inflammatory diseases can increase EAT. Clinically tested drugs that improve HFpEF may also reduce EAT accumulation. Their role in other CVD and EAT reduction remains to be further investigated.</figcaption></figure>

function has been studied across a range of populations, includ-
ing healthy individuals and patients with metabolic and CVDs. In-
creased EAT thickness has been consistently linked to higher LV
mass, worsening systolic and diastolic function, and a greater risk
of HF in these groups [36].

**Rheumatoid arthritis-** Patients with rheumatoid arthritis ex-
hibit greater EAT accumulation compared to healthy controls. This
increased EAT is accompanied by a rise in LV wall thickness and
diastolic dysfunction, with excessive EAT strongly correlating with
both diastolic dysfunction and disease duration [44]. Furthermore,
EAT accumulation has been linked to reduced global longitudinal
strain (GLS), which may contribute to LV myocardial systolic dys-
function in rheumatoid arthritis. This suggests that EAT could serve
as an imaging marker for detecting subclinical LV systolic insuffi-
ciency in these patients [45].

**Virus infection-** The novel COVID-19, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is associated with cardiac issues, including myocarditis, pericarditis, and thrombosis [46]. Visceral fat, particularly EAT, has been proposed as both a functional reservoir and amplifier of SARS-CoV-2. The inherent inflammatory environment of visceral fat may exacerbate the inflammatory response in COVID-19 patients, potentially leading to severe cardiovascular complications.

In summary, EAT is a bioactive organ that plays a crucial role in CVDs. Clinically, EAT can be measured using cardiac imaging techniques, which provide valuable insights for predicting and stratifying cardiovascular risk.

Pharmacological interventions targeting eat in cardiovascular
diseases

Promising strategies against HFpEF and arrhythmia emerge
from pathological insights in EAT, targeting the accumulation of
EAT. Recent studies have explored the effects of anti-inflammatory
and anti-hyperlipidemic drugs and diabetes medications that re-
duce the risk of several cardiovascular disorders, including HFpEF,
and exert parallel effects modifying the volume and biology of EAT
(Fig. 5).

1. Anti-cytokine Drugs: Inflammation drives the abnormal expansion of EAT. Anti-cytokine agents like IL-1 and IL-6 blockers the-

oretically could reduce EAT accumulation, yet clinical evidence
remains in debates. The D-HART trial found cardioprotective
benefits of IL inhibitors in individuals with HFpEF [47]. The sub-
sequent D-HART2 trial showed that anakinra, an IL-1 blocker.
did not improve exercise capacity in HFpEF patients [10]. More
rigorous trials are needed to clarify their effects on EAT in HF-
pEF with T2DM.

2. Anti-hyperlipidemic Drugs: Statins, 3-hydroxy-3-methylglutaryl coenzyme-A reductase inhibitors, which inhibit cholesterol production, exhibit anti-inflammatory properties. Research shows that statin therapy significantly reduces EAT thickness and inflammatory mediators. For instance, atorvastatin outperformed other lipid-lowering agents in EAT reduction [48]. New lipid-lowering agents like proprotein convertase subtilicin type 9 (PCSK9) inhibitors (alirocumab, evolocumab) also show promise, with a study reporting a 20 % reduction in EAT thickness after treatment [49]. Statins may lower mortality in HFpEF patients, yet randomized trials are essential for validation. Further research is needed to determine the role of hypolipidemic medicines in attenuation of aberrant EAT expansion in diabetic HFpEF, and HFrEF patients.

3. Anti-hyperglycemic Drugs: Metformin, an oral anti-hyperglycemic drug, is commonly used for T2DM to lower blood glucose levels by decreasing hepatic glucose production (gluconeogenesis) and to improve insulin sensitivity by increasing peripheral glucose uptake and utilization. Despite the exact mechanism by which metformin affects EAT is unclear, the role of metformin on EAT thickness reduction has already been shown [50]. Metformin also shows potential in lowering mortality and improving heart function in HFpEF [51].

Thiazolidinediones (TZDs), which are PPAR-γ agonists, acting as an insulin sensitizer, used to manage and treat T2D. TZD can reduce EAT inflammatory markers but may pose cardiovascular risks, limiting their use in HFpEF. Pioglitazone can also reduce EAT inflammatory markers (IL-6, TNF-α, resistin, and matrix metalloproteinase-9) in patients with coronary artery disease and T2DM [52]. However, the clinical use of TZDs in the treatment of HFpEF is limited, because of their potential to aggravate HF.

Dipeptidyl peptidase 4 (DPP-4) inhibitors, which inhibit
glucose-dependent insulin secretion by increasing bioactive in-
cretins, show potential in EAT management, but evidence remains
limited, because of their role in increasing risk of HF in T2DM [53].

Glucagon-like peptide-1 receptor agonists (GLP1-RAs) such as
such as liraglutide and semaglutide, which maintains glucose
homeostasis by stimulating glucose-dependent insulin secretion,
suppressing glucagon release, and inhibiting gastric emptying have
been shown by several investigations to reduce EAT and improve
glycolipid metabolism disorders, together with significant weight
loss and reduced C-reactive protein, with potential reductions in
HF hospitalizations [54–56]. However, liraglutide failed to reduce
visceral or subcutaneous fat and EAT accumulation in T2DM [57],
despite positively affecting heart function in HFpEF patients. How-
ever, liraglutide treatment improves LV stiffness and diastolic dys-
function and reduce mortality in HFpEF patients [58]. Because of
the small patient numbers of these studies, further research is still
necessary to confirm their role on EAT accumulations.

Sodium-glucose co-transporter 2 inhibitors (SGLT2-Is) such as empagliflozin, dapagliflozin, canagliflozin, ipragliflozin, and luseogliflozin are the newly developed anti-hyperglycemic agents, that bind to the SGLT2 transporter in the proximal tubule of the kidney to increase the urinary excretion of glucose [59]. Thus, SGLT2-Is promote glucose excretion and have anti-inflammatory effects. Research indicates SGLT2-Is significantly reduce EAT and improve metabolic outcomes [55,60]. These drugs, known for their glucose-lowering effects, also promote lipolysis and reduce EAT volume and improve inflammatory markers. They also enhance cardiac function and quality of life in HFpEF patients, suggesting a vital role in managing EAT expansion. As a result, SGLT2-Is show substantial prevention of abnormal EAT expansion and demonstrate promising therapeutic benefits in HFpEF, highlighting the need for further clinical validation.

In summary, while pharmacological interventions hold promise
for managing EAT and improving outcomes in HFpEF, further well-
designed studies are necessary for confirmation and validation.

Prokineticins as a potential therapeutic target for development
and accumulation of adipocyte tissues

Prokineticin and their Receptors

Prokineticins (PKs), hormones-, neuropeptides-, cytokines-like
proteins exist in two forms: prokineticin 1 (PK1) and prokineticin
2 (PK2). Both PKs interact with two closely related G protein-
coupled receptors (GPCRs), namely prokineticin receptor 1 (PKR1)
and prokineticin receptor 2 (PKR2). PKs and their receptors are
widely expressed across mammalian species, and their expression
is notably dynamic [61]. Both prokineticin receptors (PKR1 and
PKR2) are involved in distinct signaling pathways that contribute
to the development and progression of CVD.

Prokineticin 2 in Appetite Regulation

PK2 is known for its anorectic effects. Studies by Gardiner and colleagues demonstrated that intracerebroventricular administration of Bv8/PK2 in rodents leads to reduced food intake [62]. PK2 also influences food intake peripherally through brainstem activation of PKR1 [63]. PK2 injection into the olfactory bulbs (OB) induces anorexia, while PK2 short hairpin RNA injection leads to dysregulated feeding. PK2's anorectic effect is partially mediated through the melanocortin system in the hypothalamus, where it activates pro-opiomelanocortin (POMC)-expressing neurons and stimulates the release of α-melanocyte-stimulating hormone (α-MSH), which acts on the melanocortin-4 receptor to regulate feeding behavior [64].

<figure><img src="image_8.png" /><figcaption>Fig. 6. Role of PKR1 signaling in regulation of epicardial cell fate and cardiomyocyte communication.
Epicardial PKR1 signaling is involved in a switch between adipogenesis and vasculogenesis. Activation of PKR1 inhibits EAT but increases vasculogenesis by controlling EPDC fate. Additionally, epicardial PKR1 may be involved in cardiomyocyte maturation and regulation of rhythmicity. On the other hand, PKR1 signaling in cardiomyocyte induces survival pathway in a cell-autonomous fashion and increases neovascularization in a paracrine manner by increasing release of its own ligand, PK2.</figcaption></figure>

Role of Prokineticin on Insulin Resistance, and Diabetes-related disorders: cardiomyopathy and skeletal muscle dysfunction

Disruption of insulin translocation from endothelial cells (ECs) to skeletal muscle that is crucial for effective glucose uptake contributes to insulin resistance [65]. Endothelial-specific PKR1 knockout mice (EC-PKR1(-/−)) display an impaired capillary formation, reduced insulin uptake, and decreased glucose and insulin sensitivity, leading to symptoms such as polyphagia, polydipsia, and severe lipodystrophy [66]. This insulin resistance can be rescued by PKR1 gene transfection (Fig. 6). These findings are reminiscent to insulin resistance in human T2MD, characterized by impaired insulin secretion and endothelial dysfunction.

Diabetic cardiomyopathy involves heart changes due to diabetes and can lead to HF. Recent studies reveal that metformin, a common T2DM medication, provides cardioprotection via the PK2/PKR1 pathway [67]. In a model of insulin resistance, PKR1 enhances glucose uptake and GLUT4 translocation [68]. Reduced PKR1 levels are observed in obese mice and insulin-resistant human muscle cells, highlighting its role in maintaining insulin sensitivity. Targeting PKR1 could therefore be a promising strategy for improving muscle function in insulin-resistant conditions.

Prokineticin 2 in Obesity

PK signaling plays a significant role in regulating adipose tissue
growth, specifically suppressing the expansion of visceral and sub-
cutaneous fat. PK2, which is released from adipose tissue, binds to
PKR1, a receptor subtype predominantly expressed in adipocytes,
thereby reducing adipose tissue growth in visceral and epicardial
area [69,70]. Notably, lower levels of PKR1 transcripts have been
observed in the visceral and subcutaneous adipose tissues of obese
individuals, highlighting the crucial role of PKR1 in adipose tissue
regulation.

In vitro studies have shown that PK2 inhibits the proliferation of mouse preadipocytes and their differentiation into adipocytes through PKR1 signaling, as demonstrated in the preadipocyte cell line after adipogenic stimulation. Furthermore, adipose tissue-specific PKR1 knockout mice (ad-PKR1(-/−)) develop obesity due to
adipocyte hyperplasia and macrophage infiltration in their adipose tissues [70].

**PK2 levels in patient with metabolic disorders:** Wang and his colleagues investigated PK2 levels in childhood obesity and found significantly elevated fasting serum PK2 levels in obese children compared to age-matched healthy controls [71]. This positive correlation with BMI was evident in both obese children with and without nonalcoholic fatty liver disease, a common complication of obesity. No significant correlation was observed between PK2 and inflammatory markers such as IL-6, TNF-α, or white blood cell and neutrophil counts in this cohort. However, circulating PK2 levels were positively correlated with the homeostatic model assessment for insulin resistance across the cohort.

Mortreux and his colleagues found lower plasma PK2 levels in individuals with T2DM compared to nondiabetics within the D.E.S.I.R. cohort (Data from an Epidemiologic Study on the Insulin-Resistance syndrome) [72]. This association vanished after adjusting for BMI and/or caloric intake. Univariate regression analyses demonstrated significant inverse correlations between PK2 and BMI, waist circumference, fasting blood glucose, HbA1c, and low-density lipoprotein cholesterol. In their multivariable model, PK2 levels remained associated with BMI, energy intake, and plasma low-density lipoprotein cholesterol. Interestingly, the study found no significant abnormalities in sleep or circadian rhythms among obese children, suggesting that elevated PK2 levels are not primarily due to disrupted circadian rhythms. In these cohort studies, development of EAT and its correlation with PK2 levels remains to be studied.

## Prokineticin 2 in the suppression of EAT and development of vascular cells

Promising strategies against HFpEF and arrhythmia could reduce the accumulation of EAT and promote its replacement by vascular cells (Fig. 6). The PK2/PKR1 pathway inhibits the formation of EAT from human EPDCs under high-calorie intake by activating an epigenetic pathway via H3K27me3 demethylase 6A (KDM6A). This activation reduces PPARγ expression and adipogenic signaling, thereby suppressing the differentiation of EPDCs into adipocytes [69]. Although H3K27me3 demethylation typically derepresses Wnt expression, leading to Wnt/β-catenin signaling activation and inhibition of adipogenesis, Wnt expression remained unchanged in hEPDCs treated with PK2 [69]. Activated PPARγ, in high calory context, promotes the degradation of β-catenin via a proteasomal pathway, thereby counteracting the anti-adipogenic effects of β-catenin. However, when β-catenin is stabilized in the cytoplasm by inhibiting its proteasomal degradation, it blocks the interaction of other factors with PPARγ, thereby inhibiting PPARγ activity in hEPDCs treated with PK2 [69]. An association between elevated PPARγ levels and reduced β-catenin signaling has also been linked to the development of arrhythmogenic right ventricular cardiomyopathy, a rare condition characterized by the accumulation of fatty tissue in both ventricles, with prominent involvement of the right ventricular myocardium. Furthermore, genetic inactivation of PKR1 specifically in the epicardium using Gata5cre mice results in cardiomyopathy and cardiac arrhythmias in adult mice, likely due to the development of lipotoxicity in their hearts. After coronaryligation, only 20 % of these mice survived, emphasizing the link between congenital heart dysfunction and increased mortality after myocardial infarction [73].

In hEPDCs, it has been shown that PK2/PKR1 signaling is involved in a switch between EAT development and vascular replacement; PK2-mediated maintenance of epithelial stability is associated with hypomethylation of H3K27me3 by KDM6A, which is responsible for the asymmetric division. In fact, asymmetric

division by PK2/KDM6A allows regaining epithelial features with self-renewal and rapid generation of vascular precursors. Conversely, PK2/PKR1-mediated KDM6A activation suppresses respective H3K27me3 marks on vascular gene promoters, promoting vascular formation by enhancing the differentiation of endothelial cell precursors into vascular smooth muscle and ECs. Genetic inactivation of PKR1 in the epicardium using WT1 GFPcre mice leads to cardiac developmental defects during the embryogenesis [73] due to vascular refraction. These findings suggest that PK2/PKR1 signaling regulates the balance between vasculogenic and adipogenic differentiation in hEPDCs through epigenetic modifications.

Epicardium-specific PKR1(-/−) mice (Tcf21⁺-PKR1(-/−)) have exhibited reduced capillary formation [74]. However, when subjected to a high-fat diet, these mice show increased EAT and severe regression of blood vessels, leading to cardiac dysfunction. Similarly, in hEPDCs, PKR1 ablation using siRNA inhibits vasculogenic differentiation, resulting in heightened adipocyte formation when exposed to adipocyte induction medium in Tcf21⁺ cells. Tcf21⁺-PKR1(-/−) mice, with PKR1 specifically deleted in adult epicardium that mostly corresponds to Tcf21⁺ positive cardiac fibroblasts precursors, displayed significant accumulation of the pericardial adipose tissue, PVAT and atrioventricular groove adipose tissue, along with disrupted vascular networks and compromised cardiac function, when they were fed with high-fat diet [74].

Indeed, EPDCs fate can be controlled via cardiomyocyte-dependent paracrine pathways. As shown in transgenic mice, over-expression of PKR1 in their cardiomyocytes exhibited induction of neovascularization due to PK2 release from cardiomyocytes which promotes EPDC differentiation into the endothelial and smooth muscle cells [75].

Overall, the data indicate that PK2/PKR1 signaling is crucial for determining epicardial cell fate, influencing the shift from adipogenesis to vasculogenesis both in vitro and in vivo (Fig. 7).

<figure><img src="image_9.png" /><figcaption>**Fig. 7.** Targeting PKR1 for treatment of obesity and diabetes and EAT-related cardiac disorders. Additionally, prokineticin signalling, particularly through its receptor PKR1, has emerged as a key regulator of appetite, energy expenditure, insulin sensitivity, and adipose tissue development, especially in the context of high caloric intake. Moreover, PKR1 agonist protects heart against myocardial infarction and anticancer mediated cardiotoxicity. PKR1 signaling reduces EAT and increases vasculogenic differentiation. Targeting PKR1 may offer a valuable strategy for managing obesity and related metabolic disorders and EAT-mediated CVDs.</figcaption></figure>
## Summary and conclusion

The epicardium appears to be more responsive to lipogenesis than other types of visceral adipose tissue, likely due to the abundance of EPDCs and other mesenchymal cells within the epicardium. While these cells serve as sources of cardiac progenitors that differentiates into cardiac cells during foetal development, in adulthood, they mainly differentiate into adipocytes under the stress stimuli. This unique characteristic of EAT provides a novel perspective on the pathophysiology of major CVDs.

EAT plays a direct role in the development and progression of CAD through various mechanisms, primarily promoting inflammation, but also contributing to endothelial damage, oxidative stress, and lipid and glucose accumulation in the proximal coronary arteries. In AF, EAT acts as a new pathogenic substrate by locally secreting factors that induce fibrosis and disrupt the neurohormonal regulation of atrial myocytes. Additionally, in HF, EAT contributes to disease progression via pathways that include excessive fatty acid release leading to ectopic lipid accumulation, overexpression of local pro-inflammatory and pro-fibrotic cytokines with proarrhythmogenic properties, and increased β-adrenergic receptor activation. EAT accumulation is linked to myocardial fibrosis, microvascular rarefaction, and elevated fibrosis markers, all of which are associated with both atrial fibrillation and HF.

Importantly, EAT is a modifiable and measurable cardiovascular risk factor that enhances the precision of cardiovascular risk stratification. Contemporary imaging techniques, such as echocardiography, CT, and MRI, offer accessible means to assess EAT, providing valuable tools for modern cardiologists.

Pharmacological modulation of EAT has shown promising cardiometabolic benefits. Recent advances in diabetes therapies, including SGLT2-Iso and GLP-1 receptor agonists, have demonstrated their ability to reduce pro-inflammatory cytokine production and improve glucose regulation, thereby directly influencing EAT. These findings suggest EAT as a potential therapeutic target, although further research is required to clarify its role in HFpEF and other conditions.

Additionally, prokineticin signalling, particularly through its receptor PKR1, has emerged as a key regulator of appetite, energy expenditure, insulin sensitivity, and adipose tissue development, especially in the context of high caloric intake. Moreover, PKR1 agonist protects heart against myocardial infarction and anti-cancer mediated cardiotoxicity. PKR1 signaling reduces EAT and increases vasculogenic differentiation. Targeting PKR1 may offer a valuable strategy for managing obesity and related metabolic disorders and EAT-mediated cardiovascular disorders (Fig. 7).

Despite advancements, numerous questions about the role of EAT in CVDs remain unresolved. Additional research is necessary to investigate the mechanisms, diagnostic implications, and therapeutic potential of EAT in HF. This knowledge aims to enhance the treatment of CVDs and inform future clinical applications.

## Future perspectives

Future research should prioritize the following key areas:

**Accurate Identification of EAT:** Future studies must standardize the nomenclature and classification of EAT and paracardial adipose tissue that is essential to avoid confusion in clinical and research settings to improve consistency in research and therapeutic interventions.

**Comprehensive Assessment of EAT:** Future research should focus on utilizing three-dimensional assessments through non-invasive cardiac imaging techniques like echocardiography, CT, and MRI, which enable clinicians to assess cardiovascular risk more accurately to refine cardiovascular risk stratification and identify potential therapeutic targets. Elucidating the pathways involved

in EAT development will lead to the discovery of new target to reduce EAT transformation for cardiovascular health. Current evidence points to the therapeutic potential of PKR1 agonists for treating obesity and CVDs, including EAT-related conditions, marking them as promising candidates for future research.

In conclusion, the accumulation of EAT is closely linked to the development and progression of various CVDs, including HFpEF, CAD, and AF. Future research should prioritize the accurate identification and assessment of EAT, explore compartment-specific differences, and investigate novel therapeutic strategies targeting EAT. By integrating these insights, future studies can contribute to personalized and more effective treatments for CVDs, particularly in the context of increasing HFpEF prevalence.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## CRediT authorship contribution statement

Martina Vincenzi: Writing – original draft, Visualization, Validation, Software. Canan G. Nebigil: Writing – review & editing, Visualization, Validation, Supervision, Project administration, Investigation, Funding acquisition.

## Acknowledgements

This study was supported by grants Fondation de France (R20085MM and R023044MM), and MV was financially supported by Sapienza University of Rome doctoral fellowship and fellowship of Italian Society of Pharmacology (SIF).

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