Adenosine A3 Receptor: From Molecular Signaling to Therapeutic Strategies for Heart Diseases

Cardiovascular diseases (CVDs), particularly heart failure, are major contributors to early mortality globally. Heart failure poses a significant public health problem, with persistently poor long-term outcomes and an overall unsatisfactory prognosis for patients. Conventionally, treatments for heart failure have focused on lowering blood pressure; however, the development of more potent therapies targeting hemodynamic parameters presents challenges, including tolerability and safety risks, which could potentially restrict their clinical effectiveness. Adenosine has emerged as a key mediator in CVDs, acting as a retaliatory metabolite produced during cellular stress via ATP metabolism, and works as a signaling molecule regulating various physiological processes. Adenosine functions by interacting with different adenosine receptor (AR) subtypes expressed in cardiac cells, including A1AR, A2AAR, A2BAR, and A3AR. In addition to A1AR, A3AR has a multifaceted role in the cardiovascular system, since its activation contributes to reducing the damage to the heart in various pathological states, particularly ischemic heart disease, heart failure, and hypertension, although its role is not as well documented compared to other AR subtypes. Research on A3AR signaling has focused on identifying the intricate molecular mechanisms involved in CVDs through various pathways, including Gi or Gq protein-dependent signaling, ATP-sensitive potassium channels, MAPKs, and G protein-independent signaling. Several A3AR-specific agonists, such as piclidenoson and namodenoson, exert cardioprotective impacts during ischemia in the diverse animal models of heart disease. Thus, modulating A3ARs serves as a potential therapeutic approach, fueling considerable interest in developing compounds that target A3ARs as potential treatments for heart diseases.


Introduction
Adenosine initiates a wide range of cardiovascular effects upon binding to various subtypes of adenosine receptors (ARs), including A 1 AR, A 2A AR, A 2B AR, and A 3 AR, expressed within cells of the cardiovascular system [1,2].During myocardial ischemic conditions, adenosine is released in large amounts and subsequently activated by A 1 AR and A 3 AR to confer ischemic preconditioning (IPC) in the heart [3,4].Extensive research has focused on the role of A 1 AR in protecting the heart from damage, revealing that the stimulation of A 1 ARs can trigger protective responses within cardiac tissues [5,6].However, recent studies have continuously revealed that A 3 AR also induces protective responses similar to those activated by A 1 AR [7,8].However, the role of A 3 ARs is not as extensively documented in pathological cardiac tissues as other ARs.This is primarily due to the low expression of A 3 AR in the heart and disparities in its adenosine binding affinity between human and rodent models [9].
The A 3 AR subtypes are distributed throughout various tissues and organs such as the testes, lungs, kidneys, heart, brain, and spleen, although the level of expression varies among species [10].High intensities of A 3 AR localization have been observed in several immune and inflammatory cells, including neutrophils, eosinophils, and mast cells, emphasizing the pivotal role of A 3 ARs in inflammation processes [11,12].Interestingly, the differential expression of A 3 AR has been documented in both pathological and normal cells.The overexpression of A 3 ARs has been detected in several types of cancer cells, including leukemia, lymphoma, astrocytoma, melanoma, lung, breast, and renal carcinomas, whereas it is minimally expressed in normal cells [10,13].
Human A 3 AR was initially cloned and characterized in 1993 [14].In terms of sequence similarity, A 3 AR structure presents a significant difference in species homolog.Human A 3 ARs are more closely related to those in sheep than in rats, exhibiting 85% versus 74% similarity, respectively [15], and equine A 3 ARs also show high similarity to both humans and sheep, suggesting unique ligand recognition and pharmacological behaviors, which pose challenges for fully understanding and characterizing these receptors [16].The A 3 AR gene (ADORA3), a single-copy gene located on human chromosome 1p21-p13, features a coding region consisting of two exons separated by a single intron of approximately 2.2 kb, coding for 319 amino acids [17].The promoter of the A 3 AR gene lacks a TATA motif but contains a CCAAT motif and binding sites for multiple transcription factors, such as specificity protein 1 (SP1), transcription factor nuclear factor interleukin 6 (NF-IL6), GATA1, and GATA3, suggesting its significant role in immune responses [15].A 3 AR plays a complex role in the cardiovascular system, as its activation helps to limit the injury processes occurring in the heart during ischemia [4,16].Additionally, during the reperfusion phase, A 3 AR exerts an anti-inflammatory action, thereby minimizing further damage to the heart tissue [18].Studies conducted on both cardiomyocytes and intact hearts of rats subjected to ischemia/reperfusion (I/R) showed that A 3 AR agonists can reduce the infarct size associated with the upregulation of pro-survival signaling pathways such as extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphoinositide 3-kinase (PI3K)/Akt pathways [19,20].Moreover, the stimulation of A 3 ARs protects against cardiotoxic side effects induced via chemotherapy, indicating the cardioprotective effects of A 3 AR signaling [21,22].The activation of A 3 ARs has been shown to influence vascular tone in multiple ways, including the inhibition of coronary flow in isolated mouse aorta hearts [23], the induction of vasoconstriction in hamster arterioles [24], and the restoration of vascular reactivity following hemorrhagic shock in rats [25].
A balance of A 3 AR expression levels is essential for optimal cardiac function.For instance, the overexpression of A 3 ARs can lead to decreased heart rate, the preservation of energetics, and the protection of the heart against ischemic damage [26].However, higher levels of A 3 AR expression are associated with the development of dilated cardiomyopathy [27].Remarkably, A 3 AR signaling is involved in growth promotion in the heart, as the genetic ablation of A 3 AR attenuated pathological cardiac remodeling characterized by hypertrophic growth and fibrotic changes [28].While a direct cardioprotective effect mediated by A 3 AR has been observed, the A 3 AR expression level in cardiomyocytes is relatively low.Therefore, there is a potential for indirect protection, possibly by affecting the function of immune cells such as mast cells and neutrophils, which abundantly express A 3 ARs.However, species differences need to be considered in AR signaling-mediated mast cell degranulation [11,12].Overall, A 3 AR plays a complex role in the cardiovascular system, involving both direct and indirect mechanisms.Developing compounds targeting A 3 ARs, either agonists or antagonists, could potentially be manipulated to offer cardiovascular therapeutic benefits.However, it requires careful consideration due to the potential risk of adverse effects.
Building on these considerations, this review aims to elucidate the essential role of A 3 ARs in the heart.It offers an extensive review of the signal transduction pathways associated with A 3 ARs and explores the use of A 3 AR agonists and antagonists in various models of pathological heart conditions.

The Distribution of A 3 ARs in the Cardiovascular System
The cardiovascular system is composed of various cell types, including cardiomyocytes, cardiac fibroblasts, smooth muscle cells, and endothelial cells.Although A 3 AR is expressed in the atria and left ventricle of rat hearts, its mRNA levels are relatively low compared to those in other tissues (e.g., testes, kidneys, lungs, brain, liver, eyes, and spleen) (reviewed in [15]), and this expression is downregulated during hypertension [29].There are also significant differences in expression levels within and between species.For instance, high levels of A 3 AR mRNA are reported in the testes and mast cells of rats, while other rat tissues, including the cardiovascular system, exhibit low levels of this mRNA [10,30].In humans, the lungs and liver express high amounts of A 3 AR mRNA, whereas low levels have been found in the aorta and brain [14].
Additionally, A 3 AR expression has been identified in blood vessels, such as rat thoracic aorta [29] and human coronary and carotid arteries [31,32].In situ hybridization results detected A 3 AR mRNA expression in the vascular smooth muscle layer and, to a lesser extent, in the endothelial layer of the mouse aorta [33].Interestingly, a recent study has demonstrated that A 1 AR mRNA is the most abundant, followed by A 2 AR and A 3 AR in human right atrial tissue [34].In addition, the stimulation of A 3 ARs regulated spontaneous calcium release in isolated human atrial myocytes [34], indicating the role of A 3 AR in calcium homeostasis.The stimulation of A 3 AR, using its selective agonist, reduced infarction size in isolated rabbit hearts, exerting a cardioprotective effect in I/R injury [35].These findings suggest that, despite the limited data on its distribution in cardiovascular tissues, A 3 AR plays a crucial role in cardiovascular protective effects.

G i Protein-Dependent Signaling
A 3 AR is a cell surface receptor belonging to the family of G protein-coupled receptors (GPCRs).Upon binding to its agonist, A 3 AR can trigger both G protein-dependent and independent signaling pathways, which vary by cell type; these pathways are associated with anticancer, anti-inflammatory, and cardioprotective effects (reviewed in [36,37]).A 3 AR signaling is characterized by its interaction with both G i and G q proteins.This dual coupling enables A 3 AR to mediate a diverse range of biological responses depending on the type of G protein activated (reviewed in [36,37]).Notably, A 3 AR demonstrates a significant association with the G i protein, resulting in the inhibition of cyclic adenosine monophosphate (cAMP) production and thereby activating the mitogen-activated protein kinase (MAPK) pathway, including ERK1/2 and p38 [38] (Figure 1).
A 3 AR, when coupled with G i , results in the inhibition of adenylyl cyclase (AC), leading to a decrease in cAMP formation.The low levels of cAMP subsequently decrease protein kinase A (PKA) activity, which in turn attenuates the phosphorylation of target proteins and eventually affects a series of intracellular events [13].Following A 3 AR coupling with G i protein, the inhibition of cAMP formation indirectly reduces glycogen synthase kinase-3 beta (GSK-3β) activity, leading to an increase in the phosphorylation of β-catenin.This prevents β-catenin from translocating to the nucleus, resulting in the decreased expression of various target genes that regulate cell cycle progression, such as cyclin D1 and c-myc, ultimately inhibiting cell growth (reviewed in [13,15,37]).Of note, the activation of G i protein-dependent signaling can have detrimental effects on the heart.Previous studies have demonstrated that the cardiac-specific expression of a G i -coupled receptor, a synthetic receptor designed to interact with the G i protein, led to adverse outcomes in mouse hearts, including a slowing heart rate, arrhythmia, and lethal cardiomyopathy [39,40].A3AR-mediated signaling pathways for cardiovascular protective effects.The signal transduction pathway of A3ARs in the cardiovascular system involves the activation of Gαq and Gαi proteins.When A3AR is coupled with Gαq, it stimulates PLC activity and induces transcription factors EGR, leading to the increased proliferation of coronary smooth muscle cells.Additionally, the coupling of A3AR with Gαq leads to elevated levels of IP3 and DAG, with the latter activating PKC, which mediates an anti-ischemic effect in isolated hearts.A3AR can also transduce its signals independently of Gα protein mediating RhoA-PLD interaction, thereby protecting the heart from ischemic damage in cardiomyocytes.When coupled with the Gαi protein, A3AR inhibits GSK-3β activity through the PI3K pathway, improving mitochondrial function.In addition, A3AR activates mitoKATP, further enhancing mitochondrial function.Furthermore, A3AR exhibits an anti-apoptotic effect by activating the PI3K/Akt pathway, and an anti-hypertrophic effect through the Gαidependent activation of MEK1/ERK, ultimately conferring cardioprotective effects in cardiomyocytes.Abbreviations: DAG, diacylglycerol; EGR, growth response factors; ERK, extracellular signal-regulated kinase; GSK-3β, glycogen synthase kinase-3 beta; IP3, inositol triphosphate; MEK1, mitogen-activated protein kinase kinase 1; mitoKATP, mitochondrial ATPsensitive potassium channel; PKC, protein kinase C; PI3K, phosphoinositide 3-kinase; PLC, phospholipase C; PLD, phospholipase D; RhoA, Ras homolog family member A.
A3AR, when coupled with Gi, results in the inhibition of adenylyl cyclase (AC), leading to a decrease in cAMP formation.The low levels of cAMP subsequently decrease protein kinase A (PKA) activity, which in turn attenuates the phosphorylation of target proteins and eventually affects a series of intracellular events [13].Following A3AR coupling with Gi protein, the inhibition of cAMP formation indirectly reduces glycogen synthase kinase-3 beta (GSK-3β) activity, leading to an increase in the phosphorylation of β-catenin.This prevents β-catenin from translocating to the nucleus, resulting in the decreased expression of various target genes that regulate cell cycle progression, such as cyclin D1 and c-myc, ultimately inhibiting cell growth (reviewed in [13,15,37]).Of note, the activation of Gi protein-dependent signaling can have detrimental effects on the heart.Previous studies have demonstrated that the cardiac-specific expression of a Gi-coupled receptor, a synthetic receptor designed to interact with the Gi protein, led to adverse outcomes in mouse hearts, including a slowing heart rate, arrhythmia, and lethal cardiomyopathy [39,40].
The PI3K/Akt and ERK signaling cascades are recognized for their pivotal roles in cell survival and cardiac defensive mechanisms, with A3AR capable of transducing its signal through the activation of these pathways [19,20].In rat cardiomyocytes, the stimulation of A3ARs leads to the phosphorylation of Akt through the Gi/Go protein.
Simultaneously, the inhibition of PI3K prevented the activation of Akt, indicating the role of PI3K as a downstream mediator of the Gi protein-dependent signaling pathway for Akt activation [41].In addition, the role of A3ARs in cardioprotection during IPC has been A 3 AR-mediated signaling pathways for cardiovascular protective effects.The signal transduction pathway of A 3 ARs in the cardiovascular system involves the activation of G αq and G αi proteins.When A 3 AR is coupled with G αq , it stimulates PLC activity and induces transcription factors EGR, leading to the increased proliferation of coronary smooth muscle cells.Additionally, the coupling of A 3 AR with G αq leads to elevated levels of IP 3 and DAG, with the latter activating PKC, which mediates an anti-ischemic effect in isolated hearts.A 3 AR can also transduce its signals independently of G α protein mediating RhoA-PLD interaction, thereby protecting the heart from ischemic damage in cardiomyocytes.When coupled with the G αi protein, A 3 AR inhibits GSK-3β activity through the PI3K pathway, improving mitochondrial function.In addition, A 3 AR activates mitoK ATP , further enhancing mitochondrial function.Furthermore, A 3 AR exhibits an anti-apoptotic effect by activating the PI3K/Akt pathway, and an anti-hypertrophic effect through the G αi -dependent activation of MEK1/ERK, ultimately conferring cardioprotective effects in cardiomyocytes.Abbreviations: DAG, diacylglycerol; EGR, growth response factors; ERK, extracellular signal-regulated kinase; GSK-3β, glycogen synthase kinase-3 beta; IP3, inositol triphosphate; MEK1, mitogen-activated protein kinase kinase 1; mitoKATP, mitochondrial ATP-sensitive potassium channel; PKC, protein kinase C; PI3K, phosphoinositide 3-kinase; PLC, phospholipase C; PLD, phospholipase D; RhoA, Ras homolog family member A.
The PI3K/Akt and ERK signaling cascades are recognized for their pivotal roles in cell survival and cardiac defensive mechanisms, with A 3 AR capable of transducing its signal through the activation of these pathways [19,20].In rat cardiomyocytes, the stimulation of A 3 ARs leads to the phosphorylation of Akt through the G i /G o protein.Simultaneously, the inhibition of PI3K prevented the activation of Akt, indicating the role of PI3K as a downstream mediator of the G i protein-dependent signaling pathway for Akt activation [41].In addition, the role of A 3 ARs in cardioprotection during IPC has been documented, as the same group of investigators demonstrated the preconditioning effect of adenosine involved with A 1 AR-and A 3 AR-mediated anti-apoptotic effects via the mitogen-activated protein kinase kinase 1 (MEK1) and ERK1/2 pathway [20].Interestingly, A 3 AR-induced IPC also involved a PI3K-dependent pathway in rat cardiomyocytes [20].Moreover, previous studies have also reported the pharmacological preconditioning effects of resveratrol, a compound found abundantly in grapes, in protecting the heart, mainly mediated by A 3 ARs [42,43].The stimulation of A 3 ARs leads to the activation of two different signaling pathways, including the PI3K/Akt pathway and the response element-binding protein (CREB)-dependent pathway, through both Akt-dependent and -independent signaling in the heart tissues.Both pathways lead to the activation of apoptosis regulator Bcl-2 signaling, providing survival signals to the heart [42,43].Furthermore, A 3 AR signaling mediated the activity of MAPK signaling pathways, such as ERK1/2, which has been documented in various cell models [15].For instance, the triggering of A 3 ARs in Chinese hamster ovary (CHO) cells expressing human A 3 ARs resulted in the robust activation of ERK1/2 through G i/o protein-dependent signaling pathway, involving the activation of PI3K, Ras, and MEK to induce ERK1/2 activation [44,45].Moreover, in the rat heart, the triggering phase of beta-adrenergic during IPC led to the generation of adenosine and reactive oxygen species (ROS), which then stimulated A 3 ARs, resulting in the activation of specific signaling pathways, including PI3K/Akt and ERK.This may lead to the inhibition of GSK-3β activity, ultimately closing the mitochondrial permeability transition pore (mPTP) and providing cardioprotection [46].In addition, A 3 AR-mediated cardioprotection involved the activation of pro-survival signaling pathways including MEK1/2-ERK1/2 and PI3K/Akt (Figure 1), resulting in decreased caspase-3 activity, a marker of cellular apoptosis, and protecting the rat hearts from I/R injury [19].
The role of p38 MAPK has also been demonstrated in rat cardiomyocytes.The activation of p38 MAPK occurred downstream from the opening of the mitochondrial ATPsensitive potassium (mitoK ATP ) channels following A 3 AR activation, thereby participating in the intracellular signaling pathway and contributing to a protective effect on the heart against ischemic injury in rats [47].While the precise mechanism of p38 MAPK activation remained unclear, it is suggested that ROS generated upon mitoK ATP channel activation may trigger this process [47].However, in certain cell types, such as A375 human melanoma cells, A 3 AR stimulation did not promote ERK1/2 or p38 MAPK activation.Instead, it transduced its signal through the PI3K/Akt signaling pathway, resulting in a reduction in the basal levels of ERK1/2 phosphorylation and the inhibition of cell proliferation [48].Furthermore, in response to A 3 AR stimulation, the inhibition of both p38 MAPK and nuclear factor kappa B (NFκB) pathways has been observed, associated with the suppression of inflammatory processes in human synoviocytes derived from osteoarthritis patients [49].
In the heart, several downstream mediators were reported to be involved in the cardioprotective effects of A 3 ARs, including GSK-3β, mitoKATP channel, protein kinase C (PKC), ROS, connexin 43, and mPTP (reviewed in [13]).The stimulation of A 3 ARs suppresses GSK-3β activity through the PI3K/Akt pathway in isolated rat hearts, leading to the inhibition of mPTP.This, in turn, protects the heart from I/R injury by reducing infarction size [50].

G q Protein-Dependent Signaling
A 3 AR interacts with G q to activate phospholipase C (PLC), which then produces inositol triphosphate (IP 3 ), increasing intracellular Ca 2+ levels, thereby regulating various cellular activities and activating PKC [10,13,51] (Figure 1).Although the upstream signaling pathways of PKC are not fully understood, previous studies have underscored the essential role of A 3 AR and PKC in the heart.For example, the activation of A 3 ARs has been shown to induce delayed preconditioning cardioprotection in the mouse heart, specifically through the activation of PKC-δ isoform via mechanisms yet to be fully elucidated [52].It has been suggested that A 3 AR-mediated PKC activation may trigger various downstream mediators, including ERK1/2 and p38 MAPK, and contribute to cardioprotective effects [2,15].Moreover, the modulation of Ca 2+ release from the cardiac sarcoplasmic reticulum has been implicated in PKC activation, independent of PLC activation through A 3 ARs in isolated rat hearts [53].
IPC has been observed to protect guinea pig hearts by triggering a signaling cascade involving A 3 AR stimulation, and the subsequent activation of PKC-ε and mitochondrial aldehyde dehydrogenase type-2 (ALDH2) in cardiac mast cells [54].This series of reactions helps to prevent the detrimental effects associated with the renin-angiotensin system (RAS) activation during I/R injury [54].In the rat brain, the oxygen/glucose deprivation-induced depression of synaptic transmission is mediated by the coactivation of metabotropic glutamate receptor 1 (mGluR1) and A 3 AR [55].Interestingly, the blocking of A 3 AR and PKC, along with a decrease in internal Ca 2+ , has been shown to prevent this depression of synaptic transmission in CA3 pyramidal neurons, underscoring the significant role of PLC-dependent G protein signaling in ischemic conditions [55].

ATP-Sensitive Potassium Channels (K ATP )
Additionally, A 3 AR stimulation facilitated the activation of the ATP-sensitive potassium (K ATP ) channels, leading to their opening and conferring cardioprotective effects during I/R injury in rabbit hearts [56].However, it remained uncertain whether the channel opens as a direct result of A 3 AR stimulation or following the activation of PKC [56].Furthermore, the function of A 3 ARs is also involved in the activation of the sarcolemmal K ATP channels, offering protection against reperfusion injury in mouse hearts [57].However, the molecular mechanism underlying this effect is not elucidated in the study.

G Protein-Independent Signaling
Interestingly, A 3 AR signaling has been shown to mediate both G protein-dependent and -independent pathways (Figure 1).The stimulation of A 3 AR can transduce its signals independently of traditional G protein-mediated signaling pathways, such as phospholipase D (PLD) and Ras homolog family member A (RhoA) [13].In cultured chick embryo cardiomyocytes, the RhoA-PLD interaction is a key player in A 3 AR signaling, protecting heart cells from ischemic injury [58].However, the underlying mechanisms of cardioprotective effects during cardiac injury mediated by G protein-independent pathways are not quite understood.

The Role of A 3 ARs in Heart Diseases
Over time, as our understanding of the modulation of A 3 ARs has advanced, its pivotal roles in cardiac physiology and pathology have become increasingly apparent.This has led to a focused effort to explore the potential of selective A 3 AR agonists for protecting the heart.Various A 3 AR-specific agonists, including IB-MECA, Cl-IB-MECA, CP-532903, and CP-608039, have been examined for their effectiveness in treating a range of heart conditions, such as I/R injury and cardiac remodeling [5,15,37].The cardioprotective effects of A 3 AR agonists have been studied across different animal models, including rodents and rabbits, as shown in Table 1.Notably, variations in agonist profiles for treatment efficacy have been observed in accordance with interspecies differences [59].Furthermore, some A 3 AR agonists may interact with other subtypes of ARs under different conditions, emphasizing the need for the cautious interpretation of results [59].

Contributions of A 3 ARs in Ischemic Heart Disease
The activation of A 3 ARs has shown beneficial outcomes in cardiac ischemia in both in vitro and in vivo studies with various agonists targeting A 3 AR reported for cardiac ischemic protection [57,[60][61][62][63].In 1994, a study revealed that treatment with aminophenylethyladenosine (an A3AR agonist) produced protective effects similar to adenosine against I/R injury.The blocking of A 3 AR with BWA1433, an A 3 AR antagonist, abolished this protective effect in rabbit hearts, with A 1 AR not mediating this outcome [64].In mice models of I/R injury, a reduction in infarct size was observed following A 3 AR activation using Cl-IB-MECA (A 3 AR agonist).However, this beneficial effect was abolished in A 3 AR-knockout mice, confirming the essential role of A 3 ARs in ischemic heart protection [65] (Table 1).
Moreover, the modulation of A 3 AR levels in the heart is crucial for optimal cardiac function and protection against ischemia [27].By employing cardiomyocyte-specific A 3 AR transgenic mice engineered to express A 3 ARs at varying levels, including low (one copy of the transgene) and high (six copies of the transgene), the data revealed that while mice with low levels of A 3 ARs exhibited no discernible cardiac abnormalities, those with high levels experienced several cardiac issues [27].Nonetheless, both sets of transgenic mice demonstrated improved outcomes in reducing infarct size compared to wild-type mice, indicating a conferred protection against I/R injury.
The agonism of A 3 AR consistently led to a reduction in infarct size across various study models of I/R injury (Table 1).The efficacy of CP-532903 (piclidenoson; A 3 AR agonist) in protecting against I/R injury has been demonstrated in mice [60].In a rabbit model of regional ischemia, CP-532903 effectively decreased infarct size without inducing hemodynamic liabilities [61].Additionally, CP-532903 showcased its ability to reduce infarct size in an in vivo mouse model of infarction and to enhance functional recovery in isolated mouse hearts subjected to I/R injury.These protective effects were attributed to the activation of sarcolemmal K ATP channels [57], and similar cardioprotective mechanisms were also observed in adult mouse ventricular cardiomyocytes [60].
The administration of IB-MECA has shown significant promise in reducing infarct size by 61% compared to the control group in conscious rabbits, providing the cardioprotective effects mediated through the PKC-dependent pathway against I/R injury without causing significant hemodynamic changes [62].In agreement with another study, preconditioning with IB-MECA prior to low-flow ischemia in Langendorff rat hearts improved the recovery of heart function and reduced apoptosis after 150 min of reperfusion, indicating that the activation of A 3 AR signaling can reduce cell death following I/R injury [66].Moreover, CP-608039, an A 3 AR agonist, with high water solubility suitable for intravenous route administration, has been developed for preventing perioperative myocardial ischemic injury [61,67].Currently, there is controversy regarding the timing of cardioprotective effects of A 3 AR signaling, with ongoing debate on whether pre-ischemic or post-ischemic treatment with A 3 AR agonists is more beneficial [7,15], indicating the need for further research.[69].However, treatment with Cl-IB-MECA (A 3 AR agonist) during reperfusion has demonstrated the ability to reduce infarct size by decreasing leukocyte recruitment.This reduction is attributed to the inhibition of neutrophil migration and pro-inflammatory actions through the activation of A 3 ARs in bone marrow-derived cells, effectively suppressing the inflammatory response during reperfusion injury in a mouse model [18].Furthermore, the activation of A 3 ARs with 2-CL-IB-MECA (A 3 AR agonist) led to the activation of prosurvival signaling pathways, including the MEK1/2-ERK1/2 and PI3K/Akt pathways.This activation was accompanied by a decrease in apoptosis, necrosis, and caspase 3 activity in rat cardiomyocytes in response to hypoxia/reoxygenation injury [19].
The benefits of stimulating A 3 AR signaling in preconditioning the heart have been well documented in several studies [20,64,66].However, paradoxical effects of A 3 AR were observed in multiple experiments.One study revealed that while A 3 AR activation is typically beneficial in protecting the heart from I/R injury under normal circumstances, A 3 AR-knockout mice surprisingly displayed a post-ischemic recovery response compared to wild-type mice [70].Consistently, mice lacking A 3 AR exhibited smaller infarct sizes compared to wild-type mice [71].Moreover, A 3 AR exacerbated I/R injury, possibly through involvement in promoting inflammatory responses, and it is not essential for the protective mechanism of IPC [71].Indeed, the paradoxical effect of A 3 AR might be due to the species differences.A 3 AR signaling in mast cells, particularly in rodents, may trigger a pro-inflammatory response through mast cell degranulation, potentially harming the heart [11,12,54].Consequently, mice lacking A 3 AR may exhibit beneficial effects on cardiac function.This evidence highlighted the complexity of A 3 AR signaling mechanisms and suggested a potential role of inflammation in both I/R injury and IPC [59,72].

Protection of Oxidative Stress, Apoptosis, and Mitochondrial Dysfunction
The stimulation of A 3 ARs has been found to exhibit a cardioprotective effect against doxorubicin-induced cardiotoxicity.In cultured rat cardiomyocytes, treatment with Cl-IB-MECA (A 3 AR agonist) effectively mitigated the adverse effects of doxorubicin through multiple mechanisms [73].Cl-IB-MECA exerted its cardioprotective effects by inhibiting cardiac cell damage, which was mediated by the activation of cellular antioxidant enzymes, leading to a reduction in free-radical generation and lipid peroxidation [73].Furthermore, Cl-IB-MECA demonstrated the ability to protect cells from mitochondrial damage, thereby preventing a decrease in ATP levels [73].Consistent with these findings, pretreatment with Cl-IB-MECA was shown to attenuate left ventricular dysfunction in rat hearts, improve SR calcium storage capacity, and prevent mitochondrial calcium overload, thus protecting the cardiomyocytes [22].Additionally, the activation of A 3 ARs with Cl-IB-MECA was associated with the activation of pro-survival signaling pathways, leading to a reduction in caspase-3 activity in rat hearts, thereby conferring cardioprotection against I/R injury [19].

Impacts of A 3 ARs on Heart Failure
Endogenous adenosine can protect the overloaded heart against the development of pathological heart failure [74].A study showed that mice deficient in extracellular adenosine production, stemming from a deletion in the CD73 gene, showed maladaptive tissue remodeling with the induction of cardiomyocyte hypertrophy, fibrosis, and left ventricular dilation and dysfunction in contrast to their wild-type counterparts under chronic systolic overload [75].However, the role of specific AR subtypes that contribute to this pathological heart has rarely been discussed.It has been noted that alterations in A 3 AR levels exert a significant influence on both cardiac phenotypes and functions.The cardiomyocyte-specific overexpression of A 3 ARs in mice led to dilated cardiomyopathy with an elevation of hypertrophic markers and systolic dysfunction, whereas a low level of A 3 ARs showed no detectable deleterious events [27].Remarkedly, mice harboring more than six copies of the A 3 AR transgene experienced even worse outcomes, succumbing within 4 weeks of birth [27].Consistently, A 3 AR overexpression in the heart resulted in dose-dependent atrioventricular (AV) block and sinus nodal dysfunction, as well as the induction of bradycardiomyopathy in mice [76].These observations collectively underscore the deleterious impact of amplified A 3 AR signaling on cardiac functions, especially in the failing heart.Manipulating A 3 AR levels in the heart could offer possibilities for heart failure patients, and A 3 ARs could be considered a potential candidate for gene therapy in the context of heart failure.

Hypertrophic Effects
Pressure overload is a critical factor in cardiovascular diseases, particularly hypertension.The A 3 ARs have been shown to have detrimental effects in animal models of pressure overload as shown in Table 2. Following chronic pressure overload, A 3 AR-knockout mice exhibited a decrease in left ventricular hypertrophy and dysfunction, along with a reduction in myocyte size and fibrosis compared to wild-type mice [28].The antagonism of A 3 ARs with MRS1911 not only reduced oxidative stress and the elevated levels of hypertrophic markers but also potentiated the anti-hypertrophic effects of CADO, an adenosine analog, in phenylephrine-induced hypertrophy in isolated mouse cardiomyocytes [28].Additionally, MAPKs, ERK, and JNK, which are often activated and contribute to cardiac hypertrophy and heart failure, were also reduced in the presence of an A 3 AR antagonist [28].These findings collectively suggest that A 3 AR plays a significant role in mediating adverse effects in the pressure-overloaded heart.

Fibrotic Effects
There is a limited number of studies focusing exclusively on the role of A 3 ARs in cardiac fibrosis.However, a study by Lu and colleagues [28] shed light on this aspect by demonstrating that A 3 AR-knockout mice exhibited a significant reduction in left ventricular fibrosis compared to wild-type mice in a model of pressure overload (Table 2).Moreover, the previous findings showed that mice subjected to an early-life reduction in nephron numbers combined with chronic high salt intake (UNX-HS mice) developed hypertension and exhibited cardiac hypertrophy and fibrosis along with renal injury compared to controls [77].Interestingly, A 3 AR-knockout mice displayed a significant attenuation of these pathologies and showed higher baseline levels of pro-and anti-inflammatory cytokines.This elevation in immune homeostasis may contribute to the resistance of mice lacking A 3 AR to UNX-HS-induced renal and cardiac pathologies [77].In summary, these studies collectively suggested that A 3 AR signaling played a significant role in mediating the pathological effects of cardiovascular and renal injury.The lack of hemodynamic effects of A 3 AR stimulation has been documented in several models.Previous findings revealed that the agonism of A 3 AR with IB-MECA did not produce any effects on heart rate or blood pressure measured for 1 h, even at higher doses of up to 300 µg/kg, unlike other AR agonists such as CCPA (A 1 AR agonist) and CGS 21680 (A 2A AR agonist), which altered these hemodynamic parameters in the rabbit model [62].In addition, IB-MECA showed no impact on ischemic contractility, as evidenced by the absence of any significant change in the contractile rate and force following I/R injury in preconditioned rat hearts [66].In the rabbit model of I/R injury, CP-532903 (A 3 AR agonist) remarkably reduced infarct size in a dose-dependent manner without notably affecting hemodynamic variables such as mean arterial pressure, heart rate, and contractile rate and force, compared to the control group [61].Moreover, following I/R, A 3 AR-overexpressing mice showed better preservation of cardiac function and ATP energy stores, leading to cardioprotection [26].Overall, these suggested the protective role of A 3 AR in I/R injury without affecting the overall hemodynamic parameters.Nevertheless, it should be noted that mice overexpressing A 3 AR exhibited a lower basal heart rate and contractility compared to wild-type mice [26].
The role of A 3 ARs in arrhythmias is less well defined compared to other AR subtypes.However, an in vivo study has demonstrated that the overexpression of A 3 ARs in mice can considerably alter the electrophysiological function of the heart, particularly affecting the sinus and AV nodes [76].Mice overexpressing A 3 ARs exhibited various pathological changes, including complete AV block, sinus bradycardia, atrial enlargement, and fibrosis [76].Furthermore, elevated levels of A 3 AR expression were associated with the development of bradycardia-tachycardia syndrome, a type of cardiac arrhythmia, and may contribute to bradycardiomyopathy [76].

Involvement of A 3 ARs in Hypertension
A 3 AR has shown significant implications for vascular functions with extensive distributions in endothelial cells and vascular smooth muscle cells, reinforcing the idea that A 3 AR signaling may play a crucial role in hypertension and the pathophysiology of vascular diseases [78].Previous studies have demonstrated that A 3 AR signaling is involved in vascular contraction [78,79] and that A 3 AR regulates cAMP levels and influences blood pressure in response to adenosine in the mice model [33].The role of A 3 ARs on endotheliumdependent contraction was confirmed in mouse aorta [78].Treatment with Cl-IBMECA, an A 3 AR agonist, induced contractions in wild-type mouse aorta with an intact endothelium but not in endothelium-denuded tissue, with this effect being abolished in A 3 AR-knockout mice (Table 2).Interestingly, A 3 AR-mediated contraction primarily relied on the endothelial cyclooxygenase-1 (COX-1) signaling pathway, contributing to vascular tone regulation [78].Subsequent findings further corroborated the role of A 3 ARs in vascular contraction using the A 3 AR-knockout mice model, suggesting that vascular contraction involved ROS production mediated by A 3 ARs through the activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subtype 2 or NOX2 [79].In conditions of hemorrhagic shock in rats, a decrease in A 3 AR expression was observed, correlating with diminished vasoreactivity to norepinephrine-induced vascular contraction response [25].However, in the rat hemorrhagic shock model, exposure to IB-MECA considerably improved the response of the abdominal aorta to norepinephrine, potentially augmenting vasoconstriction and restoring the vascular function of vascular smooth muscle cells [25].
A 3 AR signaling was found to mediate the proliferation of vascular cells since the A 3 ARinduced proliferation of human coronary smooth muscle cells occurred through the activation of PLC and the induction of early growth response factors (EGRs), EGR2 and EGR3 [31].In aortic vascular smooth muscle cells, A 3 AR-knockout mice exhibited decreased cell proliferation alongside an increase in the level of lysyl oxidase, an enzyme crucial for maintaining the structure and function of blood vessels through its involvement in crosslinking processes of the extracellular matrix [80].However, A 3 AR deficiency alone is not significant enough to control the vascular response to injury or protect against atherogenesis in vivo [80].In addition, A 3 AR is involved in regulating vascular functions in nonvascular cell types, such as mast cells.A 3 AR induced arteriolar constriction through mast cell degranulation in hamster cheek pouch arterioles, thus mediating vasoconstriction [24].

The Role of A 3 AR Agonists in the Treatment of Heart Diseases
Although the findings from animal models of heart disease in various previous studies remain inconclusive, earlier research has shown that several A 3 AR agonists, including piclidenoson (IB-MECA) and namodenoson (2Cl-IB-MECA), effectively shield hearts from I/R injury in various animal studies (Table 1).However, their practical application in clinical settings, particularly for patients with acute myocardial infarction, is restricted as they need to be administered before the onset of ischemia.Recent research suggests that the cardioprotective impact of preconditioning manifests early in reperfusion, primarily by targeting the mPTP opening via the activation of reperfusion injury salvage kinases (RISKs) like ERK and PI3K [81,82].Similarly, past studies have shown that namodenoson and piclidenoson offer cardioprotection against I/R injury when administered upon reperfusion in animal models (Table 1).Thus, an A 3 AR agonist could potentially be suitable for patients with acute myocardial infarction; however, its tendency to induce systemic hypertension at higher doses in certain species necessitates further research to verify its safety and efficacy across various animal species [29,78].As of now, no agonist targeting A 3 ARs has been advanced into clinical trials for the treatment of heart disease, highlighting a gap in the translation of preclinical findings to potential therapeutic applications for A 3 AR agonists in cardiovascular medicine.

Clinical Studies of A 3 AR Agonists for the Treatment of Non-Cardiac Diseases
In addition to its role in cardiovascular diseases, A 3 AR has been highlighted across multiple cell types implicated in various pathological conditions, especially cancer and inflammation [15,51,83].Consequently, there is substantial interest in exploring its potential as a therapeutic target for both anti-tumor and anti-inflammatory therapies.This section provides a summary of clinical studies on two selective A 3 AR agonists, namodenoson and piclidenoson, detailing research progress and potential applications of A 3 AR agonists in non-cardiac diseases.These include non-alcoholic fatty liver disease (NAFLD), hepatocellular carcinoma (HCC), and chronic plaque psoriasis, as outlined in Table 3.

Namodenoson (2Cl-IB-MECA; CF-102)
NAFLD, characterized by excessive fat accumulation in the liver associated with metabolic dysfunction, ranks among the most prevalent chronic liver disorders globally, impacting approximately 25% of the adult population [84].NAFLD is considered the most common cause of chronic liver disease such as cirrhosis (liver fibrosis) and HCC [84,85].Besides NAFLD, HCC is also one of the major global health problems, accounting for about 75-85% of all liver cancer cases [86].Interestingly, it has been reported that the expression of A 3 ARs is higher in liver cells derived from inflammatory and tumor tissues, but not in normal liver cells [83,87].Furthermore, targeting A 3 ARs has demonstrated the ability to elicit anti-inflammatory and anti-cancer effects, emphasizing the promising prospect of developing A 3 AR agonists as a treatment option for liver diseases [88].
To assess the efficacy and safety of namodenoson, a phase II randomized, dose-finding trial was performed in NAFLD patients aged older than 18 years with or without nonalcoholic steatohepatitis (NASH) (N = 60) [89].The trial administered two distinct doses of namodenoson (12.5 or 25 mg twice daily) for 12 weeks (Table 3).Patients receiving namodenoson, particularly in the 25 mg group, showed a pronounced decrease in serum ALT levels, an indicator of liver inflammation, compared to the placebo group, with a higher proportion achieving normalization at 16 weeks.Concurrently, improvements in other liver disease-related parameters such as AST and adiponectin were evident, with both doses demonstrating good tolerability [89].Overall, namodenoson led to improvements in liver function and inflammation, particularly at the higher dose.However, this study was limited by several factors including a small number of patients, a short treatment period, and the absence of post-treatment liver biopsy to monitor the effect of the drug on the liver [89].
HCC and cirrhosis are intricately linked, with cirrhosis serving as a major risk factor for HCC, thereby affecting liver function and mutually influencing each other, presenting a particularly challenging clinical scenario [90].A phase II randomized clinical trial was conducted to evaluate the efficacy and safety of namodenoson as a second-line therapy (after failing first-line sorafenib) in advanced HCC and Child-Pugh B cirrhosis patients (N = 78) [91].Patients were randomized to receive either namodenoson (25 mg twice daily) or placebo until discontinuation (Table 3).Regrettably, namodenoson did not demonstrate an improvement in overall survival (primary endpoint) compared to placebo (median overall survival, 4.1 months vs. 4.3 months, p = 0.46).However, namodenoson was well tolerated without any adverse events, and a significant improvement in 12-month overall survival was documented in a subgroup of patients with a Child-Pugh score of 7 (44% vs. 18%, p = 0.028) [91].

Piclidenoson (IB-MECA; CF-101)
To evaluate the safety, tolerability, and pharmacokinetics of piclidenoson, a phase I clinical study in healthy men in Europe was conducted as shown in Table 3.Following oral administration, piclidenoson reached its maximum concentration (C max ) of 81.6 ng/mL from a single 5 mg dose, with a peak time (T max ) of 1-2 h and a half-life (T 1/2 ) of about 9 h in healthy men [92].Single oral doses of up to 5 mg and repeated doses of up to 4 mg every 12 h for 7 days were well tolerated, while higher doses caused side effects like flushing, tachycardia, nausea, and vomiting [92].However, given that medications are frequently administered intravenously for treating patients with acute myocardial infarction, the safety and pharmacokinetics of piclidenoson should be investigated via this route.
Piclidenoson has been elucidated in the context of psoriasis, a type of autoimmune disease characterized by the rapid proliferation of keratinocytes, resulting in itchy and red patches of skin covered with silvery scales.In phase II/III trials, CF-101 (piclidenoson) demonstrated the safety profile in patients with moderate-to-severe plaque psoriasis [93].The majority of patients were males (63.5%) and White/Caucasian (99.0%).Recently, the efficacy and safety of piclidenoson were further investigated in psoriasis patients through the COMFORT-1 study, a phase 3 clinical trial (Table 3).This study enrolled 529 patients with moderate-to-severe chronic plaque psoriasis, who were randomized to receive piclidenoson (2 or 3 mg), apremilast (30 mg), or placebo twice daily [94].Both doses of piclidenoson exhibited comparable efficacy and favorable safety profiles.However, the primary endpoint, the proportion of patients achieving ≥ 75% improvement in psoriasis area and severity index (PASI) from baseline to 16 weeks, was achieved only in 3 mg BID of piclidenoson (9.7% vs. 2.6%, piclidenoson vs. placebo, p = 0.037) [94].Furthermore, a noninferiority test comparing the efficacy of piclidenoson and apremilast revealed that neither the 3 mg nor the 2 mg doses of piclidenoson displayed comparable efficacy to apremilast in improving psoriasis, as assessed with PASI-75 after 32 weeks of treatment (17.0% vs. 20.8% vs. 26.2%,respectively; p = 0.88 and p = 0.43 for the respective comparisons vs. apremilast).Overall, although the effectiveness of piclidenoson is not comparable to that of apremilast, its beneficial effects show a trend toward improvement, supporting further development of piclidenoson for treating psoriasis [94].

Limitations and Conclusions
Today, the pharmaceutical landscape is actively engaged in the development of various drug targets for the management of CVDs, particularly heart diseases [95,96], among which A 3 AR stands out as a promising candidate, although it has not yet received approval.In recent years, increasing knowledge about the role of A 3 ARs in heart diseases has been documented in several preclinical models.Even though some findings suggest that mod-ulating A 3 AR signaling confers a cardioprotective function, the available preclinical and clinical data remain insufficient for application in patient care.The dynamic expression of A 3 ARs across different tissue contexts in pathological states, the genetic variations of A 3 AR signaling, and disparities in agonist/antagonist binding profiles between rodents and humans underscored the limitations in directly extrapolating findings from animal models to humans.Moreover, the specificity of A 3 AR-selective ligands may have been compromised, particularly at higher concentrations, leading to unintended off-target effects on other AR subtypes.Furthermore, experimental techniques for studying A 3 AR function are limited, requiring new pharmacological probes and molecular methods to validate its expression in mammalian cardiomyocytes.Gene-knockout studies have provided insights into the functional roles of A 3 ARs but may trigger compensatory mechanisms that influence experimental outcomes.It was noteworthy that while A 3 AR activation demonstrates positive effects, excessive A 3 AR activation might have contributed to pathological cardiac remodeling, including hypertrophy and fibrosis.Lastly, a significant limitation of A 3 AR agonists and antagonists was their potential adverse effects, which must be carefully considered.
In conclusion, our current review offered updated insights into the signal transduction pathways of A 3 ARs.These pathways notably involved G protein-dependent signaling cascades, particularly involving G i and G q proteins, alongside several downstream signaling effectors.Today, various A 3 AR agonists have shown protective effects and demonstrated efficacy in preventing arrhythmias, protecting against myocardial infarction, and reducing cardiac hypertrophy, fibrosis, and myocardial dysfunction in preclinical models of heart diseases.However, the role of A 3 AR antagonists in these contexts remains less explored, with limited available data.While existing A 3 AR agonists or antagonists are still in earlystage clinical trials, their potential therapeutic implications for heart diseases remain largely unexplored.Nevertheless, animal studies have provided promising insights, bringing the therapeutic application of A 3 AR-targeting compounds notably closer to realization.

Figure 1 .
Figure1.A3AR-mediated signaling pathways for cardiovascular protective effects.The signal transduction pathway of A3ARs in the cardiovascular system involves the activation of Gαq and Gαi proteins.When A3AR is coupled with Gαq, it stimulates PLC activity and induces transcription factors EGR, leading to the increased proliferation of coronary smooth muscle cells.Additionally, the coupling of A3AR with Gαq leads to elevated levels of IP3 and DAG, with the latter activating PKC, which mediates an anti-ischemic effect in isolated hearts.A3AR can also transduce its signals independently of Gα protein mediating RhoA-PLD interaction, thereby protecting the heart from ischemic damage in cardiomyocytes.When coupled with the Gαi protein, A3AR inhibits GSK-3β activity through the PI3K pathway, improving mitochondrial function.In addition, A3AR activates mitoKATP, further enhancing mitochondrial function.Furthermore, A3AR exhibits an anti-apoptotic effect by activating the PI3K/Akt pathway, and an anti-hypertrophic effect through the Gαidependent activation of MEK1/ERK, ultimately conferring cardioprotective effects in cardiomyocytes.Abbreviations: DAG, diacylglycerol; EGR, growth response factors; ERK, extracellular signal-regulated kinase; GSK-3β, glycogen synthase kinase-3 beta; IP3, inositol triphosphate; MEK1, mitogen-activated protein kinase kinase 1; mitoKATP, mitochondrial ATPsensitive potassium channel; PKC, protein kinase C; PI3K, phosphoinositide 3-kinase; PLC, phospholipase C; PLD, phospholipase D; RhoA, Ras homolog family member A.

Figure 1 .
Figure1.A 3 AR-mediated signaling pathways for cardiovascular protective effects.The signal transduction pathway of A 3 ARs in the cardiovascular system involves the activation of G αq and G αi proteins.When A 3 AR is coupled with G αq , it stimulates PLC activity and induces transcription factors EGR, leading to the increased proliferation of coronary smooth muscle cells.Additionally, the coupling of A 3 AR with G αq leads to elevated levels of IP 3 and DAG, with the latter activating PKC, which mediates an anti-ischemic effect in isolated hearts.A 3 AR can also transduce its signals independently of G α protein mediating RhoA-PLD interaction, thereby protecting the heart from ischemic damage in cardiomyocytes.When coupled with the G αi protein, A 3 AR inhibits GSK-3β activity through the PI3K pathway, improving mitochondrial function.In addition, A 3 AR activates mitoK ATP , further enhancing mitochondrial function.Furthermore, A 3 AR exhibits an anti-apoptotic effect by activating the PI3K/Akt pathway, and an anti-hypertrophic effect through the G αi -dependent activation of MEK1/ERK, ultimately conferring cardioprotective effects in cardiomyocytes.Abbreviations: DAG, diacylglycerol; EGR, growth response factors; ERK, extracellular signal-regulated kinase; GSK-3β, glycogen synthase kinase-3 beta; IP3, inositol triphosphate; MEK1, mitogen-activated protein kinase kinase 1; mitoKATP, mitochondrial ATP-sensitive potassium channel; PKC, protein kinase C; PI3K, phosphoinositide 3-kinase; PLC, phospholipase C; PLD, phospholipase D; RhoA, Ras homolog family member A.

Table 1 .
Protective effects of A 3 AR agonists in preclinical models of heart diseases.
■Reduced infarct size and necrosis■ ■ Improved contractile function against ischemic preconditioning

Table 2 .
Detrimental effects of A 3 ARs in heart diseases.

Table 3 .
Clinical studies of A 3 AR agonists in non-cardiac diseases.