Cyclic AMP-dependent regulation of ryanodine receptors in healthy and diseased hearts

Abstract

Cyclic adenosine monophosphate (cAMP) is a critical second messenger in cardiomyocytes, regulating essential cellular functions. Upon G-protein-coupled receptor stimulation, adenylyl cyclase (AC) synthesizes cAMP, which phosphodiesterase (PDE) enzymes subsequently degrade. Recent studies challenge the traditional view of uniform cAMP signaling, revealing nanodomain-specific regulation within cardiomyocytes. This localized cAMP signaling modulates key Ca²⁺-handling proteins, including ryanodine receptor type-2 (RyR2), through channel-bound protein kinases and PDEs. Additionally, nucleoside-diphosphate kinases (NDPKs), particularly NDPK-C, contribute to cAMP synthesis and RyR2 regulation. Elevated NDPK-C levels in failing hearts correlate with increased cAMP levels, enhanced sarcoplasmic reticulum Ca²⁺ release, and cardiac arrhythmias. Furthermore, cAMP influences the expression of Ca²⁺-handling proteins. This review examines the mechanisms governing cAMP levels in the sarcoplasmic reticulum nanodomain and their role in regulating RyR2 function in healthy and diseased hearts.

1. Introduction

Cyclic 3′,5′-adenosine monophosphate (cAMP) is a pivotal second messenger in cardiomyocytes, mediating the response to external stimuli such as epinephrine through adrenoceptor signaling pathways critical for cardiac function. Since its Nobel prize-winning discovery by Dr. Earl Sutherland, Jr. [1], the cAMP signaling cascade has been extensively studied. Sutherland elucidated that hormones like epinephrine activate G-protein-coupled receptors (GPCRs), triggering G-protein binding to guanosine-5′-triphosphate (GTP) and dissociation into Gα and Gβγ subunits. The GTP-bound Gα subunit activates adenylyl cyclase (AC), catalyzing cAMP synthesis. This cAMP then activates downstream effectors, including protein kinase A (PKA) and exchange protein activated by cAMP (Epac).

In cardiomyocytes, PKA phosphorylates key Ca²⁺-handling proteins, such as L-type voltage-gated Ca²⁺ channel (LTCC) [2], ryanodine receptor type-2 (RyR2) [3], and phospholamban (PLN) [4], thereby regulating cardiac function. Advances in cAMP sensors [5,6] and imaging technologies have revealed that cAMP signaling occurs in a nanodomain-specific manner, localized at organelles like the sarcoplasmic reticulum (SR) and mitochondria [7,8], enabling differential regulation of LTCC at the plasma membrane and RyR2 at the SR [9].

While global GPCR signaling and RyR2-mediated Ca²⁺ handling are covered well in the existing literature, this review focuses on localized cAMP signaling and cAMP-dependent Ca²⁺ handling in the heart. It discusses studies advancing mechanistic insights into localized RyR2 regulation via novel concepts: highly localized cAMP nanodomains; NDPKs (particularly NDPK-C) in localized cAMP synthesis; and PKA and Epac/CaMKII pathway impacts on RyR2 function and arrhythmogenesis. These discussions offer new perspectives on therapeutic strategies targeting RyR2-mediated Ca²⁺ handling in cardiac diseases.

2. The synthesis of cAMP

cAMP is synthesized by AC, an enzyme first identified in the liver and later confirmed to be critical for cardiac muscle function [10,11]. AC, primarily a transmembrane enzyme complex, converts one molecule of adenosine triphosphate (ATP) into one molecule of cAMP [11]. Mammals express ten AC isoforms [12,13], nine of which (AC1 to AC9) are transmembrane, while the tenth, soluble AC (sAC), resides in the cytoplasm [14]. All isoforms except AC8 [15] are abundant in the heart [16], with AC5 and AC6 being the most prevalent in cardiomyocytes [17,18]. Cardiac fibroblasts show prominent expression of the AC2 to AC7 isoforms [19]. However, since this review is primarily focused on the cAMP regulation of RyR2, we will be discussing the role of ACs in cardiomyocytes.

Okamura et al. [20] reported no change in basal heart function, whereas Tang et al. [21] observed increased left ventricular contractility at baseline in AC5 knockout (KO) mice. Both studies, however, noted elevated heart rates, likely due to impaired parasympathetic regulation of cAMP production in AC5 KO mice [22]. Additionally, AC5 and AC6 are subject to Ca²⁺-mediated inhibition, and acetylcholine binding to Gi subunits in GPCRs suppresses cAMP synthesis by inhibiting AC. [22] Binding of acetylcholine, the predominant neurotransmitter regulating the heart rate, to the M2 muscarinic receptor activates the inhibitory G_i protein, causing its α_i subunit to break off and inhibit AC5/6 [23]. This results in lower cAMP levels, which in turn reduces RyR2 activity.

Recent research has highlighted AC1’s role in regulating sinoatrial (SA) node automaticity through compartmentalized cAMP production in response to Ca²⁺ dynamics [24]. AC1 co-localizes with key Ca²⁺-handling proteins, such as LTCC and RyR2, in cardiomyocyte nanodomains [24]. SA node-specific AC1 silencing via CRISPR/Cas9 induced SA nodal dysfunction, sinus arrhythmias, and diminished cAMP responses to isoproterenol at the plasma membrane, cytosol, and SR [24]. Furthermore, SA1-deficient mice exhibited RyR2-mediated Ca²⁺ leak from the SR, consistent with AC1-RyR2 co-localization [24]. These findings underscore the critical role of AC isoforms in localized cAMP production within distinct nanodomains of cardiomyocytes and nodal cells.

Finally, Li et al. [25] reported that AC9 can associate with KCNQ1 in adult cardiomyocytes and, in complex with PKA, PP1, PDE4D3, can regulate the I_Ks current in a localized regulation of β-adrenergic signaling. While there are reports of low levels of expression of AC2 and AC4, they are not reported to be major regulators of cAMP signaling in adult cardiomyocytes [26].

2.1. The degradation of cAMP

The degradation of cAMP is an essential control mechanism in cells, ensuring precise regulation of downstream signaling pathways. Phosphodiesterases (PDEs), first identified by Dr. Sutherland Jr. during his studies of GPCR pathways [27], inactivate cAMP without releasing inorganic phosphate. PDEs are activated by magnesium (Mg²⁺) and inhibited by caffeine [27]. They are metallic phosphohydrolases that cleave the 3′,5′-cyclic phosphate bond of cAMP, rendering it unable activate downstream effectors such as PKA (PKA) [28,29].

Mammals express 11 PDE families (PDE1-PDE11), encoded by 21 genes, producing over 100 isoforms. In the heart, seven PDE families are expressed: PDE1-PDE5, PDE8, and PDE9 [30]. Expression varies by cardiac region and subcellular domain; for instance, PDE4A and PDE4D are more abundant in the atria than the ventricles [31]. Initially, PDE4D was implicated in localized cAMP regulation at RyR2 [32], while PDE4B and PDE4D regulated LTCCs. [33] Recent studies using transgenic mice with Förster Resonance Energy Transfer (FRET)-based cAMP-biosensors targeted to caveolin-rich plasma membrane, SERCA2a, and RyR2 nanodomains revealed a nuanced pattern: PDE4B predominantly controls cAMP dynamics in caveolin-rich plasma membrane and RyR2 nanodomains, whereas PDE4D primarily regulates the SERCA2a nanodomain [34].

Precise cAMP regulation near Ca²⁺-handling proteins is critical to prevent SR Ca²⁺ leak, which can impair cardiac contractility or trigger fatal arrhythmias [35]. PDE4D deficiency, for example, promotes heart failure (HF) and ventricular arrhythmias by enhancing SR Ca²⁺ leak [32]. In the atria, PDE4 accounts for 15 % of PDE activity in humans but 60 % of cAMP hydrolysis in mice, highlighting species differences [31]. PDE4 inhibition increases atrial L-type Ca²⁺ current (I_Ca,L) and Ca²⁺ sparks in human atrial and nodal cardiomyocytes [31,36]. Reduced PDE4 activity was also observed in patients with atrial fibrillation (AF) compared to those in normal sinus rhythm [31,37]. Genome-wide association studies (GWAS) have linked single nucleotide polymorphisms in PDE4 (rs153312 and SNP45) to cardioembolic stroke [38,39]. Furthermore, PDE3, but not PDE4, modulates the inotropic response to serotonin in permanent AF, with unaltered PDE3 activity in sinus rhythm or paroxysmal AF [40]. PDE3 contributes to atrial arrhythmias by altering cAMP levels in nanodomains, amplifying I_Ca,L, and increasing spontaneous SR Ca²⁺ release events [31]. Collectively, PDEs play a pivotal role in regulating localized cAMP levels within Ca²⁺-handling nanodomains, essential for normal cardiomyocyte function [41].

PDE8, encompassing PDE8A and PDE8B isoforms, is also a critical regulator of cAMP degradation in cardiomyocytes [42]. PDE8A, highly expressed in human and murine hearts, governs cAMP signaling in response to β-adrenoceptor stimulation, modulating LTCC, SR Ca²⁺ handling, and contractility. Its role in cAMP compartmentation, potentially via AKAP12 scaffolding, ensures localized signaling [43]. Notably, PDE8A knockouts exhibit enhanced Ca²⁺ transients and contractility, while PDE8B2 upregulation in AF may contribute to arrhythmias [42]. Insensitive to 3-isobutyl-1-methylxanthine (IBMX), PDE8 requires specific inhibitors like PF-04957325 for studies [44]. Further research into PDE8’s regulatory domains and pathological roles in cardiac disease states is warranted.

PDE1, PDE2, PDE5, and PDE9 also contribute to cAMP degradation in specific contexts. PDE1, activated by Ca²⁺/calmodulin (CaM), regulates cAMP in response to intracellular Ca²⁺ elevations, particularly in pathological hypertrophy [45]. PDE2, a cGMP-stimulated PDE, provides crosstalk between cAMP and cGMP signaling, modulating cAMP levels in response to nitric oxide signaling [46]. PDE5, primarily cGMP-specific, indirectly affects cAMP by inhibiting PDE3, which degrades both cyclic nucleotides, and is targeted by sildenafil in HF treatment [47]. PDE9, though less studied, is upregulated in HF and may regulate cAMP in specific nanodomains [48]. The interplay between these PDE families and cAMP-synthesizing enzymes, such as ACs, ensures tight control of cAMP levels. For instance, AC5 and AC6 inhibition by Ca²⁺ or Gα_i signaling counterbalances PDE-mediated cAMP hydrolysis, maintaining nanodomain-specific signaling [22]. Emerging evidence also suggests that nucleoside diphosphate kinases (NDPKs), through their role in G-protein activation, may enhance cAMP production, potentially overwhelming PDE activity in pathological states like HF and AF [46].

Therapeutic modulation of PDEs holds promise for treating cardiac diseases. PDE3 inhibitors (e.g., milrinone) enhance contractility in HF but are limited by proarrhythmic effects due to excessive cAMP elevation [49]. PDE4 inhibitors (e.g., rolipram) increase cAMP in atrial nanodomains, potentially exacerbating AF, but selective inhibition may restore sinus rhythm in specific contexts [31]. PDE5 inhibitors (e.g., sildenafil) improve cardiac function in HF by modulating cGMP-cAMP crosstalk, though their efficacy varies with disease stage [47]. Species-specific differences in PDE expression, such as the dominant role of PDE4 in murine atria compared to humans, complicate the translation of preclinical findings to clinical therapies [31]. For example, PDE4 contributes 60 % to cAMP hydrolysis in mice versus 15 % in humans, suggesting that PDE4-targeted therapies may have different effects in human patients. Future studies should focus on developing nanodomain-specific PDE inhibitors to precisely modulate cAMP signaling without off-target effects.

In summary, PDEs are critical regulators of cAMP degradation in cardiomyocytes, with distinct families (PDE1–PDE9) governing compartment-specific signaling at the SR, plasma membrane, and other nanodomains. Their interplay with ACs and NDPKs maintains cAMP homeostasis, directly influencing RyR2 activity and Ca²⁺ handling. Dysregulation of PDE activity in HF and AF promotes SR Ca²⁺ leak and arrhythmias, underscoring their therapeutic potential. However, species-specific differences and the complexity of nanodomain signaling necessitate further research to optimize PDE-targeted therapies. This intricate regulation of cAMP degradation sets the stage for exploring additional modulators, such as NDPKs, which further shape cAMP signaling in cardiomyocytes.

Finally, PDEs may regulate cAMP signaling indirectly through another second messenger, as cGMP (cyclic 3′,5′-guanosine monophosphate). Kodja et al. [50] showed a putative synergy between cAMP and cGMP signaling downstream of nitric oxide (NO) stimulation, possibly as a result of the inhibition of PDE3. However, a recent study showed that cGMP can negatively regulate local cAMP levels via PDE2 [51]. Collectively, these studies suggest a possibility of interplay between cAMP and cGMP signaling in the cardiomyocytes.

2.2. Nucleoside diphosphate kinases (NDPKs) in cAMP regulation in cardiomyocytes

NDPKs, encoded by the Nme gene family, are critical regulators of cAMP signaling in cardiomyocytes, influencing cardiac function and pathology [52]. The Nme family in humans in divided into two groups, group I (NDPK-A, NDPK-B, NDPK-C, and NDPK-D) is ubiquitously expressed and forms catalytically active hexamers, while group II is associated with cilia and flagella and is not discussed here [53]. This section focuses on group I NDPKs, particularly their role in cAMP regulation and their potential impact on RyR2 activity in the heart.

Group I NDPKs share high sequence homology and primarily function to transfer terminal phosphates from nucleoside triphosphates (NTPs) to nucleoside diphosphates (NDPs), maintaining nucleoside homeostasis [54]. NDPK-D is the only isoform expressed in mitochondria, whereas all NDPK-A through NDPK-D isoforms are expressed in the cytosol and at the plasma membrane of various tissues, including the heart. This phospho-transfer activity supports cellular processes, such as providing GTP for G-protein activation, a prerequisite for AC stimulation and cAMP production [55].

Beyond their canonical role, NDPKs exhibit a noncanonical histidine kinase activity, discovered in 2003, which enables receptor-independent G-protein activation [56,57]. NDPK-B, in the presence of ATP, auto-phosphorylates at His-118 and forms complexes with the Gβγ subunits of G-proteins, transferring phosphate to Gβ at His-266. This phosphate is subsequently relayed to the Gα subunit, activating G-proteins without the classical GDP/GTP exchange [56,57]. NDPK-C enhances this process by directly binding G-proteins via its N-terminal hydrophobic domain, anchoring the NDPK-B/G-protein complex to the plasma membrane [58]. In contrast, NDPK-A does not interact with G-proteins, underscoring isoform specificity [59]. In healthy cardiomyocytes, NDPK-C preferentially associates with Gαs, increasing cAMP levels and sensitizing cells to isoproterenol-accelerated contractility [60]. However, in end-stage HF, NDPK-C shift affinity toward Gα_i2, reducing cAMP levels [58].

NDPKs were first implicated in cardiac cAMP regulation in the early 1990s in frog and guinea pig atrial tissues, though specific isoforms were not identified [61]. Recent studies have elucidated the roles of NDPK-B and NDPK-C in cardiomyocytes, particularly in pathological contexts [58,62,63]. These isoforms preferentially interact with Gα_s over Gα_i complexes, promoting Gα_s-mediated AC activation and elevating cAMP levels [58]. In HF and arrhythmogenic right ventricular cardiomyopathy, NDPK-B and NDPK-C levels are increased at the cardiomyocyte plasma membrane [58,62,63]. In chronic AF, elevated NDPK-B and NDPK-C levels correlate with enhanced cAMP production, potentially contributing to arrhythmogenesis [64].

Although direct co-localization of NDPKs with RyR2 in cardiomyocytes has not been demonstrated, NDPK-mediated cAMP elevation likely influences RyR2 activity indirectly through downstream effectors such as PKA and Epac. For instance, NDPK-C’s interaction with Gα_s may increase local cAMP levels in nanodomains near the SR, enhancing PKA-mediated phosphorylation of RyR2 at serine 2808 (S2808), which increases channel open probability and SR Ca²⁺ release [3,58]. Similarly, elevated cAMP could activate Epac, triggering CaMKII phosphorylation of RyR2 at S2814 via the Epac-CaMKII pathway, further promoting SR Ca²⁺ leak [65,66]. This is supported by studies showing that NDPK overexpression in canine atrial cardiomyocytes increases cAMP levels and frequency of spontaneous SR Ca²⁺ release events, indicative of enhanced SR Ca²⁺ leak [64]. These findings are consistent with increased NDPK levels in canine models of pacing-induced AF remodeling and in human chronic AF samples compared to sinus rhythm controls [64]. Thus, NDPK-mediated cAMP elevation likely enhances RyR2 activity, promoting Ca²⁺-dependent arrhythmias in AF.

The precise localization of NDPKs relative to RyR2 remains a critical knowledge gap. Advanced imaging techniques, such as super-resolution microscopy, could reveal whether NDPK-B or NDPK-C resides in SR nanodomains, potentially forming signaling complexes with ACs, PKA, or Epac near RyR2 [67]. Co-immunoprecipitation studies could further confirm physical interactions between RyR2 and mAKAP or PDE4D, which scaffold cAMP signaling complexes [34,68]. Such studies will be essential to establish whether NDPKs directly or indirectly modulate RyR2 activity and to elucidate their role in nanodomain-specific cAMP signaling.

Therapeutic strategies targeting NDPKs hold promise for mitigating their pathological effects in heart failure and AF. Specific inhibitors of NDPK histidine kinase activity, such as small molecules targeting His-118 autophosphorylation, could reduce receptor-independent Gα_s activation, thereby lowering cAMP levels and attenuating RyR2 hyperactivity [69]. Alternatively, modulators disrupting NDPK-C’s interaction with Gαs, such as peptides mimicking its N-terminal domain, could selectively dampen cAMP production in diseased hearts [70]. These approaches could complement existing therapies, such as PDE inhibitors or RyR2 stabilizers, by addressing upstream cAMP dysregulation. However, the ubiquitous expression of NDPKs and their role in nucleoside homeostasis necessitate careful design to avoid off-target effects.

In summary, NDPK-B and NDPK-C play a pivotal role in regulating cAMP levels in cardiomyocytes by facilitating receptor-independent Gα_s activation, with implications for RyR2 activity and arrhythmia susceptibility. Their increased expression in HF and AF underscores their pathological relevance, likely through PKA- and Epac-mediated enhancement of SR Ca²⁺ release. Future studies using advanced imaging and biochemical approaches are critical to confirm NDPK-RyR2 co-localization and elucidate their precise mechanisms. Targeting NDPKs offers a novel therapeutic avenue to restore cAMP homeostasis and mitigate RyR2 dysfunction in cardiac disease, warranting further investigation.

2.3. cAMP and RyR2 regulation

RyR2 is a critical regulator of excitation–contraction coupling in cardiomyocytes, controlling Ca²⁺ release from the SR. RyR2 is part of a well-organized junctional membrane complex, in which junctophilin-2 helps maintain a fixed distance between the plasmalemmal and SR membranes [71,72]. As a homotetrameric Ca²⁺ release channel with a cytoplasmic head and SR-embedded transmembrane stalk, RyR2 is modulated by multiple mechanisms, including protein interactions, small molecules, and post-translational modifications (Fig. 1, Table 1). cAMP activates protein kinase A (PKA), which is targeted to various Ca²⁺ handling proteins within cardiomyocytes, including the voltage-gated L-type Ca²⁺ channel (LTCC; top right), sarco/endoplasmic reticulum ATPase 2a (SERCA2a), and RyR2 (middle right). cAMP also binds to Epac, which in turn activates CaMKII. Activated CaMKII phosphorylates RyR2, resulting in increased Ca²⁺ leak [73]. In addition, PKA can phosphorylate the cAMP-response element-binding protein (CREB) in the nucleus, enabling CREB to bind to cAMP Response Elements (CREs) in the promoter regions of Ca²⁺ handling genes [74]. Phosphorylation by PKA and Ca²⁺/CaM-dependent protein kinase II (CaMKII) plays a central role in RyR2 regulation under physiological and pathological conditions, enhancing SR Ca²⁺ release [3,35,73,75].

Fig. 1.

Cartoon of cAMP-Dependent and cAMP-Independent Regulators of RyR2.

Table 1.

Roles of AC isoforms, PDE families, and NDPKs in cAMP regulation in cardiomyocytes.

Protein Family	Expression	Nanodomain Localization	Physiological Function	Pathological Roles
Adenylyl Cyclase (AC) Isoforms
AC1	High in sinoatrial (SA) node; present in cardiomyocytes [16,24]	Co-localizes with LTCC and RyR2 in SA node and cardiomyocyte nanodomains [24]	Regulates SA node automaticity via localized cAMP production; enhances Ca2+-dependent cAMP signaling [24]	SA-node-specific AC1 silencing causes sinus arrhythmias and RyR2-mediated SR Ca2+ leak [24]
AC5, AC6	Predominant in cardiac tissue; abundant in ventricles [17,18]	Plasma membrane and SR nanodomains [16]	Mediates cAMP synthesis in response to β-adrenergic stimulation; regulates contractility and heart rate [20,21]	AC5 KO mice show elevated heart rates and impaired parasympathetic regulation; linked to HF progression [20,22]
Soluble AC (sAC)	Cytoplasmic, expressed in cardiomyocytes [14]	Cytosol, not specifically localized to SR or plasma membrane [14]	Contributes to basal cAMP production; less studied in cardiac context [14]	Limited data; potential role in HF unclear
Phosphodiesterase (PDE) Families
PDE4 (PDE4A, PDE4B, PDE4D)	PDE4A/PDE4D higher in atria; PDE4B/PDE4D at ventricles [31,34]	PDE4B: caveolin-rich plasma membrane, RyR2 nanodomains; PDE4D: SERCA2a nanodomains [34]	Regulates localized cAMP hydrolysis; PDE4B controls LTCC and RyR2 signaling; PDE4D modulates SERCA2a [31,33,34]	PDE4D deficiency promotes HF and ventricular arrhythmias via SR Ca2+ leak; reduced PDE4 activity in AF increases Ca2+ sparks [31,32,37]
PDE3	Expressed in atria and ventricles [30,40]	Near LTCC and SR nanodomains [31]	Modulates inotropic responses to serotonin; hydrolyzes cAMP to prevent overaccumulation [40]	Enhances atrial arrhythmias in permanent AF by amplifying ICa,L and SR Ca2+ release [31,40]
PDE8 (PDE8A, PDE8B)	High in human and murine hearts [42]	Associated with LTCC and SR via AKAP12 scaffolding [43]	Governs cAMP signaling for β-adrenergic responses; regulates LTCC and SR Ca2+ handling [42,43]	PDE8A KO enhances Ca2+ transients; PDE8B2 upregulation linked to arrhythmias in HF [42]
Nucleoside Diphosphate Kinase (NDPKs)
NDPK-B, NDPK-C	Expressed in cytosol and plasma membrane of cardiomyocytes [58]	Plasma membrane; no direct co-localization with RyR2 demonstrated [58,64]	NDPK-B/C activate Gαs via histidine kinase activity, increasing cAMP; enhance isoproterenol-induced contractility [58,60]	Upregulated in HF and AF; NDPK-C shifts to Gαi2 in end-stage HF, reducing cAMP; promotes arrhythmogenic SR Ca2+ leak in AF [58,64]

Abbreviations: AC, adenylyl cyclase; AF, atrial fibrillation; cAMP, cyclic adenosine monophosphate; HF, heart failure; I_Ca,L, L-type Ca²⁺ current; KO, knockout; LTCC, L-type Ca²⁺ channel; PDE, phosphodiesterase; RyR2, ryanodine receptor type-2; SA, sinoatrial; SERCA2a, sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase 2a; SR, sarcoplasmic reticulum.

cAMP activates PKA, which phosphorylates key Ca²⁺ handling proteins: LTCC, increasing Ca²⁺ influx; RyR2, enhancing systolic Ca²⁺ release; and PLN, promoting SERCA2a-mediated SR Ca²⁺ uptake during diastole [35]. Interestingly, PKA also phosphorylates Rad, a monomeric G protein that inhibits LTCCs, which results in dissociation of Rad from the CaV1.2 complex and increased Ca²⁺ influx [76]. This coordinated phosphorylation elevates cytoplasmic Ca²⁺ levels and boosts cardiac contractility [73,77]. PKA’s catalytic subunit is anchored to RyR2 via mAKAP [3,68]. Studies identified S2808 as a primary PKA phosphorylation site, though debate persists regarding Ser2030 [3,32,78]. Research by Bers and colleagues detected phosphorylation at S2808 and S2814 (by PKA and CaMKII) but not S2030 in cardiomyocytes [79]. PKA phosphorylation at S2808 was shown to trigger dissociation of the channel-stabilizing subunit FKBP12.6 (calstabin-2) [80], increasing RyR2 activity in response to β-adrenoceptor stimulation during stress [3,75]. However, studies with S2808 phospho-mimic (S2808D) mice suggest that channel oxidation also contributes FKBP12.6 dissociation [81].

cAMP also regulates RyR2 through Epac, a guanine nucleotide exchange factor highly expressed in the heart with cAMP-binding affinity comparable to PKA [82,83]. cAMP forms a signaling complex with mAKAP, PKA, and PDE4D3 to modulate RyR2 [32,84,85]. In rat cardiomyocytes, Epac stimulation by a PKA-insensitive cAMP analogue (e. g., 8-CPT-2’-O-Me-cAMP) increases Ca²⁺ spark frequency and SR Ca²⁺ leak via CaMKII-mediated phosphorylation at S2814, an effect blocked by CaMKII inhibitor KN93. [65,66]. This Epac2-CaMKII pathway mimics β1-adrenoceptor-induced SR Ca²⁺ leak, which is inhibited by nitric oxide synthase 1 (NOS1) blockade, as NOS1 blockade enhances leak [86]. Excessive S2814 phosphorylation is linked to AF and ventricular arrhythmias in HF, contributing to pathological diastolic SR Ca²⁺ leak that impairs contractility and promotes arrhythmias [87–90].

Epac1 and Epac2 exhibit distinct roles in cardiomyocytes, with Epac2 primarily implicated in β1-adrenoceptor-induced SR Ca²⁺ leak, as demonstrated by Pereira et al. [66]. Epac2, localized at the SR, activates CaMKII through a pathway involving Rap1, a small GTPase, which amplifies Ca²⁺-dependent signaling [91]. In contrast, Epac1 is more abundant at the plasma membrane and regulates hypertrophic signaling and LTCC activity via Rap1 and protein kinase C (PKC) [92]. Epac1’s role in RyR2 regulation is less clear but may involve indirect effects through NOS1 modulation, as Epac1 activation enhances NOS1 activity, potentially stabilizing RyR2 by increasing S-nitrosylation [93]. Epac’s interactions with Rap1 and NOS1 highlight its role in integrating cAMP signaling with other pathways, such as NO-mediated redox regulation. Compartment-specific cAMP signaling is critical, as Epac2’s localization near RyR2 enables rapid CaMKII activation, whereas Epac1’s plasma membrane localization modulates LTCC and hypertrophic responses [66]. Dysregulation of Epac signaling in HF and AF, particularly Epac2’s enhancement of SR Ca²⁺ leak, underscores its pathological significance.

The therapeutic potential of targeting Epac is promising, particularly with selective inhibitors like ESI-09, which block Epac1 and Epac2 without affecting PKA-mediated contractility [94]. ESI-09 reduces SR Ca²⁺ leak and arrhythmic events in preclinical models of AF and HF by inhibiting Epac2-CaMKII signaling, offering a targeted approach to mitigate RyR2 hyperactivity [95]. Unlike PDE inhibitors, which broadly increase cAMP and risk proarrhythmic effects, Epac inhibitors provide specificity by preserving PKA’s inotropic effects. However, challenges remain, including potential off-target effects of ESI-09 and the need to develop isoform-specific inhibitors to distinguish Epac1 and Epac2 functions. Future studies using cardiomyocyte-specific Epac1 or Epac2 knockout models or advanced FRET-based cAMP sensors could further elucidate their roles and optimize therapeutic strategies. Targeting Epac, particularly Epac2, represents a novel avenue to reduce pathological SR Ca²⁺ leak while maintaining cardiac contractility, complementing existing therapies like RyR2 stabilizers or CaMKII inhibitors [96,97].

CaMKII, predominantly the delta isoform in the heart, is activated by intracellular Ca²⁺ and CaM [98]. It phosphorylates RyR2 at S2814, increasing RyR2 Ca²⁺ sensitivity and subsequently channel open probability [73]. PDE4D hydrolyzes cAMP in the RyR2 nanodomain, reducing PKA activity and RyR2 Ca²⁺ sensitivity to maintain signaling balance [32,34].

Protein phosphatases PP1 and PP2A play a critical role in regulating RyR2 as a counter-mechanism to cAMP-dependent regulation in cardiac muscle. While cAMP activates PKA, which phosphorylates RyR2, PP1 and PP2A dephosphorylate the channel, restoring RyR2 to a less active state. This dephosphorylation is essential for maintaining Ca²⁺ homeostasis and preventing excessive SR Ca²⁺ leak, which can lead to arrhythmias or contractile dysfunction, particularly in HF. PP1 and PP2A are targeted to RyR2 via anchoring proteins, such as spinophilin (PPP1R9B) [99] and PPP1R3A [100] for PP1, PR130 [99] and B56α [101] for PP2A, and mAKAP [99] that scaffolds phosphatase complexes, ensuring precise spatial regulation [102]. In healthy hearts, this balance between PKA phosphorylation and PP1/PP2A dephosphorylation fine-tunes RyR2 activity. However, in diseased states, reduced PP1/PP2A activity or disrupted anchoring can lead to RyR2 hyperphosphorylation, exacerbating Ca²⁺ mishandling [103–105]. cAMP/PKA signaling can influence CaM’s interaction with RyR2, affecting channel stability [106]. This interaction highlights the potential bridge between cAMP-dependent and -independent mechanisms, as CaM binding is modulated by both phosphorylation and direct Ca²⁺ interactions.

Finally, cAMP may regulate RyR2 through another indirect manner, particularly in the cardiac conduction system (CCS) cells. cAMP is known to directly bind and regulate the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels [107,108], which regulate the funny current (I_f) in the cells of the CCS. Increased I_f results in diastolic depolarization, which activates the influx of Ca²⁺ through LTCC, eventually activating RyR2.

2.4. cAMP-independent regulation of RyR2

While cAMP-dependent pathways are central to RyR2 regulation, cAMP-independent mechanisms also play key roles in modulating RyR2 activity, particularly in pathological conditions such as HF and AF. These mechanisms include post-translational modifications like oxidation and nitrosylation [81,109], protein-protein interactions such as FKBP12.6, and CaM binding [110,111], phosphorylation by striated muscle preferentially expressed protein kinase (SPEG) [112,113], and modulation by ions like Mg²⁺ [114]. These processes, often dysregulated in disease, contribute to SR Ca²⁺ leak and arrhythmogenesis, synergizing with cAMP-dependent pathways [115].

2.5. cAMP-dependent gene expression of calcium handling proteins

cAMP is a critical second messenger that regulates the transcription of genes encoding Ca²⁺-handling proteins in cardiomyocytes, profoundly influencing cardiac function and disease states [116]. Elevated cAMP levels activate PKA, which translocates its catalytic subunits to the nucleus. There, PKA phosphorylates the cAMP response element-binding protein (CREB) at Ser-133, enabling CREB to bind to cAMP response elements (CREs) in the promoter regions of target genes, thereby enhancing their transcription [116,117]. This cAMP/PKA/CREB axis orchestrates the expression of multiple Ca²⁺-handling proteins, including RyR2, LTCC (CACNA1C), SERCA2a (ATP2A2), and PLN, which has significant implications for cardiac physiology and pathology [118].

Direct evidence of CREB-mediated regulation of RyR2 expression in cardiomyocytes is limited, though studies in non-cardiac cells suggest its potential. For example, Ziviani et al. [119] demonstrated that cAMP-driven CREB activation upregulates RyR2 gene expression in neurons, establishing a positive feedback loop under nicotine treatment [119]. In the heart, CREB regulates genes involved in cardiac contractility and hypertrophy, but whether it directly governs RyR2 transcriptions remains unexplored [118]. Matus et al. [116] showed that cardiomyocyte-specific CREB inactivation in mice impairs contractility, hinting at a broader role for CREB in Ca²⁺-handling protein expression, including RyR2. This gap underscores the need for cardiac-specific studies to confirm RyR2’s transcriptional regulation.

PDE4D, which modulates cAMP levels in RyR2 nanodomains, is another target of CREB-mediated transcription [120]. Liu et al. [120] reported that chronic cAMP elevation via forskolin increases PDE4D1 and PDE4D2 expression in vascular smooth muscle cells, a mechanism likely conserved in cardiomyocytes given PDE4D’s role in cardiac cAMP signaling [34]. Other PDE isoforms, including PDE7A, PDE7B, and PDE8A, are similarly regulated by CREB, forming feedback loops to fine-tune cAMP levels [121–124]. Additionally, PKA’s regulatory and catalytic subunits are transcriptionally upregulated by cAMP, reinforcing signaling [125]. In pathological states like HF and AF, dysregulated cAMP signaling disrupts the expression of Ca²⁺-handling proteins, exacerbating disease. In HF, reduced SERCA2a expression and increased CACNA1C transcription, driven by chronic β-adrenoceptor stimulation and elevated cAMP, contribute to Ca²⁺ overload and contractile dysfunction [126]. CaMKII, a key RyR2 regulator, is also transcriptionally upregulated via CREs in HF, amplifying pathological remodeling [127,128].

Despite these insights, the transcriptional regulation of Ca²⁺-handling proteins in cardiomyocytes remains underexplored compared to post-translational mechanisms. Future studies are required to confirm CREB’s role in RyR2 expression, elucidate the mechanisms underlying cAMP-driven gene dysregulation in HF and AF, and explore therapeutic strategies, such as CREB modulation, to restore Ca²⁺ homeostasis.

3. Discussion

cAMP serves as a pivotal second messenger in cardiomyocytes, orchestrating physiological function through tightly regulated synthesis by ACs and degradation by PDEs within distinct nanodomains. Emerging evidence underscores cAMP nanodomains at the SR, where localized signaling finely tunes RyR2 activity via downstream effectors, including PKA, CaMKII, Epac, and PDEs. NDPKs, particularly NDPK-C, further modulate cAMP production, potentially influencing SR Ca²⁺ release in pathological states. Dysregulation of cAMP within SR nanodomains disrupts RyR2 function, promoting aberrant Ca²⁺ release, cardiac arrhythmias, and contractile dysfunction in HF and AF through interconnected molecular pathways.

A critical component of cAMP-mediated RyR2 regulation is its phosphorylation by kinases like PKA and CaMKII, and dephosphorylation by phosphatases like PP1 and PP2. Studies discussed in this review used techniques such as mouse models with mutated residues, direct phosphorylation assessment at RyR2 sites, and deletion or overexpression of regulatory subunits. While these studies are widely accepted, advanced technology may uncover new phosphorylation residues or regulators, enhancing our understanding of cAMP’s role in RyR2 regulation. Similarly, many studies on cAMP nanodomains relied on fluorescent cAMP sensors [129], potentially limited by their access to nanodomains and real-time signaling accuracy. Improved sensors, reporters, and imaging techniques could refine our knowledge of cAMP nanodomains and their regulation of RyR2.

The therapeutic potential of targeting cAMP signaling and RyR2 dysregulation in HF and AF is significant but complex, given the nanodomain-specific nature of these pathways. PDE inhibitors, such as milrinone (PDE3 inhibitor), enhance cAMP levels to improve contractility in acute HF but are limited by proarrhythmic effects and increased mortality in chronic use [130]. Selective PDE4 or PDE8 inhibitors, which modulate cAMP in specific nanodomains, show preclinical promise for reducing SR Ca²⁺ leak but require further development to avoid off-target effects [131]. RyR2 stabilizers, such as JTV519 and dantrolene, enhance FKBP12.6 binding to RyR2, reducing pathological Ca²⁺ leak in HF and catecholaminergic polymorphic ventricular tachycardia (CPVT) models [132,133]. However, their efficacy in AF and specificity for diseased hearts remain under investigation. CaMKII inhibitors, like KN93, attenuate SR Ca²⁺ leak and arrhythmias by reducing RyR2 phosphorylation at S2814, but newer, more selective compounds are needed to minimize systemic toxicity [89,97]. Emerging Epac inhibitors, such as ESI-09, show potential in preclinical studies to suppress arrhythmogenic Ca²⁺ sparks by blocking Epac-mediated CaMKII activation, though their cardiac specificity requires optimization [134].

A major hurdle across these therapies is the precise targeting of nanodomain-specific cAMP signaling. Current drugs often lack spatial specificity, risking disruption of physiological cAMP gradients. Advances in nanodomain-targeted delivery systems, such as nanoparticle-based inhibitors or biosensors for real-time cAMP monitoring, are critical for developing precision therapeutics [135,136]. Ongoing research, including preclinical studies of PDE8 inhibitors and Epac modulators, and clinical trials of RyR2 stabilizers for HF, aims to address these challenges [42,137]. Future studies should focus on elucidating NDPK-RyR2 interactions, validating CREB as a therapeutic target for RyR2 and Ca²⁺-handling protein expression, and developing nanodomain-specific interventions to restore Ca²⁺ homeostasis. These efforts will enhance our understanding of cAMP signaling in cardiac physiology and unlock novel treatments for HF and AF, mitigating the burden of arrhythmias and contractile dysfunction.

4. Conclusion

In conclusion, this review summarizes recent studies on the regulation of RyR2 activity by localized cAMP nanodomains and their broader implications for Ca²⁺ handling in cardiac diseases. These investigations advance our understanding of the field beyond global GPCR-RyR2 signaling to nanoscale domains. In doing so, they propose several therapeutic strategies, including targeting cAMP nanodomains, developing NDPK inhibitors, and selectively inhibiting specific isoforms of AC or PDEs, to enable compartment-, cell-type-, and disease-specific modulation of RyR2 function.