6-Nitrodopamine (6-ND) is a novel endogenous catecholamine that exerts potent and long-lasting positive chronotropic and inotropic responses in the isolated rat heart (Britto-Júnior et al., 2022; 2023a; Zatz and De Nucci, 2024). In rat isolated right atria, 6-ND at a concentration as low as 10 fM markedly potentiates the positive chronotropic effects induced by the classical catecholamines dopamine, noradrenaline, and adrenaline (Britto-Júnior et al., 2023b). Interestingly, the low concentration (100 nM) of selective β1-adrenoceptor antagonists atenolol, betaxolol, and metoprolol significantly reduced both basal atrial rates and 6-ND-induced positive chronotropism (Britto-Júnior et al., 2022). This result implies that these drugs act as 6-ND receptor antagonists (Britto-Júnior et al., 2023b). More recently, 4-nitropropanolol (Sparaco et al., 2022) has been identified as a more selective 6-ND receptor antagonist in the rat isolated right atrium (Oliveira et al., 2024b); however, the receptor of 6-ND is yet to be identified.

Human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (Di Baldassarre et al., 2018) reproduce the molecular mechanisms involved in heart disorders such as long QT syndrome (Keller et al., 2005), catecholamine polymorphic ventricular tachycardia (Itzhaki et al., 2012), and Brugada syndrome (Davis et al., 2012), thus offering a suitable translational model for investigating the expression of 6-ND receptors. Therefore, we employed hiPSC-derived cardiomyocytes to explore the potential targets for 6-ND, using a chemical pulldown approach, in which 6-ND was immobilized on agarose beads and incubated with cardiomyocyte membrane extracts. The purified proteins were then fractionated by SDS-PAGE; the bands were cut and hydrolyzed with trypsin, and the peptide mixtures were separated through UPLC and analyzed using mass spectrometry. Protein identification was carried out using the MaxQuant search-engine to query the UniProt Homo Sapiens database. In the competitive assay, 4-nitropropranolol, here referred to as a selective 6-ND antagonist, was added to the protein extract, and the proteins retained on the beads were subtracted from that obtained in the absence of 4-nitropropranolol. Three proteins that modulate the adenylyl cyclase (AC) activity were identified. Because cyclic 3′,5′-cyclic adenosine monophosphate (cAMP) plays a fundamental role in the positive inotropic effects of classical catecholamines (Guellich et al., 2014), we demonstrated the synergistic effects of 6-ND with noradrenaline on the positive inotropic response by using the Langendorff isolated heart perfusion model.

Mammalian hearts express β1- and β2-adrenoceptor subtypes, both of which are involved in the increases in tissue cAMP due to AC activation (Brodde, 2007). Catecholamines such as noradrenaline and adrenaline bind to G-protein–β adrenoceptors, releasing the stimulatory G-protein subunit (Gas) inside the cardiomyocyte, activating AC (Motiejunaite et al., 2021). The generated cAMP binds to protein kinase A (PKA)–R subunits, leading to PKA activation (Liu et al., 2022). Increased PKA activity increases Ca²⁺ levels, leading to enhanced cardiac muscle contractility. Our study using the Langendorff’s preparation clearly demonstrated that 1-min infusion of the perfused heart with a very low concentration of 6-ND (0.1 pM) markedly potentiated the positive chronotropic and inotropic responses induced by noradrenaline. Thus, these pharmacological data seem to be of great value in identifying the 6-ND receptor. Because cAMP-activated PKA is a central regulator of heart chronotropism and inotropism, it is possible that remarkable potentiation caused by 6-ND could be due to AC pathway activation.

The 116 proteins were identified as potential receptors for this novel endogenous catecholamine by using a chemical proteomics approach based on the affinity purification procedure coupled to mass spectrometry for protein identification. The experiments were performed to purify and identify the cardiomyocyte membrane proteins, obtained from cell lysates, bound to 6-ND agarose and inhibited by the selective 6-ND antagonist 4-nitropropranolol (Sparaco et al., 2022; Oliveira et al., 2024a). The number is not surprising since the ligand concentration (in the micromolar or millimolar range) is high to facilitate protein uptake. The AP-MS-based methods present many advantages; they are not time-consuming, do not require the use of specific tools such as antibodies, and allows direct identification of proteins without any bias or prediction. However, it entails limitations since it is an in vitro method, and therefore, it can lead to identification of physically real but not physiologically meaningful interactions (Ziegler et al., 2013). Furthermore, false-positive interacting proteins can be extracted upon binding with matrix or with the linker between the beads and the molecule, which can also partially affect the binding process (Tabana et al., 2023). Nevertheless, this approach remains one of the most widely used preliminary and unbiased exploration strategies for exploring protein–ligand interactions and characterizing protein binders to small molecules in various contexts (Babak et al., 2015; Smith et al., 2023; Saltzman et al., 2024). A similar affinity purification method was used to identify dopamine targets in a human embryo kidney cell line (HEK293; Weigert Muñoz et al., 2023). A comparison of the 205 interactors identified in this study with the 869 interactors here reported revealed 30 common proteins. It is worth mentioning that CAP-1, CAP-2, and STIM-1 were absent in the dopamine interactomes.

In the rat isolated right atrium, 6-ND, as a positive chronotropic agent, is 100 times more potent than noradrenaline and adrenaline and 10,000 times more potent than dopamine (Britto-Júnior et al., 2022). As a positive inotropic agent in the rat isolated heart, 6-ND was 1,000 times more potent than noradrenaline and 10,000 times more potent than adrenaline (Britto-Júnior et al., 2022). Thus, the results of the functional pharmacological approaches are of paramount importance to identify the 6-ND receptor.

One distinct characteristic of 6-ND action in the heart, as compared to the classical catecholamines, is its remarkable ability to potentiate the positive chronotropic (Britto-Júnior et al., 2022) and inotropic (Britto-Júnior et al., 2023a) effects induced by noradrenaline, adrenaline, and dopamine and, as shown here, the positive inotropic effect induced by noradrenaline. As mentioned above, binding to transmembrane β-adrenoceptors, to stimulate cAMP-dependent PKA activation in cardiomyocytes, is considered the initial step in cardiomyocyte activation by the classical catecholamines (Liu et al., 2022). It is unlikely that 6-ND acts as a partial agonist on β-adrenoceptors since the increases in the atrial rate induced by PDE-3 inhibitors such as dipyridamole, cilostazol, and milrinone are virtually abolished by pre-incubation with 6-ND, whereas those induced by dopamine, noradrenaline, and adrenaline are unaffected (Britto-Júnior et al., 2023a). Another finding that indicates a lack of effect on β-adrenoceptors is that the pre-incubation of the atria with the protein kinase inhibitor H-89 abolished the increases in the heart rate induced by dopamine, noradrenaline, and adrenaline but only attenuated the increase induced by 6-ND (Britto-Júnior et al., 2023a). The absence of adrenoceptor proteins bound to the 6-ND agarose under our experimental conditions is notable. Thus, modulation of cAMP levels could be a mechanism by which 6-ND could synergize with the classical catecholamines.

Cyclic nucleotide phosphodiesterases (PDEs) modulate cyclic nucleotide signaling by degrading cAMP and 3′,5′-cyclic guanosine monophosphate (cGMP). There are 11 PDE superfamilies [PDE1–11 (Bender and Beavo, 2006)], and the heart/myocytes express mRNA for all but PDE6 (Hashimoto et al., 2018). The major PDE expressed by human cardiomyocytes is PDE1C (Bork et al., 2023), which is a dual PDE substrate, metabolizing both cAMP and cGMP. Inhibition of PDE3 and PDE4 activity increases the atrial rate (Dolce et al., 2021), and PDE1 inhibition enhances cardiomyocyte contractility through a PKA-dependent mechanism (Muller et al., 2021). Therefore, considering that 6-ND does not increase cAMP levels in human platelets (Nash et al., 2022) and of the 817 cardiomyocyte proteins that are bound to 6-ND, none were identified as cAMP- or cGMP-PDE signaling, advocating that 6-ND does not directly modulate PDE activity. It is interesting that the three membrane proteins directly involved in the modulation of AC, namely, cyclase associated protein-1 (CAP-1; Kakurina et al., 2018), CAP-2 (Pelucchi et al., 2023), and STIM-1 (El Assar et al., 2022), are bound to 6-ND agarose, with the binding selectively blocked by 4-nitropropranol, being candidates for the 6-ND receptor.

Purification of the adenylyl cyclase complex from the yeasts Saccharomyces cerevisiae (Field et al., 1990) and Schizosaccharomyces pombe (Kawamukai et al., 1992) identified the presence of a 70-kDA protein (CAP), whose amino-terminal domain is associated with the AC catalytic area. This protein interaction with AC allows the enzyme to respond appropriately to its regulatory proteins (Vojtek and Cooper, 1993). Homologs for CAP1 and CAP2 have been identified in Homo sapiens (Matviw et al., 1992; Yu et al., 1994) and in Rattus norvegicus (Swiston et al., 1995); however, the patterns of CAP1 and CAP2 expression varied significantly in adult rat tissues. Interestingly, mRNA levels for CAP2 were significantly higher than those for CAP1 in the rat heart (Swiston et al., 1995).

CAP-1 binds and activates adenylyl cyclase in mammalian cells (Zhang et al., 2021). In PCCL3 thyroid follicular cells, overexpression of CAP-1 caused a marked leftward shift in the forskolin dose–response curve, whereas negative modulation of CAP-1 resulted in a significant rightward shift (Zhang et al., 2021). It is interesting that although 6-ND potentiates the positive chronotropic effect of classical catecholamines, it strongly reduces the positive chronotropic (Kaumann et al., 2009) and inotropic (Christ et al., 2009) effects induced by PDE3 inhibitors, indicating a dual ability to modulate AC. Ablation of CAP2 in mice causes dilated cardiomyopathy associated with severe reduction in the heart rate (Peche et al., 2013). It is interesting that the two human CAP proteins are distinct enough to suggest that they may have different regulatory roles (Yu et al., 1994). Whether they have different ability to modulate AC in the heart is under current investigation. Direct quantification of cAMP levels following stimulation with 6-ND and classical catecholamines may yield additional insights into 6-ND signaling within cardiomyocytes. Furthermore, silencing CAP-1 and CAP-2 mRNA expressions should elucidate the modulatory functions of these proteins in the mechanism of action of 6-ND.

Stromal interaction protein (STIM1) is expressed in cardiomyocytes (Liu et al., 2023) and has a single transmembrane domain (Hooper et al., 2000); it is located in the sarcoplasmic reticulum and plasma membrane (Soboloff et al., 2012), and it is also associated with adenylyl cyclase activation (Motiani et al., 2018). Changes in cytosolic Ca²⁺ levels are known to either enhance or depress cAMP production through various Ca²⁺-sensitive AC isoforms (Willoughby et al., 2012). Of the nine transmembrane AC isoforms described so far, AC1 and AC8 are the major Ca²⁺-activated isoforms, whereas AC5 and AC6 are subjected to direct inhibition by physiological Ca²⁺ in the cytosol. Cardiac-specific deletion of STIM1 in mice causes a reduction in the heart rate and sinus arrest, together with a potentiation of the autonomic response to cholinergic signaling (Zhang et al., 2016). Although the negative chronotropism is compatible with the lack of the 6-ND effect, there is no evidence that 6-ND potentiates cholinergic effects (Oliveira et al., 2024b). It is interesting that STIM1 can also act as a Ca²⁺ sensor by activating store-operated calcium channels (SOCCs) following sarcoplasmic/endoplasmic reticulum Ca²⁺ store depletion (Zhang et al., 2005). STIM1 can bind Ca²⁺ under resting conditions, but, after store depletion, STIM1 can interact with Orai channels to activate the store-operated Ca²⁺ entry (Rosenberg et al., 2021), raising the possibility that 6-ND may modulate calcium transport. Indeed, one of the identified interactors with 6-ND is dysferlin, a 238-kDa transmembrane protein with multiple Ca²⁺-binding domains, which mediates Ca²⁺-dependent membrane fusion in striated muscle cells. Dysferlin-knockout (KO) mice develop a dilated cardiomyopathy characterized by decreased left ventricular ejection fraction and reduced heart rate (Paulke et al., 2024). Although both cardiac phenotypes are compatible with a reduction in 6-ND action, our findings that potentiation by 6-ND on catecholamine action on rat perfused heart is also observed in smooth muscle (Britto-Júnior et al., 2023a) and that the expression of dysferlin is restricted to the skeletal muscle (Chernova et al., 2022) exclude dysferlin as the primary 6-ND target in the heart. Indeed, the cardiac phenotypes in dysferlin-KO mice only become evident in mice over 32 weeks of age (Paulke et al., 2024), whereas the effects reported here are observed acutely. Calpains are 6-ND interactors; they constitute a conservative family of Ca²⁺-dependent intracellular cysteine proteases commonly expressed in all cells (Goll et al., 2003). Two ubiquitous forms of calpains have been identified (Taneike et al., 2011), namely, m- (CAP1) and m-(CAP2), which are activated by micromolar and millimolar calcium concentrations, respectively. Calpain-1 is the primary isoform expressed in cardiomyocytes (Zhang et al., 2024) and plays a critical role in normal heart function by cleaving several target proteins (Patterson et al., 2011). Both Capn1 and Capn2 are heterodimers presenting an 80-kDa catalytic subunit and a common 28-kDa regulatory subunit (calpain 4). Although cardiac-specific deletion of calpain 4 resulted in decreased protein levels of Capn1 and Capn2, no cardiac phenotypes under baseline conditions were observed, a finding not compatible with the acute effects reported here for 6-ND.

Noradrenaline is rapidly degraded by momoamino oxidases (Manzoor and Hoda, 2020), and inhibition of these enzymes by 6-ND could cause potentiation of the inotropic effect of NA. However, 6-ND caused 20% and 30% of inhibition of MAO-A and MAO-B, respectively, at 1 mM; at 100 nM, no inhibition of either enzyme was observed (Fuguhara et al., 2025). Inhibition of catecholamine uptake by 6-ND could potentiate both the positive chronotropic and inotropic effects induced by classical catecholamines. Transporter-mediated uptake plays a major role in determining both the magnitude and duration of the catecholamine effect (Gasser, 2021). There are two types of monoamine transporters, namely, a high-affinity with low capacity to transport monoamines, such as NET (Engelhartet et al., 2020), a low-affinity with high capacity like organic cation transporters (OCT 1–3, Amphoux et al., 2006; Bacq et al., 2012), and the plasma membrane monoamine transporter (PMAT; Torres et al., 2003; Engel et al., 2004). However, it is unlikely that 6-ND could interact with catecholamine uptake since, in our experimental settings, none of these proteins were bound to the 6-ND-derivatized agarose.

The use of human-induced pluripotent stem cell-derived cardiomyocytes is associated with some constraints, such as limited capacity to evaluate contractility, altered maturation properties, and reduced survivability (Shead et al., 2024). Purification of the 6-ND receptor from rat neonatal ventricular myocytes may provide further indication of the importance of the cyclase-associated proteins CAP-1 and CAP-2.

6-Nitrodopamine-induced potentiation of the catecholamines’ chronotropic and inotropic effects is due to the modulation of adenylyl cyclase activity, probably via direct interactions with CAP-1 and CAP-2.