Intrinsic cardiac adrenergic cells

In early 1990s, Huang et al. [24] pioneered the idea of an intrinsic cardiac adrenergic system within the mammalian heart, supplementing the well-established sympathoadrenal system. This groundbreaking work involved comprehensive in situ and in vitro immunohistochemistry experiments of neonatal and adult rat hearts, as well as fetal human hearts. In their analysis, the researchers identified a distinct class of cardiac cells, unrelated to cardiac sympathetic axons or neurites, which exhibited positive staining with an anti-TH antibody in both rat and human hearts. These cells contained the major catecholamine biosynthetic enzymes TH, DBH, and PNMT [24]. These cells were subsequently named intrinsic cardiac adrenergic (ICA) cells.

To unravel the intricacies of ICA cells within the cardiac microenvironment, previous researchers have developed two distinct mouse models, the Pnmt-GFP knock-in mouse model and the Pnmt-reporter mouse model [13, 25, 26]. The first genetic modified mouse model designed for the investigation of ICA cells was the Pnmt-GFP knock-in mouse model. Mice harboring this Pnmt-GFP mutation exhibit green fluorescence in cells expressing the Pnmt gene, thereby facilitating the identification, isolation, and characterization of all Pnmt-expressing cells, including ICA cells. The second genetically modified mouse model, the Pnmt-Cre/R26R mouse model, was generated by crossing the Pnmt-Cre mouse model with the Rosa26 reporter (R26R) mouse model. The R26R mouse model involves a specific targeted insertion of the LacZ reporter gene into the Rosa26 locus, which is ubiquitously expressed but initially silenced by a loxP-flanked STOP cassette [27]. When Pnmt-Cre mice are crossed with R26R mice, the offspring have both genetic modifications. In cells expressing Pnmt, Cre recombinase is produced, which excises the STOP cassette at the Rosa26 locus. This leads to the activation of the LacZ reporter gene specifically in Pnmt-expressing cells. The expression of LacZ results in the production of β-galactosidase, which can be detected using histochemical staining with XGAL, producing a blue color in Pnmt-expressing cells. Due to the irreversible genetic change induced by the Cre recombinase enzyme, continuous expression is unnecessary for activating LacZ expression. Cells can be permanently marked as β-galactosidase positive, even with transient Cre recombinase expression. As the Cre recombinase enzyme is introduced into the Pnmt locus, cells transiently expressing Pnmt during mouse development exhibit β-galactosidase positivity. Consequently, the Pnmt-Cre/R26R mouse model serves as a valuable tool for conducting genetic fate-mapping experiments for ICA cells [13].

Characteristics of ICA cells

To elucidate the morphology and ultrastructure of ICA cells, a series of electron microscopic investigations have been undertaken, primarily focusing on primary isolates of ICA cells. Huang et al. contributed significantly to this understanding, demonstrating that isolated ICA cells, when stained positively with anti-Th and anti-neuron-specific enolase (Nse) antibodies in the adult rat heart, exhibited a distinct roundish-shaped gross morphology (Fig. 1A). Expanding the scope beyond adult rat hearts, the same researchers identified ICA cells with a similar roundish-shaped gross morphology, stained positively with an anti-TH antibody, in two fetal human hearts [24]. Building upon these observations, Natarajan et al. not only validated the roundish shape of ICA cells but also introduced a new dimension to their morphology. In their study on neonatal rat hearts, ICA cells were not only co-stained positively with anti-Pnmt, anti-Th, and anti-Dbh antibodies but also exhibited a triangular-shaped gross morphology. This discovery has expanded the understanding of ICA cell diversity, revealing distinct shapes in different developmental stages [28]. These electron microscopic studies collectively provide a comprehensive insight into the morphological variations of ICA cells, laying the foundation for a nuanced understanding of their structural characteristics and potential functional implications in various cardiac contexts.

Fig. 1

Morphology, distribution, co-staining, and noradrenaline (NA) uptake of ICA cells. (A) Isolated ICA cells in the adult rat heart stained positively with anti-Th antibodies (left) and anti-Nse antibodies (right). Scale bar: 10 μm. Pictures were reprinted from ref. [24] with permission obtained from the copyright holder. (B) The co-localizing immunoreactivity of noradrenaline transporter (Nat; left) and Th (middle) in fetal rat ICA cell–myocyte co-cultures. The nuclei represent ICA cells and myocytes (right). Scale bar: 20 μm. (C) [3H] noradrenaline uptake in control conditions and in the presence of exogenous noradrenaline (1 μM) and nisoxetine (1 μM). * P < 0.05 and ** P < 0.01 (ANOVA). (B and C) were reprinted from ref. [30] with permission obtained from the copyright holder. (D) Co-expression of XGAL and Hcn4 in the sinoatrial node (SAN) in the embryonic Pnmt-Cre/R26R mouse heart at E15.5. XGAL staining (left), Hcn4 staining (middle), and overlay graph (right) of the SAN. Scale bar: 0.1 mm. Pictures were reprinted from ref. [26] with permission. (E) Distribution pattern of the XGAL staining in the adult Pnmt-Cre/R26R mouse heart. Left is a 2D section at 20 μm. Right is 3D image generated from the 2D section. Pictures were reprinted from ref. [35] with permission obtained from the copyright holder. (F) Co-expression of Hcn4 and Th in TGAC8 and wild-type mouse models. Scale bar: 20 μm. Pictures were reprinted from ref. [34] with permission obtained from the copyright holder. (G) Exclusive expression of CGRP mRNA in ICA cells and co-expression of CGRP and DOR in both human and rat LV myocardium. Human section (i–vi): (i) TH and CGRP mRNA in left ventricular myocardium. H, human LV myocardium; Neu, human neuroblastoma cell line. (ii) ICA cells (arrow) stained positively with anti-CGRP antibody (brown). (iii) The expression of CGRP mRNA (green) in an ICA cell cluster. (iv) An ICA cell co-stained positively with anti-CGRP (green) and anti-TH (red) antibodies. (v) Another ICA cell co-stained positively with anti-CGRP (green) and anti-TH (red) antibodies. (vi) An ICA cell co-stained positively with anti-DOR (red) and anti-TH (green) antibodies. (vii) A rat ICA cell co-stained positively with anti-Cgrp (green) and anti-Th (red) antibodies. Scale bar: 10 μm. Pictures were reprinted from ref. [44] with permission obtained from the copyright holder. (H) (i and ii) Immunofluorescent dual labeling of TH and C-KIT (a progenitor cell marker) highlights their colocalization (insert) in dividing (binucleated) ICA cells from an autopsy-derived LV myocardial sample. (iii) Immunoperoxidase labeling of TH (brown DAB staining) in two ICA cell clusters in another autopsy-derived LV sample of a human heart allograft, with the upper cluster located in close proximity to an arteriole (A) and a venule (v). (iv) High-magnification (100×) image of the ICA cell cluster shown in (iii). Scale bar in C: 50 mm, all others 10 mm. (Pictures are unpublished observations of Minghe Huang)

Distribution of ICA cells

To elucidate the spatial organization of ICA cells within the intact heart, researchers have conducted immunofluorescent histochemical staining experiments utilizing antibodies specific to catecholamine biosynthetic enzymes, including TH, DBH, and PNMT, alongside the cardiomyocyte-specific marker, sarcomeric α-actinin. A consistent distribution pattern emerged across studies, revealing a predominant presence of ICA cells in perivascular and intramyocardial regions. Specifically, Huang et al. observed clusters of ICA cells in close proximity to coronary microvasculature, including venules, arterioles, and capillaries, as well as clusters adjacent to atrial and ventricular myocytes in the adult rat heart. This observation aligns with the findings of Natarajan et al., who demonstrated a similar spatial arrangement of neonatal rat ICA cells, frequently situated alongside cardiomyocytes within ventricular myocardium. Using immunofluorescent staining techniques, Ebert et al. studied the distribution pattern of ICA cells in the embryonic rat heart across various embryonic stages. At E10.5, ICA cells were found in truncus arteriosus as well as atrial and ventricular chambers, and dorsal venous valve and atrioventricular canal regions at E11.5. Subsequently, at E12.5–E13.5, they turned out to be in the atrioventricular node (AVN) and then in the crest of the interventricular septum, particularly the dorsal limb (early bundle of His), at E16.5 [29]. The perivascular distribution of ICA cells implies a potential role as cardiac chemoreceptors, where neurotransmitters or circulating molecules may modulate the function of ICA cells, regulating the release of endogenous catecholamines from these cells. Owing to the unique perivascular and intramural distribution of ICA cells, it is conceivable that they may participate in regulating coronary blood flow, especially in scenarios involving compromised blood supply, such as in ischemic heart diseases [24, 28].

Huang et al. revealed the diffuse distribution of ICA cells throughout the entire rat heart, with a notable concentration in the left atrium. A similar distribution pattern was observed in both neonatal and adult stages, where ICA cells exhibited a diffuse and sporadic presence across all four chambers of the rat heart [30], as documented by Huang et al. and Natarajan et al. However, these experiments do not comprehensively capture the dynamic distribution pattern of ICA cells within intact heart tissue throughout a more integrated life cycle, spanning from the embryonic stage to the neonatal stage and progressing into the adult stage.

Catecholamine biosynthesis, release, and uptake by ICA cells

ICA cells, recognized as constitutively active entities, possess neuroendocrine properties, including the biosynthesis, release, and uptake of catecholamines. Specifically, Huang et al. conducted seminal studies revealing that the levels of endogenous noradrenaline and adrenaline in rat ICA cells closely resembled those found in sympathetic neurons of the rat stellate ganglion. However, the dopamine content in ICA cells was notably lower compared with sympathetic neurons, with noradrenaline being the predominant catecholamine. Analysis of endogenous catecholamine levels in ICA cells and whole cardiac tissue indicated that ICA cells contributed to approximately 16%, 13%, and 18% of the total cardiac contents of dopamine, noradrenaline, and adrenaline, respectively [13]. The substantial catecholamine content in ICA cells implies their potential for catecholamine synthesis, a notion reinforced by the presence of catecholamine biosynthetic enzymes in these cells. The same researchers conducted comprehensive northern and western blot analyses, revealing the existence of both mRNA and proteins for key catecholamine biosynthetic enzymes, including Th, Dbh, and Pnmt, in adult rat ICA cells [24]. Similarly, in fetal rat ICA cells, Huang et al. observed the presence of Th and Pnmt mRNA at E16.0, even in the absence of Th-positive sympathetic nerve endings, indicating the absence of cardiac sympathetic innervation at this developmental stage [13]. Collectively, these findings underscore the unique catecholamine biosynthetic system of ICA cells within the heart, operating independently of cardiac sympathetic innervation. This distinctive feature suggests a potential role for ICA cells in regulating cardiac adrenergic function during the early developmental stages [30].

In addition to their capacity for catecholamine biosynthesis, ICA cells exhibit the ability to release catecholamines in vitro, as demonstrated by Huang et al. Notably, the release ratio of noradrenaline to adrenaline in these cells differed from their content ratio, hinting at potential differences in storage and secretion mechanisms for each catecholamine. The intricacies of these mechanisms warrant further investigation in future experiments. The catecholamines released by ICA cells have the potential to stimulate G protein-coupled receptors in both autocrine and paracrine manners [31]. Significantly, Huang et al. highlighted the co-localized immunoreactivity of TH and noradrenaline transporter (NAT) in ICA cell–myocyte co-cultures, indicating the presence of noradrenaline uptake mechanisms in these cells. [3H] noradrenaline uptake assays demonstrated that exogenous noradrenaline and the NAT inhibitor nisoxetine partially inhibited [3H] noradrenaline uptake in ICA cells by 20% and 36%, respectively (Fig. 1B, C). This suggests that ICA cells are capable of noradrenaline uptake via NAT, but the partial inhibition by nisoxetine suggests potential structural or functional distinctions in NAT compared with sympathetic nerve endings. Future experiments are required to explore the specific structure and function of ICA-cell-specific NAT and to investigate the presence of possible NAT subtypes [30]. Consistent with previous studies, Huang et al. reaffirmed that ICA cells spontaneously released [3H] noradrenaline following uptake, further supporting the conclusion that ICA cells possess neuroendocrine properties, encompassing the biosynthesis, release, and uptake of catecholamines [13].

Cardiac development and potential progenitor roles of ICA cells

Several studies have contributed evidence highlighting that certain subsets of cardiomyocytes have developmentally adrenergic progenitors. It is interesting to consider whether a subset of ICA cells may undergo transdifferentiation to become pacemaker and conduction system cells during cardiac development. In a seminal study, Ebert et al. employed genetic fate-mapping experiments utilizing the Pnmt-Cre/R26R reporter mouse model, revealing that approximately 50% of pacemaker cells in the SAN, identified by positive staining with the anti-hyperpolarization-activated cyclic nucleotide-gated channel isoform 4 (Hcn4) antibody, co-stained positively with blue XGAL in the embryonic mouse heart at E15.5 (Fig. 1D) [26]. The XGAL staining served to detect LacZ expression, marking cells with a history of Pnmt expression, including ICA cells [32]. Hcn4, a pivotal pacemaker channel protein, exhibits its highest expression in the SAN [33]. Although Hcn4 staining is not exclusive to SAN pacemaker cells, the predominant Hcn4 expression in the SAN region, supports the conclusion that a subset of pacemaker cells in the SAN originates from adrenergic cells, possibly ICA cells given their high concentration in the embryonic SAN [13]. In a recent study, Moen et al. introduced an innovative genetically modified mouse model, termed the TGAC8 mouse model, designed to exclusively overexpress adenylyl cyclase type 8 (Ac8) in the heart. Ac8 is a key regulator of intrinsic cyclic AMP–protein kinase A (PKA)–Ca2+-mediated pacemaker function. Using this mouse model, the researchers demonstrated a noteworthy increase in the concentrations of Th in the TGAC8 mouse’s Hcn4-stained SAN cells when compared with the wild-type HCN4-stained SAN cells (Fig. 1F). This intriguing finding further supports the prevalence of cardiomyocyte catecholamine production and its dynamic response to physiological and pathophysiological stressors. It is tempting to theorise whethera subset of catecholaminergic pacemaker cells within the adult SAN may even trace their origin back to ICA cells [34].

Some have also proposed that a fraction of conduction system cells may also derive from ICA cells. Ebert et al. found that in Pnmt-Cre/Rosa26R mouse embryos, LacZ-positive cells appeared in specific areas of the heart from E8.5 onward. By E10.5, LacZ staining was present in all parts of the heart, especially at the atrial and ventricular junction [26]. Osuala et al. [35] found that in hearts separated at 8–10 weeks of age after hybridization of Pnmt-Cre-Rosa26 Lacz mice, XGAL staining was found primarily in the left atrium and left ventricle. The left atrial myocardium had extensive XGAL staining, while the staining in the left ventricle was more concentrated, the apical XGAL staining was stronger, the dorsal section was stronger, and the staining inside the heart was more pronounced than around it. In the most dorsal part, there was more extensive staining throughout the bottom of the left ventricle (Fig. 1E) [35].

In 2017, by crossing Pnmt-Cre mice with channelrhodopsin-2 (ChR2)-tdTomato mice, our group generated a mouse model in which Pnmt+ cells not only expressed tdTomato fluorescence but also ChR2 channels. On the basis of morphology, surprisingly, we found another kind of elongated, rod-shaped Pnmt+ cells, other than the small triangular-shaped ICA cells, that expressed α-actinin. These were termed Pnmt+ cell-derived cardiomyocytes (PdCMs). Cardiomyocyte-like cells that expressed tdTomato fluorescence were mainly found in the left side of the heart and conduction system. To be specific, 50% of the total myocytes in left atrium (LA), 21% in left ventricle (LV), 7% in right atrium (RA), and 2% in right ventricle (RV). In the AVN region, these cells showed partial co-expression with HCN4, but in the SAN they existed in peripheral regions and showed little co-expression with HCN4 [36]. Ren et al. performed real-time 3D cardiac imaging reconstruction by combining a modified cardiac transparency technology with high-resolution light-sheet fluorescence microscopy (LSFM) using the Zeiss Zen program. Using heart samples from transgenic mouse models (HCN4-DreER/tdTomato and Pnmt-Cre/ChR2-tdTomato), they successfully reconstructed the 3D spatial distribution of HCN4+ pacemaker cells and PdCMs. They found that the distribution patterns of HCN4+ cells in HCN4-DreER/tdTomato heart and Pnmt+ cells in Pnmt-Cre/ChR2-tdTomato heart had an identical expression pattern [37]. In general, PdCMs are preferentially distributed in the left side of the adult heart and partially co-expressed with HCN4 [36, 38]. This expression pattern closely aligns with the anticipated distribution of ventricular conduction system cells, providing further, albeit limited, support for the hypothesis that a subset of these conduction system cells might originate from ICA cells [26, 39]. These findings collectively highlight the close relationship between the developing cardiac conduction system and endogenous cardiac catecholamines - of which ICA cells may likely be an important source of catecholamines, and potentially even act as progenitors during the cardiac development.

Physiological and pathological roles of ICA cellsICA cells regulate cardiac contractile and pacemaker function

Given the proximity of ICA cells to clusters of atrial and ventricular myocytes within the intramyocardial distribution, they are believed to play a pivotal role in regulating the cardiac contractile and pacemaker functions of cardiomyocytes. In a seminal study, Huang et al. demonstrated that the application of the β-adrenoreceptor (β-AR) antagonist timolol (1 μM) to neonatal rat ventricular myocyte primary isolates, which included ICA cells, significantly reduced the spontaneous beating rate of these myocytes by approximately 58% in vitro. While the specific signalling mechanism underlying this reduction remains unclear, this groundbreaking study marked the first instance of showcasing the role of ICA cells in regulating the beating rate of neonatal cardiomyocytes [24].

During the neonatal stage, the sympathetic nervous system has a minimal impact on the beating rate of rat cardiomyocytes, as the maturation of cardiac sympathetic innervation is completed after the second week of rat birth [13, 40]. Nonetheless, the beating rate of neonatal cardiomyocytes can be stimulated by both α-adrenergic receptor (α-AR) agonists and β-AR agonists, indicating the presence of functional α-ARs and β-ARs in the neonatal rat heart [13, 41]. Subsequently, as cardiac sympathetic innervation matures, the beating rate of cardiomyocytes becomes predominantly under β-AR control, as observed in the adult stage [42]. Building on these insights, Natarajan et al. expanded upon Huang’s research and demonstrated that the beating rate of neonatal rat cardiomyocyte cultures containing ICA cells was significantly decreased in the presence of the catecholamine-depleting agent reserpine, the α1-AR antagonist prazosin, and the β-AR antagonist timolol in a concentration-dependent manner. Intriguingly, the reduction induced by reserpine could be reversed by the addition of exogenous noradrenaline [13, 28]. These results collectively suggest that ICA cells, as catecholamine-synthesizing cells, can regulate the beating rate of neonatal cardiomyocytes by releasing endogenous catecholamines to stimulate α-AR or β-AR in the developing heart before the establishment of cardiac sympathetic innervation [13]. The presence of ICA cells underscores their importance in early fetal heart development. Fetal blood carries less oxygen than postnatal blood because it is oxygenated by the placenta rather than the lungs. The fetal heart is small and relatively weak, limiting the amount of blood it can pump with each beat. To maintain adequate cardiac output (CO = heart rate × stroke volume), the fetal heart compensates by beating at a much faster rate, reaching a peak of 170–180 bpm by 9–10 weeks of pregnancy, to ensure sufficient delivery of oxygen and nutrients. In the absence of a fully developed sympathetic nervous system, ICA cells likely play a pivotal role in supporting this elevated cardiac activity. Consistent with cardiac adrenergic gene expression [23], Ebert et al. demonstrated that, in mice, ICA cells emerge as early as E8.5, 2 days prior to the development of SAN pacemaker cells and continue to accumulate in substantial numbers through birth [43].

In the adult heart, the coexistence of intrinsic and extrinsic cardiac catecholaminergic systems likely holds distinct physiological significance. Under resting parasympathetic-dominance, cardiac sympathetic nerves release minimal noradrenaline. Instead, constitutive catecholamine release from ICA cells may suffice to support basal heart function. During stress, ICA cells and sympathetic nerves may act synergistically to achieve optimal cardiac augmentation. It is intriguing that despite the crucial role of catecholamines, widely used clinical therapies involving β-blockers do not significantly impair cardiac performance in patients with cardiovascular diseases. This is because β-blockers are predominantly selective for β1-AR, with a lower affinity for β2-AR. As a result, β2-mediated chronotropic and inotropic effects of ICA cell-derived adrenaline remain largely intact during β-blocker therapy. In addition, ICA cells secrete calcitonin gene-related peptide (CGRP) [44], a neuropeptide also known to enhance contractility in ventricular myocytes [45]. Adrenaline can further stimulate CGRP production from ICA cells via β2-AR-mediated gene upregulation [46]. These mechanisms highlight the human heart’s remarkable and multi-layered neurohormonal backup systems that preserve cardiac performance under stress.

Nonetheless, the specific signalling mechanism responsible for triggering the release of endogenous catecholamines from ICA cells remains unknown. Given that the release of neurotransmitters from neuroendocrine cells typically requires Ca2+ influx [47], it is reasonable to assume that ICA cells similarly necessitate Ca2+ influx for the release of endogenous catecholamines. In support of this hypothesis, Huang et al. demonstrated that ICA cells generated spontaneous Ca2+ influx-mediated [Ca2+]i transients characterized by a lower basal frequency (compared with canonical cardiomyocytes) and a significantly irregular rhythm in ICA cell–myocyte co-cultures. Their experiments further revealed that the addition of the voltage-gated sodium channel blocker tetrodotoxin abolished the [Ca2+]i transients of ICA cells, and the L-type calcium channel blocker nifedipine significantly decreased the amplitude of ICA cell [Ca2+]i transients [13, 30]. This implies that the generation of [Ca2+]i transients by ICA cells involves the activation of both membrane voltage-gated sodium channels and L-type calcium channels. This, in turn, stimulates the release of endogenous catecholamines, activating α-AR or β-AR in the developing heart before cardiac sympathetic innervation [13].

ICA cells possess an oxygen-sensing function

ICA cells possess the remarkable ability to sense various oxygen tensions, including hypoxia and reoxygenation. Hypoxia, characterized by reduced oxygen levels, typically leads to increases in heart rate. However, in certain situations, there can be decreases in heart rate despite hypoxia. For instance, elite divers experience rapid reductions in heart rate during deep breath-hold dives at sea, as observed by Kiviniemi et al. [48]. However, the specific molecular mechanisms underlying bradycardia despite hypoxia remain a subject of debate and likely involve multiple interacting organ systems. Shedding light on this mechanism, Huang et al. demonstrated that acute hypoxia promptly inhibited the generation of [Ca2+]i transients by ICA cells in co-cultures with myocytes [30]. This inhibition suggests a potential mechanism for hypoxic bradycardia, possibly mediated by the reduced release of endogenous catecholamines from ICA cells. Interestingly, this hypoxia-induced inhibitory response of ICA cells contrasts with the behavior of adrenal chromaffin cells, which increase catecholamine release during hypoxia [49].

Following hypoxia, reoxygenation often triggers a surge in myocardial interstitial catecholamine concentrations. Killingsworth et al. demonstrated a significant increase in noradrenaline and adrenaline concentrations after reperfusion in anesthetized pigs. This surge, which far exceeds the increase achieved through cardiac sympathetic stimulation alone, suggests the involvement of an alternative mechanism [50]. Further elucidating this mechanism, Huang et al. showed that reoxygenation after hypoxia substantially increased the frequency of [Ca2+]i transients in ICA cells in co-cultures with myocytes, with a sustained response [30]. This heightened activity, unique to ICA cells, caused temporal summation of [Ca2+]i transients upon reoxygenation, potentially contributing to the surge in catecholamine concentrations observed after reperfusion in pigs. Collectively, these findings highlight the oxygen-sensing capabilities of ICA cells and their pivotal role in regulating cardiac function under both resting and stress conditions. By modulating their [Ca2+]i transient activities, ICA cells can dynamically adjust the release of endogenous catecholamines, thereby influencing cardiac contractility and pacemaker functions.

In response to hypoxia–reoxygenation, cardiac tissues must deploy various mechanisms to enhance oxygen delivery, one of which involves the activation of myocardial angiogenesis [51]. Given the oxygen-sensing capabilities of ICA cells, it is reasonable to speculate that these cells may play a role in angiogenesis underlying myocardial infarction, in addition to their established roles in regulating in cardiac contractility and pacemaker functions. ICA cells might potentially trigger signaling pathways involved in the promotion of angiogenesis, leading to the formation of new blood vessels in ischemic cardiac tissues. This hypothesis is supported by emerging evidence suggesting that catecholamines, which are synthesized and released by ICA cells, can modulate angiogenesis in various physiological and pathological contexts [52]. Furthermore, studies have demonstrated that catecholamines can influence endothelial cell function, including proliferation, migration, and tube formation, all of which are crucial steps in angiogenesis [53]. In addition, catecholamines have been implicated in the regulation of vascular endothelial growth factor (VEGF) expression, a key player in angiogenesis [54]. Therefore, it is conceivable that ICA cells, through their catecholamine production and release, may exert paracrine effects on endothelial cells, thereby promoting angiogenesis in the ischemic heart. Alternatively, ICA cells may serve as progenitor cells that directly transdifferentiate into vascular-specific cells, including smooth muscle cells and endothelial cells. However, further research is warranted to elucidate the precise mechanisms by which ICA cells contribute to angiogenesis in the ischemic heart. Understanding the role of ICA cells in this process may pave the way for novel therapeutic strategies aimed at harnessing the angiogenic potential of these cells to promote cardiac repair and regeneration following myocardial infarction.

ICA cells mediate cardiac protection

Indeed, ICA cells, endowed with their oxygen-sensing function, are believed to play a crucial role in mediating cardioprotective mechanisms against ischemia–reperfusion injury in the heart. Numerous studies have implicated the stimulation of the δ-opioid receptor (DOR) in mediating the cardioprotective effects of ischemic preconditioning across various experimental models [13, 55]. Similarly, activation of the β2-AR during ischemia has been shown to confer cardioprotection by reducing infarct size and improving LV function in murine hearts [56, 57]. Interestingly, the cardioprotective effects mediated by DOR are partly attributed to β2-AR activation within the heart [