During myocardial ischemia, the cardiac tissues become profoundly hypoxic. This is caused by an attenuated supply of oxygen and metabolites to the area at risk by the occluded coronary artery. Several previous studies have shown that even very short episodes of myocardial ischemia (as short as only 5 min) are associated with the stabilization of HIF ^[1][23]. These transcription factors were discovered in the early 1990s in studies of the erythropoietin promoter ^[2][3][4][24,25,26], a discovery that was subsequently awarded the Nobel Prize in 2019 ^[5][27]. HIF are heterodimeric transcription factors with a constitutively expressed beta unit (HIF1B) ^[6][28]. In contrast, the alpha unit (HIF1A or HIF2A) is substantially regulated on the post-translational level ^{[7][8][9][10]}[29,30,31,32]. During normal oxygen availability, HIF1A/HIF2A are targeted for proteasomal degradation through a molecular pathway that involves oxygen-sensing HIF prolyl hydroxylases (PHD1, PHD2, or PHD3) ^{[11][12][13][14]}[33,34,35,36]. PHDs use oxygen as a co-factor to promote hydroxylation of a conserved prolyl-residue with the HIF1A/HIF2A subunit, which subsequently promotes binding of the Von-Hippel-Lindau gene product, polyubiquitination, and proteasomal degradation ^{[9][15][16][17]}[31,37,38,39]. However, if oxygen levels fall, PHDs are functionally inactivated. In addition, other metabolic changes in the microenvironment ^[10][18][19][32,40,41], or oxygen-independent mechanisms of PHD inhibition, have been demonstrated previously (e.g., elevations of succinate levels) ^[20][21][42,43]. These changes in metabolic supply and demand lead to the stabilization of HIF1A or HIF2A, which form a transcriptionally active complex with the HIF1B subunit ^[22][44]. This transcriptionally active complex can bind to hypoxia-response elements (HREs) within the promoter region of hypoxia-responsive genes, and promote changes in the transcription rate of the specific gene products. Famous HIF target genes include, for example, erythropoietin or vascular endothelial growth factor. However, studies in genetic models show that approximately 570 genes are transcriptionally altered by the activity of HIF, and most likely more than that ^[23][45]. In many instances, HIF binding to HREs will cause transcriptional increases for specific gene products ^[24][25][46,47], but, very frequently, this can also cause repression of a specific gene product ^{[23][26][27][28]}[21,45,48,49]. Repression of a specific gene product by HIF is often related to the induction of HIF-dependent microRNAs (miRNAs), which promote the subsequent repression of an indirect HIF target gene ^[29][30][50,51]. For example, a recent study demonstrated that HIF-dependent induction of miRNA miR122 causes repression of PHD1 as an indirect HIF1A-target gene ^[30][51].

Many studies on the consequences of HIF stabilization during acute myocardial ischemia-reperfusion injury highlight the protective functions of HIF. These studies include evidence that both HIF1A or HIF2A stabilization can have cardioprotective functions, but most likely involve different tissue-compartments and different hypoxia-dependent target genes ^{[31][32][33][34][35]}[52,53,54,55,56]. Similarly, pharmacologic studies using small-molecular inhibitors of PHDs (PHD inhibitors) demonstrate that pre-treatment approaches are associated with attenuated myocardial ischemia-reperfusion injury ^[1][23]. Importantly, orally available HIF activators have recently been used in phase 3 clinical trials for the treatment of renal anemia. These studies showed that HIF activator treatment is at least equally potent to promote hemoglobin levels through the induction of erythropoietin as compared to treatment with recombinant erythropoietin ^{[36][37][38][39]}[57,58,59,60]. These pharmacologic HIF activators have rarely been explored in clinical trials for cardioprotection. However, there is strong experimental evidence that those compounds (e.g., vadadustat or roxadustat) could potentially be used to attenuate myocardial ischemia-reperfusion injury in patients with acute myocardial infarction (MI) or for cardioprotection during cardiac surgery ^[9][31].

Several previous studies have proposed linkages between hypoxia, HIF, and extracellular adenosine signaling as a means to providing tissue-adaptation, or to dampen hypoxia-driven inflammation ^[40][41][42][61,62,63]. In the extracellular compartment, adenosine is generated from precursor nucleotides, such as ATP or ADP ^{[43][44][45][46]}[64,65,66,67]. Once adenosine is generated, it can signal through four distinct adenosine receptors, including the adenosine A₁ receptor (ADORA1), the adenosine A₂A receptor (ADORA2A), the adenosine A₂B receptor (ADORA2B), and the adenosine A₃ receptor (ADORA3) ^[47][48][68,69]. These G-protein coupled receptors have all been implicated in cardio-adaptive responses ^[32][53]. For example, the Adora1 is known to mediate the heart-rate slowing effects of intravenous adenosine, used for the treatment of supraventricular tachycardia ^[49][70]. In particular, the Adora2a and the Adora2b have been shown to dampen inflammatory responses ^{[50][51][52][53]}[71,72,73,74]. For example, Adora2a signaling has been discovered on polymorphonuclear neutrophils (PMNs) ^[54][75] and contributes to attenuated inflammatory responses ^[55][56][76,77]. The subsequent uptake of adenosine from the extracellular compartment ^[28][57][49,78], and metabolism to inosine ^{[58][59][60][61]}[13,79,80,81] or AMP is implicated in terminating extracellular adenosine signaling. In the next section, we will discuss studies on how HIF-dependent alterations of gene transcription can alter extracellular adenosine signaling during myocardial injury, and how these responses have functional implications on cardioprotection during myocardial ischemia-reperfusion injury.

During conditions of hypoxia, inflammation, or cellular stress, different cells release nucleotides, particularly in the form of ATP or ADP. For example, ATP can be released from inflammatory cells through specific molecular pathways ^{[62][63][64][65][66][67][68]}[16,82,83,84,85,86,87]. The extracellular release of ADP has been described extensively from platelets ^[47][68]. ATP or ADP can function as precursor molecules for the extracellular production of adenosine. This process is a two-step, enzymatically controlled pathway. As the first step, the ectonucleotidase CD39 converts extracellular ATP or ADP to AMP ^[69][88]. Studies in gene-targeted mice for cd39 (cd39^−/− mice) ^[70][89] show that these mice experience larger myocardial infarct sizes in the context of diminished levels of AMP and adenosine ^[71][90]. Moreover, cd39^−/− mice are not protected by ischemic preconditioning, where one or more preceding cycles of myocardial ischemia are associated with the attenuated size of injury ^[71][72][73][90,91,92]. Importantly, several studies demonstrate that the transcript and protein levels, and also the enzymatic function of CD39 are increased during ischemia, inflammation, or hypoxia ^{[71][74][75][76][77][78]}[15,90,93,94,95,96]. Studies on the transcriptional mechanism controlling CD39 expression during limited oxygen availability link the increased CD39 levels to transcriptional control of Sp1 ^[79][80][97,98].

The second step for the extracellular production of adenosine is under the control of the ecto-5′-nucleotidase CD73 ^[81][99]. This enzyme promotes the extracellular conversion of AMP to adenosine and can be considered as a “pace-maker” for extracellular adenosine generation. Similar to cd39^−/− mice, gene-targeted mice for cd73 ^[82][100] experience increased myocardial injury and are not protected by ischemic preconditioning ^[83][101]. As would be expected based on its enzymatic function, cd73^−/− mice experience attenuated concentrations of cardiac adenosine in conjunction with elevated cardiac AMP concentrations during myocardial injury ^[83][101]. Moreover, transcript, protein, and functional levels of CD73 are increased under hypoxia ^[83][101]. Several studies link these increases to a transcriptional program under the control of HIF1A. Studies with transcription factor binding assays and promoter constructs had demonstrated that CD73 is a classic HIF target gene and implicate HIF-dependent induction of CD73 in hypoxia-adaptive responses ^[75][76][82][93,94,100]. When exposed to myocardial ischemia-reperfusion, cd73^−/− mice experience larger infarct sizes and higher elevations of cardiac injury markers (troponin I) compared to control animals ^[83][101]. Subsequent studies during myocardial injury demonstrate that the protective effects of pharmacologic HIF activator treatment is attenuated in cd73^−/− mice ^[1][23], thereby directly implicating HIF-dependent CD73 regulation in cardioprotection. Together, these studies indicate that during myocardial ischemia, hypoxia signaling through Sp1 and HIF1A coordinate the transcriptional induction of CD39 and CD73, which leads to the increased production of extracellular adenosine and thereby contributes to attenuated myocardial infarct sizes (Figure 1).

As described above, myocardial ischemia-reperfusion injury is associated with increased production of extracellular adenosine. Adenosine acts on four different receptor subtypes, including Adora1, Adora2a, Adora2b, and Adora3. All these receptors have been implicated in providing cardioprotection ^{[83][84][85][86]}[101,102,103,104]. However, only the Adora2a and the Adora2b are transcriptionally regulated by HIF. They are highly expressed on a variety of different cellular sources, for example on cells of the innate immune system ^[78][87][88][96,105,106], erythrocytes ^[89][90][18,20], cardiac myocytes ^[35][56], stromal or epithelial cells ^{[91][92][93][94]}[107,108,109,110], regulatory T-cells ^[95][96][97][111,112,113], and other immune cells ^[98][114]. Several previous studies have shown that the Adora2b promoter contains an HRE and can be directly induced by HIF1A during conditions of hypoxia ^[99][115], inflammation ^[92][100][108,116], or during myocardial ischemia-reperfusion injury ^[1][35][23,56]. Similarly, the Adora2a has been previously identified as a target for hypoxia-signaling through the HIF2A isoform ^[101][117]. Studies in murine models of myocardial ischemia-reperfusion injury implicate both Adora2a and Adora2b in cardioprotection from ischemia-reperfusion. For example, murine studies demonstrate that infarct size-reducing effects of treatment with an Adora2a agonist are linked to Adora2a signaling on bone-marrow-derived T or B lymphocytes ^[102][118], which were subsequently identified to be most likely CD4+ T cells ^[103][119]. Similar to the Adora2a, several studies implicate the Adora2b in cardioprotection from ischemia-reperfusion injury. For example, Adora2b^−/− mice are not protected by ischemic preconditioning and exhibit larger myocardial infarct sizes ^[83][101]. Moreover, treatment with a specific agonist for the Adora2b is associated with a significant reduction in infarct sizes in murine ^[83][101] or rat ^[104][120] models of myocardial ischemia-reperfusion injury. Studies using treatment approaches with the pharmacologic HIF activator dimethyloxalylglycine (DMOG) demonstrate abolished cardioprotection by this treatment in Adora2b^−/− mice, thereby directly linking HIF1A and Adora2b signaling during cardioprotection ^[1][23]. Studies on the cellular source of the Adora2b receptor implicate myeloid-dependent Adora2b signaling in cardioprotection from ischemia-reperfusion injury ^[105][106][121,122]. Other studies suggest that Adora2b signaling on cardiac myocytes or inflammatory cells can interface with the stabilization of circadian rhythm signaling molecules, thereby contributing to the circadian oscillation of myocardial injury ^{[32][35][107][108][109]}[53,56,123,124,125]. In addition, a recent study demonstrated a regulatory function for Adora2b signaling in promoting epicardial stromal cells′ HIF stabilization after myocardial infarction as an additional crosstalk between Adora2b and HIF implicated in cardioprotection after myocardial infarction ^[93][109]. Taken together, these findings demonstrate HIF-dependent control of adenosine receptor expression and signaling in attenuating myocardial injury during ischemia-reperfusion (Figure 2).

Several studies implicate the neuronal guidance molecule netrin-1 ^[110][126] in alternative mechanisms of adenosine receptor activation, particularly for the Adora2b ^[111][112][127,128]. Netrin-1 was discovered as a neuronal guidance molecule. Its function was originally described as netrin-1 secreted from cells of the floor plate of the mammalian embryonic neural tube ^{[113][114][115]}[129,130,131]. Its secretion sets up a circumferential gradient of netrin-1, which in some instances attracts or in other instances repels other axons to the ventral midline ^[110][126]. Receptors for secreted netrin-1 include, for example, the receptor DCC (deleted in colorectal cancer) ^[116][132] and the UNC5 homologs (UNC5A, B, C, and D) ^[117][133] and neogenin-1 ^[118][134]. Importantly, the profound ability of the netrin-1 in guiding the repulse or outgrowth of neuronal cells makes it an ideal candidate molecule for the coordination of inflammatory cell migration ^[119][135].

Studies utilizing a two-hybrid screen of a human brain cDNA library discovered a previously unreported interaction of netrin-1 with the Adora2b adenosine receptor ^[120][136]. Several studies using genetic and pharmacologic approaches demonstrate that netrin-1 can function to promote Adora2b signaling during inflammatory conditions outside of the brain, including acute lung injury ^{[25][112][121][122]}[47,128,137,138], inflammatory peritonitis ^[111][127], intestinal inflammation ^[123][124][139,140], inflammatory kidney disease ^[125][141], corneal wound healing ^[126][142], and also myocardial ischemia-reperfusion injury ^[127][143]. However, one study found inconsistent results by showing that the Adora2b is actually not expressed in neurons, and is functionally not required for commissural axon guidance in the context of netrin-1 signaling ^[128][144]. At present, it is not well understood how netrin-1 and the Adora2b interact, including the possibility that netrin-1 could directly bind to Adora2b as its ligand, a role of netrin-1-dependent enhancement of extracellular adenosine levels, or indirect effects of netrin-1 by binding to a classic netrin-1 receptor and enhancing intracellular signaling cascades under the control of the Adora2b. A recent study found that netrin-1 levels were up-regulated in samples of patients who experienced myocardial ischemia-reperfusion injury ^[127][143]. Subsequent studies in mice with deletion of netrin-1 in the myeloid lineage (Ntn1^loxp/loxp LyzM Cre+ mice) revealed selectively larger infarct sizes and higher troponin levels, while other mouse lines with conditional deletion of netrin-1 in other tissues didn’t experience a similar phenotype. Importantly, treatment studies with recombinant netrin-1 demonstrated that the interaction of netrin-1 with myeloid-dependent Adora2b signaling is critical in this pathway, suggesting an autocrine signaling pathway ^[127][143]. Previous studies have found that the promoter of netrin-1 contains an HRE and that HIF1A binding to the netrin-1 promoter dramatically increases netrin-1 expression of transcript and protein levels ^[129][145]. Subsequent studies in myeloid cells confirmed that finding ^[130][146], including recent studies showing that Hif1a-deficient myeloid cells fail to induce netrin during injury ^[25][47]. In conjunction with these studies, it is conceivable that hypoxia-signaling coordinates netrin-1 and Adora2b signaling in an autocrine loop where neutrophil-derived netrin-1 attenuates myocardial injury through signaling events on Adora2b receptors expressed on myeloid cells of the innate immune system (Figure 3).

Previous studies have implicated HIF in modulating extracellular adenosine uptake and metabolism. In this context, the consequences of HIF transcriptional activity function towards attenuating extracellular adenosine uptake and intracellular metabolism, thereby enhancing extracellular adenosine signaling events. Adenosine signaling is terminated through equilibrative nucleoside transporters (ENTs), particularly ENT1 and ENT2 ^{[26][57][131]}[21,78,147]. Those are channels that allow the bidirectional flow of adenosine across the cell membrane following its gradient. Extracellular production of adenosine is dramatically increased and the gradient for adenosine is directed from the extracellular compartment towards the intracellular side during ischemia-reperfusion injury. Therefore, deletion or inhibition of adenosine transporters with an ENT inhibitor such as dipyridamole will result in increased extracellular adenosine levels. Due to its function as an ENT inhibitor, dipyridamole treatment has been used clinically for many decades during pharmacologic stress echocardiography, where it increases coronary adenosine levels, and can unmask coronary artery stenosis ^[132][133][148,149]. Importantly, for the hypoxia-adenosine link during myocardial injury, previous studies have shown that HIF functions to repress both ENT1 and ENT2 during conditions of hypoxia or inflammation, and thereby functions to increase extracellular adenosine levels ^{[26][90][92][134][135][136]}[17,19,20,21,108,150]. Interestingly, mice with global deletion of Ent1 experience elevated plasma levels of adenosine, which can contribute to cardioprotection ^[137][151]. However, the individual contributions of ENT1 versus ENT2 during myocardial ischemia-reperfusion injury have not been addressed, for example by using genetic murine models. Nevertheless, global inhibition of ENTs with dipyridamole has been implicated in cardioprotection from ischemia-reperfusion injury ^[138][139][152,153]. Taken together, these studies highlight the likelihood that HIF-dependent repression of ENTs contributes to cardioprotection from ischemia-reperfusion injury. However, it would be important to define the individual contributions of ENT1 versus ENT2, as well as their tissue-specific functions and adenosine receptor signaling events in experimental studies of myocardial ischemia-reperfusion injury (Figure 4).

In addition to the repression of ENT1 and ENT2, HIF1A has also been shown to repress a key metabolic process in intracellular adenosine metabolism. Adenosine can be metabolized intracellularly to AMP by the adenosine kinase (AK). Studies on hypoxia responses of AK demonstrate that hypoxia is associated with attenuated transcript and protein levels of AK. Moreover, studies in genetic models directly implicate HIF1A in its repression and demonstrate increased adenosine responses with AK repression ^[140][22]. Several studies implicate this pathway in cardioprotection. For example, experimental studies in rats treated with the AK inhibitor iodotubercidin demonstrate attenuated myocardial infarct sizes ^[141][154]. Together, these studies indicate the likelihood that HIF1A-dependent repression of AK contributes to adenosine-dependent cardio- protection from ischemia-reperfusion injury (Figure 4).