Role and Contribution of A2 Adenosine Receptors in the Regulation of Coronary Flow

One out of three individuals suffer from one form of cardiovascular disease, out of which, about 6,000,000 individuals suffer from coronary heart disease. Adenosine has long been known to play a role in coronary flow (CF) regulation; however, the individual role of A2A and A2B adenosine receptors (AR) and their contribution is yet to be fully elucidated. The purpose of this study was to characterize the pharmacology of both A2 ARs in coronary arteries (CAs), in addition, to identifying their role in reactive hyperemia, and their signaling mechanisms. We hypothesized that A2 ARs mediate an increase in CF, in addition, to being involved in metabolic control of CF. Therefore, in this study, we investigated CF changes due to exogenous activation of A2A and A2B ARs, in addition, to their endogenous activation during coronary reactive hyperemia, when metabolic factors, such as adenosine, play a pivotal role. We used the well established Langendorff isolated heart system as well as selective and non-selective AR agonists and antagonists, and A2A and A2B AR single and double knockout mice. We found that activation of both A2A and A2B ARs induces an increase in CF, albeit with different pharmacological profile. Further, in A2BKO mice, A2AARs are up-regulated in order to compensate for deletion of A2BARs. These findings may suggest that both A2ARs are capable of increasing the CF in conditions when a sufficient level of adenosine is available. However, we also found that only A2AARs contribute to CF changes in coronary reactive hyperemia, which may suggest that A2BARs may be activated in more severe conditions such as longer ischemic conditions, where adenosine levels are significantly augmented. We next investigated signaling pathways involved in A2AR-mediated effects. We found that non-mitochondrial K ATP channels are a major end effectors in A2AR-induced increase in CF. What is more interesting is that we illustrated that H2O 2 mediates adenosine\u27s effect on CF and that is coupled to adenosine-mediated effect on KATP channels. From these data, it can be concluded that A2A and A2B ARs may regulate CF in different conditions, albeit, maybe through the same signaling pathway

Contributions of A2A and A2B adenosine receptors in coronary flow responses in relation to the KATP channel using A2B and A2A/2B double-knockout mice

Adenosine plays a role in physiological and pathological conditions, and A2 adenosine receptor (AR) expression is modified in many cardiovascular disorders. In this study, we elucidated the role of the A2BAR and its relationship to the A2AAR in coronary flow (CF) changes using A2B single-knockout (KO) and A2A/2B double-KO (DKO) mice in a Langendorff setup. We used two approaches: 1) selective and nonselective AR agonists and antagonists and 2) A2AKO and A2BKO and A2A/2BDKO mice. BAY 60-6583 (a selective A2B agonist) had no effect on CF in A2BKO mice, whereas it significantly increased CF in wild-type (WT) mice (maximum of 23.3 ± 9 ml·min-1·g-1). 5'-N-ethylcarboxamido adenosine (NECA; a nonselective AR agonist) increased CF in A2BKO mice (maximum of 34.6 ± 4.7 ml·min-1·g-1) to a significantly higher degree compared with WT mice (maximum of 23.1 ± 2.1 ml·min-1·g-1). Also, CGS-21680 (a selective A2A agonist) increased CF in A2BKO mice (maximum of 29 ± 1.9 ml·min-1·g-1) to a significantly higher degree compared with WT mice (maximum of 25.1 ± 2.3 ml·min-1·g-1). SCH-58261 (an A2A-selective antagonist) inhibited the NECA-induced increase in CF to a significantly higher degree in A2BKO mice (19.3 ± 1.6 vs. 0.5 ± 0.4 ml·min-1·g-1) compared with WT mice (19 ± 3.5 vs. 3.6 ± 0.5 ml·min-1·g-1). NECA did not induce any increase in CF in A2A/2BDKO mice, whereas a significant increase was observed in WT mice (maximum of 23.1 ± 2.1 ml·min-1·g-1). Furthermore, the mitochondrial ATP-sensitive K(KATP) channel blocker 5-hydroxydecanoate had no effect on the NECAinduced increase in CF in WT mice, whereas the NECA-induced increase in CF in WT (17.6 ± 2 ml·min-1·g-1), A2AKO (12.5 ± 2.3 ml·min-1·g-1), and A2BKO (16.2 ± 0.8 ml·min-1·g-1) mice was significantly blunted by the KATP channel blocker glibenclamide (to 0.7 ± 0.7, 2.3 ± 1.1, and 0.9 ± 0.4 ml·min-1·g-1, respectively). Also, the CGS-21680-induced (22 ± 2.3 ml·min-1·g-1) and BAY 60-6583-induced (16.4 ± 1.60 ml·min-1·g-1) increase in CF in WT mice was significantly blunted by glibenclamide (to 1.2 ± 0.4 and 1.8 ± 1.2 ml·min-1·g-1, respectively). In conclusion, this is the first evidence supporting the compensatory upregulation of A2AARs in A2BKO mice and demonstrates that both A2AARs and A2BARs induce CF changes through KATP channels. These results identify AR-mediated CF responses that may lead to better therapeutic approaches for the treatment of cardiovascular disorders. © 2011 the American Physiological Society.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Interactions between A2A adenosine receptors, hydrogen peroxide, and KATP channels in coronary reactive hyperemia

Myocardial metabolites such as adenosine mediate reactive hyperemia, in part, by activating ATP-dependent K+ (KATP) channels in coronary smooth muscle. In this study, we investigated the role of adenosine A2A and A2B receptors and their signaling mechanisms in reactive hyperemia. We hypothesized that coronary reactive hyperemia involves A2A receptors, hydrogen peroxide (H2O2), and KATP channels. We used A2A and A2B knockout (KO) and A2A/2B double KO (DKO) mouse hearts for Langendorff experiments. Flow debt for a 15-s occlusion was repaid 128 ± 8% in hearts from wild-type (WT) mice; this was reduced in hearts from A2A KO and A2A/2B DKO mice (98 ± 9 and 105 ± 6%; P < 0.05), but not A2B KO mice (123 ± 13%). Patch-clamp experiments demonstrated that adenosine activated glibenclamide-sensitive KATP current in smooth muscle cells from WT and A2B KO mice (90 ± 23% of WT) but not A2A KO or A2A/A2B DKO mice (30 ± 4 and 35 ± 8% of WT; P < 0.05). Additionally, H2O2 activated KATP current in smooth muscle cells (358 ± 99%; P < 0.05). Catalase, an enzyme that breaks down H2O2, attenuated adenosine-induced coronary vasodilation, reducing the percent increase in flow from 284 ± 53 to 89 ± 13% (P < 0.05). Catalase reduced the repayment of flow debt in hearts from WT mice (84 ± 9%; P < 0.05) but had no effect on the already diminished repayment in hearts from A2A KO mice (98 ± 7%). Our findings suggest that adenosine A2A receptors are coupled to smooth muscle KATP channels in reactive hyperemia via the production of H2O2 as a signaling intermediate. © 2013 the American Physiological Society.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Absence of adenosine-mediated aortic relaxation in A2A adenosine receptor knockout mice

Adenosine mediates vascular responses through four receptor subtypes: A1, A2A, A2B, and A3. The role of A2A receptors in aortic vascular tone was investigated using A2A adenosine receptor (AR) knockout (A2AKO) and corresponding wild-type (A2AWT) mice. Isolated aortic rings from A2AWT and A2AKO mice were precontracted with phenylephrine (10−7 M), and concentration responses for adenosine analogs and selective agonists/antagonists were obtained. Nonselective adenosine analog (NECA; EC50 = 6.78 μM) and CGS-21680 (A2AAR selective agonist; EC50 = 0.013 μM) produced concentration-dependent relaxation (maximum of 25% and 28% relaxation at 10−5 M NECA and CGS-21680, respectively) in A2AWT aorta. In A2AKO aorta, NECA (EC50 = 0.075 μM) induced concentration-dependent contraction (maximum contraction of 47% at 10−6 M; P < 0.05 compared with A2AWT), whereas CGS-21680 produced no response. SCH-58261 (10−6 M; A2AAR selective antagonist) abolished both NECA- and CGS-21680-mediated vasorelaxation in A2AWT (P < 0.05), whereas no change was observed in A2AKO. When DPCPX (10−5 M; A1 selective antagonist) was used in NECA concentration response, greater vasorelaxation was observed in A2AWT (50% vs. 25% in controls at 10−5 M; P < 0.05), whereas lower contraction was seen in A2AKO tissues (5% vs. 47% in controls at 10−6 M; P < 0.05). Aortic endothelial function, determined by response to acetylcholine, was significantly higher in WT compared with KO (66% vs. 51%; P < 0.05). BAY 60–6583 (A2B selective agonist) produced similar relaxation in both KO and WT tissues. In conclusion, A2AAR KO mice had significantly lower aortic relaxation and endothelial function, suggesting that the A2AAR plays an important role in vasorelaxation, probably through an endothelium-dependent mechanism

Single Stem Cell Imaging and Analysis Reveals Telomere Length Differences in Diseased Human and Mouse Skeletal Muscles

Muscle stem cells (MuSCs) contribute to muscle regeneration following injury. In many muscle disorders, the repeated cycles of damage and repair lead to stem cell dysfunction. While telomere attrition may contribute to aberrant stem cell functions, methods to accurately measure telomere length in stem cells from skeletal muscles have not been demonstrated. Here, we have optimized and validated such a method, named MuQ-FISH, for analyzing telomere length in MuSCs from either mice or humans. Our analysis showed no differences in telomere length between young and aged MuSCs from uninjured wild-type mice, but MuSCs isolated from young dystrophic mice exhibited significantly shortened telomeres. In corroboration, we demonstrated that telomere attrition is present in human dystrophic MuSCs, which underscores its importance in diseased regenerative failure. The robust technique described herein provides analysis at a single-cell resolution and may be utilized for other cell types, especially rare populations of cells

Reversal of Right Ventricular Hypertrophy and Dysfunction by Prostacyclin in a Rat Model of Severe Pulmonary Arterial Hypertension

Prostacyclin analogs are among the most effective and widely used therapies for pulmonary arterial hypertension (PAH). However, it is unknown whether they also confer protection through right ventricle (RV) myocardio-specific mechanisms. Moreover, the use of prostacyclin analogs in severe models of PAH has not been adequately tested. To further identify underlying responses to prostacyclin, a prostacyclin analogue, treprostinil, was used in a preclinical rat Sugen-chronic hypoxia (SuCH) model of severe PAH that closely resembles the human disease. Male Sprague&ndash;Dawley rats were implanted with osmotic pumps containing vehicle or treprostinil, injected concurrently with a bolus of Sugen (SU5416) and exposed to 3-week hypoxia followed by 3-week normoxia. RV function was assessed using pressure&ndash;volume loops and hypertrophy by weight assessed. To identify altered mechanisms within the RV, tissue samples were used to perform a custom RNA array analysis, histological staining, and protein and transcript level confirmatory analyses. Treprostinil significantly reduced SuCH-associated RV hypertrophy and decreased the rise in RV systolic pressure, mean pulmonary arterial (mPAP), and right atrial (RAP) pressure. Prostacyclin treatment was associated with improvements in RV stroke work, maximum rate of ventricular pressure change (max dP/dt) and the contractile index, and almost a complete reversal of SuCH-associated increase in RV end-systolic elastance, suggesting the involvement of load-independent improvements in intrinsic RV systolic contractility by prostacyclin treatment. An analysis of the RV tissues showed no changes in cardiac mitochondrial respiration and ATP generation. However, custom RNA array analysis revealed amelioration of SuCH-associated increases in newly identified TBX20 as well as the fibrotic markers collagen1&alpha;1 and collagen 3&alpha;1 upon treprostinil treatment. Taken together, our data support decreased afterload and load-independent improvements in RV function following prostacyclin administration in severe PAH, and these changes appear to associate with improvements in RV fibrotic responses

Yes-Associated Protein (Yap) Is Up-Regulated in Heart Failure and Promotes Cardiac Fibroblast Proliferation.

Left ventricular (LV) heart failure (HF) is a significant and increasing cause of death worldwide. HF is characterized by myocardial remodeling and excessive fibrosis. Transcriptional co-activator Yes-associated protein (Yap), the downstream effector of HIPPO signaling pathway, is an essential factor in cardiomyocyte survival; however, its status in human LV HF is not entirely elucidated. Here, we report that Yap is elevated in LV tissue of patients with HF, and is associated with down-regulation of its upstream inhibitor HIPPO component large tumor suppressor 1 (LATS1) activation as well as upregulation of the fibrosis marker connective tissue growth factor (CTGF). Applying the established profibrotic combined stress of TGFβ and hypoxia to human ventricular cardiac fibroblasts in vitro increased Yap protein levels, down-regulated LATS1 activation, increased cell proliferation and collagen I production, and decreased ribosomal protein S6 and S6 kinase phosphorylation, a hallmark of mTOR activation, without any significant effect on mTOR and raptor protein expression or phosphorylation of mTOR or 4E-binding protein 1 (4EBP1), a downstream effector of mTOR pathway. As previously reported in various cell types, TGFβ/hypoxia also enhanced cardiac fibroblast Akt and ERK1/2 phosphorylation, which was similar to our observation in LV tissues from HF patients. Further, depletion of Yap reduced TGFβ/hypoxia-induced cardiac fibroblast proliferation and Akt phosphorylation at Ser 473 and Thr308, without any significant effect on TGFβ/hypoxia-induced ERK1/2 activation or reduction in S6 and S6 kinase activities. Taken together, these data demonstrate that Yap is a mediator that promotes human cardiac fibroblast proliferation and suggest its possible contribution to remodeling of the LV, opening the door to further studies to decipher the cell-specific roles of Yap signaling in human HF