Relaxation Of Coronary Arteries Research Articles

Female mice with menopause exhibit coronary microvascular disease

Menopause is a natural process as women age, and women with menopause are at higher risk of having cardiovascular diseases than men at the same age. Coronary microvascular disease (CMD) is one of the causes of angina, and the prevalence of CMD is higher in women than in men, and more postmenopausal women have CMD than premenopausal women. However, the causes of CMD in postmenopausal women are not clear. In this study, we investigate whether and how menopause leads to coronary microvascular disease in female mice. Menopausal mice were generated by administering a 20-day injection of 4-Vinylcyclohexene diepoxide (VCD, 160 mg/kg/day in sesame oil, i.p.). VCD gradually destroys primordial and primary follicles in ovaries and leads to ovary failure while keeping the ovary tissues intact; this model is close to the natural progression of human menopause. The vehicle, sesame oil, was administered in control mice. Coronary flow velocity reserve (CFVR) was measured to assess coronary microvascular function. Isometric tension was conducted in third-order coronary arteries (CAs). Capillary density was measured and compared in the left ventricle. Experiments were performed at 6 weeks (perimenopause period) and 8 weeks (post-menopause period) after the injection of VCD or vehicle in female mice. There was no significant difference in CFVR between VCD-injected mice and vehicle-injected mice 6 weeks after the injection. However, at 8 weeks after the injection, VCD-injected mice exhibited a significant decline in CFVR compared to the control mice. Coronary microvascular dysfunction can be driven by attenuated vascular relaxation in small coronary arteries and/or capillary rarefaction. We first conducted isometric tension and found that endothelium-dependent relaxation was significantly attenuated in VCD-injected mice at 8 weeks after the injection, but not at 6 weeks, compared to the controls. There was no significant difference in EC-independent relaxation between the two groups at any age. Interestingly, there was no significant difference in capillary density between VCD-injected mice and vehicle-injected mice at 6 weeks and 8 weeks after the injection. Postmenopausal mice, but not perimenopausal mice, develop CMD due to attenuated endothelium-dependent relaxation. This suggests that improving endothelium-dependent relaxation is the potential mechanism to treat CMD in postmenopausal women. Supported by NIH R01HL142214 and DOD W81XWH2110472. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Intracellular pathways of calcitonin gene-related peptide-induced relaxation of human coronary arteries: A key role for Gβγ subunit instead of cAMP.

Calcitonin gene-related peptide (CGRP) is a potent vasodilator. While its signalling is assumed to be mediated via increases in cAMP, this study focused on elucidating the actual intracellular signalling pathways involved in CGRP-induced relaxation of human isolated coronary arteries (HCA). HCA were obtained from heart valve donors (27 M, 25 F, age 54 ±2years). Concentration-response curves to human α-CGRP or forskolin were constructed in HCA segments, incubated with different inhibitors of intracellular signalling pathways, and intracellular cAMP levels were measured with and without stimulation. Adenylyl cyclase (AC) inhibitors SQ22536 + DDA and MDL-12330A, and PKA inhibitors Rp-8-Br-cAMPs and H89, did not inhibit CGRP-induced relaxation of HCA, nor did the guanylyl cyclase inhibitor ODQ, PKG inhibitor KT5823, EPAC1/2 inhibitor ESI09, potassium channel blockers TRAM-34 + apamin, iberiotoxin or glibenclamide, or the Gαq inhibitor YM-254890. Phosphodiesterase inhibitors induced a concentration-dependent decrease in the response to KCl but did not potentiate relaxation to CGRP. Relaxation to forskolin was not blocked by PKA or AC inhibitors, although AC inhibitors significantly inhibited the increase in cAMP. Inhibition of Gβγ subunits using gallein significantly inhibited the relaxation to CGRP in human coronary arteries. While CGRP signalling is generally assumed to act via cAMP, the CGRP-induced vasodilation in HCA was not inhibited by targeting this intracellular signalling pathway at different levels. Instead, inhibition of Gβγ subunits did inhibit the relaxation to CGRP, suggesting a different mechanism of CGRP-induced relaxation than generally believed.

Empagliflozin Induces Vascular Relaxation in Rat Coronary Artery Due to Activation of BK Channels.

The aim of this study was to investigate the effects and mechanisms of SGLT2 inhibitor empagliflozin on diabetic coronary function. A rat diabetic model was established by injection of streptozotocin. Rats in the treated group were administered empagliflozin by gavage and rat coronary vascular tensions were measured after eight weeks. Large conductance calcium activated K+ channel currents were recorded using a patch clamp technique, while human coronary artery smooth muscle cells were used to explore the underlying mechanisms. After incubation with empagliflozin (10, 30, 100, 300, 1000 μmol/L), the Δ relaxation % of rat coronary arteries were 2.459 ± 1.304, 3.251 ± 1.119, 6.946 ± 3.407, 28.36 ± 11.47, 86.90 ± 3.868, respectively. Without and with empagliflozin in the bath solution, BK channel opening probabilities at a membrane potential of +60 mV were 0.0458 ± 0.0517 and 0.3413 ± 0.2047, respectively (p < 0.05, n = 4 cells). After incubation with iberiotoxin, the Δ tensions of rat coronary arteries in the control (Ctrl), untreated (DM), low empagliflozin (10 mg/kg/d)-treated (DM+L-EMPA) and high empagliflozin (30mg/kg/d)-treated (DM+H-EMPA) group were 103.20 ± 5.85, 40.37 ± 22.12, 99.47 ± 28.51, 78.06 ± 40.98, respectively (p < 0.01 vs Ctrl, n = 3-7; p < 0.001 vs DM+L-EMPA, n = 5-7). Empagliflozin restored high glucose-induced downregulation of Sirt1, Nrf2, and BK-β1, while the effect of empagliflozin disappeared in the presence of EX-527, a Sirt1 selective inhibitor. Empagliflozin has a vasodilation effect on the coronary arteries in a concentration-dependent manner and can activate BK channels via the Sirt1-Nrf2 mechanism.

Urocortin2 attenuates diabetic coronary microvascular dysfunction by regulating macrophage extracellular vesicles

Diabetic patients develop coronary microvascular dysfunction (CMD) and exhibit high mortality of coronary artery disease. Methylglyoxal (MGO) largely accumulates in the circulation due to diabetes. We addressed whether macrophages exposed to MGO exhibited damaging effect on the coronary artery and whether urocortin2 (UCN2) serve as protecting factors against such diabetes-associated complication. Type 2 diabetes was induced by high-fat diet and a single low-dose streptozotocin in mice. Small extracellular vesicles (sEV) derived from MGO-treated macrophages (MGO-sEV) were used to produce diabetes-like CMD. UCN2 was examined for a protective role against CMD. The involvement of arginase1 and IL-33 was tested by pharmacological inhibitor and IL-33-/- mice. MGO-sEV was capable of causing coronary artery endothelial dysfunction similar to that by diabetes. Immunocytochemistry studies of diabetic coronary arteries supported the transfer of arginase1 from macrophages to endothelial cells. Mechanism studies revealed arginase1 contributed to the impaired endothelium-dependent relaxation of coronary arteries in diabetic and MGO-sEV-treated mice. UCN2 significantly improved coronary artery endothelial function, and prevented MGO elevation in diabetic mice or enrichment of arginase1 in MGO-sEV. Diabetes caused a reduction of IL-33, which was also reversed by UCN2. IL-33-/- mice showed impaired endothelium-dependent relaxation of coronary arteries, which can be mitigated by arginase1 inhibition but can’t be improved by UCN2 anymore, indicating the importance of restoring IL-33 for the protection against diabetic CMD by UCN2. Our data suggest that MGO-sEV induces CMD via shuttling arginase1 to coronary arteries. UCN2 is able to protect against diabetic CMD via modulating MGO-altered macrophage sEV cargoes.

Varied effects of tobacco smoke and e-cigarette vapor suggest that nicotine does not affect endothelium-dependent relaxation and nitric oxide signaling

Chronic smoking causes dysfunction of vascular endothelial cells, evident as a reduction of flow-mediated dilation in smokers, but the role of nicotine is still controversial. Given the increasing use of e-cigarettes and other nicotine products, it appears essential to clarify this issue. We studied extracts from cigarette smoke (CSE) and vapor from e-cigarettes (EVE) and heated tobacco (HTE) for their effects on vascular relaxation, endothelial nitric oxide signaling, and the activity of soluble guanylyl cyclase. The average nicotine concentrations of CSE, EVE, and HTE were 164, 800, and 85 µM, respectively. At a dilution of 1:3, CSE almost entirely inhibited the relaxation of rat aortas and porcine coronary arteries to acetylcholine and bradykinin, respectively, while undiluted EVE, with a 15-fold higher nicotine concentration, had no significant effect. With about 50% inhibition at 1:2 dilution, the effect of HTE was between CSE and EVE. Neither extract affected endothelium-independent relaxation to an NO donor. At the dilutions tested, CSE was not toxic to cultured endothelial cells but, in contrast to EVE, impaired NO signaling and inhibited NO stimulation of soluble guanylyl cyclase. Our results demonstrate that nicotine does not mediate the impaired endothelium-dependent vascular relaxation caused by smoking.

Suxiao Jiuxin Pill attenuates acute myocardial ischemia via regulation of coronary artery tone.

Suxiao Jiuxin Pill (SJP) is a well-known traditional Chinese medicine drug used to manage heart diseases. This study aimed at determining the pharmacological effects of SJP in acute myocardial infarction (AMI), and the molecular pathways its active compounds target to induce coronary artery vasorelaxation. Using the AMI rat model, SJP improved cardiac function and elevated ST segment. LC-MS and GC-MS detected twenty-eight non-volatile compounds and eleven volatile compounds in sera from SJP-treated rats. Network pharmacology analysis revealed eNOS and PTGS2 as the key drug targets. Indeed, SJP induced coronary artery relaxation via activation of the eNOS-NO pathway. Several of SJP's main compounds, like senkyunolide A, scopoletin, and borneol, caused concentration-dependent coronary artery relaxation. Senkyunolide A and scopoletin increased eNOS and Akt phosphorylation in human umbilical vein endothelial cells (HUVECs). Molecular docking and surface plasmon resonance (SPR) revealed an interaction between senkynolide A/scopoletin and Akt. Vasodilation caused by senkyunolide A and scopoletin was inhibited by uprosertib (Akt inhibitor) and eNOS/sGC/PKG axis inhibitors. This suggests that senkyunolide A and scopoletin relax coronary arteries through the Akt-eNOS-NO pathway. In addition, borneol induced endothelium-independent vasorelaxation of the coronary artery. The Kv channel inhibitor 4-AP, KCa2+ inhibitor TEA, and Kir inhibitor BaCl2 significantly inhibited the vasorelaxant effect of borneol in the coronary artery. In conclusion, the results show that Suxiao Jiuxin Pill protects the heart against acute myocardial infarction.

Abstract 424: Arachidonic Acid Synergizes With Aspirin In Cardioprotection Following Acute Myocardial Infarction

Introduction: Membrane-released arachidonic acid (AA) is metabolized via cyclooxygenase pathway to form prostanoids, including thromboxane, prostacyclin and prostaglandin E2. Aspirin suppresses platelet synthesis of thromboxane, rendering its first-line use for anti-thrombosis in patients with acute myocardial ischemia. We report that endogenous PGE2 protects against myocardial ischemia-reperfusion (MI/R) injury (Nat Commun. 2019;10(1):1888). The impact of AA administration while suppressing thromboxane on MI/R injury is unknown. Methods and Results: C57BL/6J mice were treated intravenously with vehicle, AA, aspirin, or their combination and then subjected to 30min coronary artery ligation and 24hr reperfusion. Cardiac function was assessed by ultrasonography and infraction size quantified by TTC staining. Cardiac microcirculation was assessed by laser doppler flow and vasomotor reactivity determined with isolated coronary arteries. Single treatment with AA at an optimized dose of 0.1mg/Kg, or combined with aspirin at a dose of 10mg/Kg that allowed sufficient suppressing of platelet activation over 24hr, decreased infarct size and improved heart function. Impressively, combination of AA with aspirin provided additional cardioprotection compared with either single treatment. While aspirin, not AA, ameliorated microvascular obstruction following MI/R injury, co-administration of AA and aspirin further increased coronary microcirculatory blood perfusion with improved vascular permeability compared with aspirin alone. MI/R impaired endothelium-dependent dilatation of coronary arteries, and the dual treatment, not AA or ASA alone, increased coronary artery relaxation. Cardiac infiltrated neutrophils and platelet-neutrophil aggregates were also reduced by the combination treatment, consistent with a suppressed leukocyte-ECs adhesion as assessed in vitro. Conclusions: AA and aspirin synergistically protect against MI/R injury and this is attributable to suppressed neutrophil inflammation and improved microvascular obstruction. AA might be used as an adjunct therapy to aspirin in patients with acute myocardial infarction.

The Long-Term Effects of Prenatal Hypoxia on Coronary Artery Function of the Male and Female Offspring.

Prenatal hypoxia predisposes the offspring to the development of cardiovascular (CV) dysfunction in adult life. Using a rat model, we assessed the effect of prenatal hypoxia on vasoconstrictive and vasodilative mechanisms in left anterior descending coronary arteries of 4- and 9.5-month-old offspring. Endothelium-dependent relaxation to methylcholine and vasoconstriction responses to endothelin-1 (ET-1) were assessed by wire myography. Prenatal hypoxia impaired endothelium-dependent vasodilation in 4- and 9.5-month-old offspring. Inhibition of nitric oxide (NO) synthase prevented coronary artery relaxation in all groups. Inhibition of prostaglandin H synthase (PGHS) improved relaxation in prenatally hypoxic males and tended to improve vasorelaxation in females, suggesting that impaired vasodilation was mediated via increased PGHS-dependent vasoconstriction. An enhanced contribution of endothelium-dependent hyperpolarization to coronary artery vasodilation was observed in prenatally hypoxic males and females. No changes in endothelial NO synthase (eNOS) and PGHS-1 expressions were observed, while PGHS-2 expression was decreased in only prenatally hypoxic males. At 4 months, ET-1 responses were similar between groups, while ETB inhibition (with BQ788) tended to decrease ET-1-mediated responses in only prenatally hypoxic females. At 9.5 months, ET-1-mediated responses were decreased in only prenatally hypoxic females. Our data suggest that prenatal hypoxia has long-term similar effects on the mechanisms of impaired endothelium-dependent vasodilation in coronary arteries from adult male and female offspring; however, coronary artery contractile capacity is impaired only in prenatally hypoxic females. Understanding the mechanistic pathways involved in the programming of CV disease may allow for the development of therapeutic interventions.

Apelin-Induced Relaxation of Coronary Arteries Is Impaired in a Model of Second-Hand Cigarette Smoke Exposure.

Apelin, an endogenous ligand for APJ receptors, causes nitric oxide (NO)-dependent relaxation of coronary arteries. Little is known about the effects of apelin/APJ receptor signaling in the coronary circulation under pathological conditions. Here, we tested the hypothesis that the vasorelaxing effect of apelin is impaired by cigarette smoke extract (CSE), an established model for second-hand smoke exposure. Isolated rat coronary arteries were treated with 2% CSE for 4 hours. Apelin-induced relaxation of coronary arteries was abolished by CSE exposure, while relaxations to acetylcholine (ACh) (endothelium-dependent relaxation) and to diethyl amine NONOate (NO donor) were similar in control and CSE-treated arteries. Immunoblot analysis demonstrated that apelin increased eNOS ser1177 phosphorylation under control conditions but had no effect after exposure to CSE. Moreover, GRK2 expression was increased in CSE-exposed coronary endothelial cells. Pretreatment with CMPD101, a GRK2 inhibitor, improved the relaxation response to apelin in CSE-exposed coronary arteries. CSE treatment failed to inhibit relaxations evoked by CMF-019, an APJ receptor biased agonist that has little effect on GRK2. In arteries exposed to CSE, apelin impaired the response to ACh but not to diethyl amine NONOate. ACh-induced relaxation was unaffected by CMF-019 in either control or CSE-treated coronary arteries. The results suggest that APJ receptor signaling using the GRK2 pathway contributes to both loss of relaxation to apelin itself and the ability of apelin to inhibit endothelium-dependent relaxation to ACh in CSE-exposed coronary arteries, likely because of impaired production of NO from endothelial cells. These changes in apelin/APJ receptor signaling under pathological conditions (eg, exposure to second-hand smoke) could create an environment that favors increased vasomotor tone in coronary arteries.

Activation of APJ Receptors by CMF‐019, but not Apelin, Causes Endothelium‐Dependent Relaxation of Spontaneously Hypertensive Rat Coronary Arteries

Hypertension is a major risk factor for coronary artery disease, but the pathophysiological mechanisms linking these diseases are not completely understood. Apelin is a novel vasoactive adipokine. Receptors for apelin (APJ receptors) are G‐protein‐coupled receptors and are widely expressed throughout the cardiovascular system. APJ receptors can also signal via G‐protein‐independent pathways, including G‐protein‐coupled‐receptor kinase 2 (GRK2), which inhibits nitric oxide synthase (eNOS) activity and nitric oxide (NO) production in endothelial cells. Apelin causes endothelium‐dependent, NO‐mediated relaxation of coronary arteries from normotensive animals, but the effects of activating APJ receptor signaling pathways in hypertensive coronary arteries are unknown. Since endothelium‐dependent relaxations are often blunted in arteries from hypertensive animals, we tested the hypothesis that apelin‐induced relaxation is impaired in coronary arteries from spontaneously hypertensive rats (SHR). Moreover, we hypothesized that endothelium‐dependent relaxations in response to CMF‐019, an APJ receptor G‐protein biased agonist with little effect on GRK2, would be unaffected in SHR coronary arteries. Western blot and RT‐qPCR analysis demonstrated that APJ receptor protein and mRNA expression did not differ between normotensive WKY (Wistar‐Kyoto) and SHR coronary arteries. eNOS protein expression was also similar between SHR and WKY coronary arteries, but GRK2 expression was significantly increased in SHR coronary endothelial cells, as compared to WKY. In myograph studies, apelin (10‐9‐10‐6 M) caused endothelium‐dependent relaxation of isolated WKY coronary arterial rings (pD2= 7.00 ± 0.11; Emax= 54 ± 4% relaxation; n=6), but had no effect in arteries from SHR. By contrast, CMF‐019 (10‐9‐10‐6 M)‐induced relaxation did not differ between normotensive and hypertensive coronary arteries. The relaxation response to CMF‐019 was abolished by the APJ receptor antagonist, F13A (10‐7 M), by removal of the endothelium, and by inhibition of eNOS with nitro‐ L‐arginine (3 x 10‐5 M). Moreover, the GRK2 inhibitor, CMPD 101 (3 x 10‐5 M), partially restored the relaxation response to apelin in SHR coronary arteries (pD2= 6.56 ± 0.22; Emax= 46 ± 6% relaxation; n=6). Immunoblot analysis of SHR coronary endothelial cells demonstrated that CMF‐019 (10‐7 M) significantly increased eNOS phosphorylation whereas apelin (10‐7 M) had no effect. We conclude that apelin failed to cause relaxation in hypertensive coronary arteries, likely due to impaired eNOS activity resulting from APJ receptor signaling via the GRK2 pathway and decreased eNOS phosphorylation. APJ receptor activation by the biased agonist, CMF‐019, caused similar endothelium‐dependent relaxations in WKY and SHR coronary arteries. Thus, APJ receptor‐biased agonists, such as CMF‐019, may be more effective than apelin in causing vasodilation of hypertensive coronary arteries.

Anti-spasmolytic effects of BAY 60-2770, a soluble guanylate cyclase activator, in isolated coronary arteries

This study investigated whether soluble guanylate cyclase (sGC) activators (activating the heme-oxidized and heme-free sGC) have potential as therapeutic drugs for coronary artery spasm. Isolated canine and porcine coronary arteries were suspended in organ chambers for isometric tension recording. The contractile responses of canine coronary arteries to potassium chloride (3×10-2 M), endothelin-1 (3×10-11 to 3×10-8 M), prostaglandin F2α (10-8 to 10-5 M), and 5-hydroxytryptamine (10-9 to 10-6 M) were suppressed by previous exposure to the sGC activator BAY 60-2770 (10-10, 10-9 and 10-8 M). In porcine coronary arteries, the addition of BAY 60-2770 (10-10, 10-9 and 10-8 M) concentration-dependently prolonged the cycle length of 3,4-diaminopyridine (10-2 M)-induced phasic contractions and reduced the peak tension. As for vessel-size-dependent difference in vasoreactivity, BAY 60-2770 (10-12 to 10-7 M) caused a greater relaxation of porcine coronary arteries precontracted with endothelin-1 (3×10-8 M) in small arteries (#9 in AHA classification) than in large arteries (#6 in AHA classification). These findings suggest that sGC activators are beneficial for the treatment of vasospastic angina. In addition, anti-spasmolytic efficacy of sGC activators may be expected to be observed even in microvascular angina.

Relaxatsion Responses To Extracts of Polygonum Hydropiper (L) In Porcine Coronary Artery: Role of Potassium Channel

Polygonum hydropiper (L) is commonly known as “smart weed” that is used traditionally for the management of hypertension. Therefore, the aim of this study was to determine the effect of P. hydropiper extracts on vasorelaxation using porcine coronary artery rings.Segments of porcine coronary artery were mounted for isometric tension recording in isolated tissue baths and pre-contracted with the thromboxane A2 analog U46619.After pre-contraction, cumulative concentrations of P. hydropiper extracts were added to the tissues to determine the fraction with greatest activity. A variety of inhibitors of intracellular signaling pathways were then utilized in order to determine the mechanism of relaxation. P. hydropiper extracts produced a concentration dependent relaxation of the porcine coronary artery, with the butanol soluble fraction producing the greatest response. The relaxation to the butanol soluble extract was not reduced by removal of endothelium indicating endothelium-independent mechanism of relaxation. Furthermore, the relaxation was unaffected by removal of extracellular calcium, and pre-incubation with the extract had no effect on contractions due to influx of extracellular calcium or release of intracellular calcium. However, relaxation responses were decreased in the presence of potassium channel blockers. Relaxation responses to sodium nitroprusside and forskolin were enhanced by the presence of the butanol soluble extract, suggesting phosphodiesterase inhibitory action. Indeed, relaxation responses to the PDE 4 and PDE 5 inhibitors rolipram and sildenafil were similarly inhibited by potassium channel blockers, indicating that the effect of the butanol extract on potassium channels may be related to phosphodiesterase activity. Theobromine and gallic acid identified as constituents of butanol soluble fraction of P. hydropiper, also produced a relaxation that was inhibited by potassium channel blockers, suggesting that the vascular activity of butanol soluble fraction of P. hydropiper may be due to the presence of theses compound. In conclusion, butanol soluble fraction of P. hydropiper produces relaxation in porcine coronary artery, which is dependent upon opening of potassium channel, potentially downstream of phosphodiesterase inhibition.

Cardioprotective and Vasoprotective Effects of Corticotropin-Releasing Hormone and Urocortins: Receptors and Signaling.

Despite the recent progress in research and therapy, cardiovascular diseases are still the most common cause of death worldwide, thus new approaches are still needed. The aim of this review is to highlight the cardioprotective potential of urocortins and corticotropin-releasing hormone (CRH) and their signaling. It has been documented that urocortins and CRH reduce ischemic and reperfusion (I/R) injury, prevent reperfusion ventricular tachycardia and fibrillation, and improve cardiac contractility during reperfusion. Urocortin-induced increase in cardiac tolerance to I/R depends mainly on the activation of corticotropin-releasing hormone receptor-2 (CRHR2) and its downstream pathways including tyrosine kinase Src, protein kinase A and C (PKA, PKCε) and extracellular signal-regulated kinase (ERK1/2). It was discussed the possibility of the involvement of interleukin-6, Janus kinase-2 and signal transducer and activator of transcription 3 (STAT3) and microRNAs in the cardioprotective effect of urocortins. Additionally, phospholipase-A2 inhibition, mitochondrial permeability transition pore (MPT-pore) blockade and suppression of apoptosis are involved in urocortin-elicited cardioprotection. Chronic administration of urocortin-2 prevents the development of postinfarction cardiac remodeling. Urocortin possesses vasoprotective and vasodilator effect; the former is mediated by PKC activation and prevents an impairment of endothelium-dependent coronary vasodilation after I/R in the isolated heart, while the latter includes both cAMP and cGMP signaling and its downstream targets. As CRHR2 is expressed by both cardiomyocytes and vascular endothelial cells. Urocortins mediate both endothelium-dependent and -independent relaxation of coronary arteries.

Selective Phosphodiesterase 1 Inhibition Ameliorates Vascular Function, Reduces Inflammatory Response, and Lowers Blood Pressure in Aging Animals.

Diminished nitric oxide-cGMP-mediated relaxation plays a crucial role in cardiovascular aging, leading to decreased vasodilation, vascular hypertrophy and stiffening, and ultimately, cardiovascular dysfunction. Aging is the time-related worsening of physiologic function due to complex cellular and molecular interactions, and it is at least partly driven by DNA damage. Genetic deletion of the DNA repair enzyme ERCC1 endonuclease in Ercc1Δ/- mice provides us an efficient tool to accelerate vascular aging, explore mechanisms, and test potential treatments. Previously, we identified the cGMP-degrading enzyme phosphodiesterase 1 as a potential treatment target in vascular aging. In the present study, we studied the effect of acute and chronic treatment with ITI-214, a selective phosphodiesterase 1 inhibitor on vascular aging features in Ercc1Δ/- mice. Compared with wild-type mice, Ercc1Δ/- mice at the age of 14 weeks showed decreased reactive hyperemia, diminished endothelium-dependent and -independent responses of arteries in organ baths, carotid wall hypertrophy, and elevated circulating levels of inflammatory cytokines. Acute ITI-214 treatment in organ baths restored the arterial endothelium-independent vasodilation in Ercc1Δ/- mice. An 8-week treatment with 100 mg/kg per day ITI-214 improved endothelium-independent relaxation in both aorta and coronary arteries, at least partly restored the diminished reactive hyperemia, lowered the systolic and diastolic blood pressure, normalized the carotid hypertrophy, and ameliorated inflammatory responses exclusively in Ercc1Δ/- mice. These findings suggest phosphodiesterase 1 inhibition would provide a powerful tool for nitric oxide-cGMP augmentation and have significant therapeutic potential to battle arteriopathy related to aging. SIGNIFICANCE STATEMENT: The findings implicate the key role of phosphodiesterase 1 in vascular function and might be of clinical importance for the prevention of mortalities and morbidities related to vascular complications during aging, as well as for patients with progeria that show a high risk of cardiovascular disease.

Apelin Does Not Impair Coronary Artery Relaxation Mediated by Nitric Oxide-Induced Activation of BKCa Channels.

Apelin-APJ receptor signaling regulates vascular tone in cerebral and peripheral arteries. We recently reported that apelin inhibits BKCa channel function in cerebral arteries, resulting in impaired endothelium-dependent relaxations. In contrast, apelin causes endothelium-dependent relaxation of coronary arteries. However, the effects of apelin on BKCa channel function in coronary arterial myocytes have not yet been explored. We hypothesized that apelin-APJ receptor signaling does not have an inhibitory effect on coronary arterial BKCa channels and hence does not alter nitric oxide (NO)-dependent relaxation of coronary arteries. Patch clamp recording was used to measure whole cell K+ currents in freshly isolated coronary smooth muscle cells. Apelin had no effect on the increases in current density in response to membrane depolarization or to NS1619 (a BKCa channel opener). Moreover, apelin did not inhibit NO/cGMP-dependent relaxations that required activation of BKCa channels in isolated coronary arteries. Apelin-APJ receptor signaling caused a marked increase in intracellular Ca2+ levels in coronary arterial smooth muscle cells, but failed to activate PI3-kinase to increase phosphorylation of Akt protein. Collectively, these data provide mechanistic evidence that apelin has no inhibitory effects on BKCa channel function in coronary arteries. The lack of inhibitory effect on BKCa channels makes it unlikely that activation of APJ receptors in coronary arteries would adversely affect coronary flow by creating a vasoconstrictive environment. It can be expected that apelin or other APJ receptor agonists in development will not interfere with the vasodilator effects of endogenous BKCa channel openers.

Apelin‐Induced Relaxation is Impaired in Coronary Arteries from Spontaneously Hypertensive Rats

Hypertension is a major modifiable risk factor for coronary artery disease. The pathophysiological mechanisms underlying hypertension as a risk factor for coronary artery disease are not fully understood. Apelin is a vasoactive peptide that binds to APJ receptors, which are highly expressed throughout the cardiovascular system, including coronary arteries. APJ receptors signal via G-protein-dependent and -independent pathways, including activation of G-protein-coupled-receptor kinase 2 (GRK2), which can potentially lead to inhibition of eNOS and nitric oxide (NO) production. We have previously shown that apelin causes endothelium-dependent, NO-mediated relaxation of coronary arteries from normotensive animals (JPET 366:265, 2018), but its effects in hypertensive coronary arteries are unknown. Here, we tested the hypothesis that apelin-induced relaxation is impaired in coronary arteries from spontaneously hypertensive rats (SHR). Western blot analysis demonstrated increased expression of GRK2 in cultured coronary endothelial cells from SHR in comparison with normotensive WKY (control) rats. Isolated coronary arterial rings were mounted in wire myographs for isometric tension recording and contracted with 5-HT (10-7 M). Apelin (10-9-10-6 M) caused endothelium-dependent relaxation of WKY coronary arteries (pD2 = 7.00 ± 0.11; Emax = 54 ± 4% relaxation; n=6), but had no effect in rings from SHR. Conversely, endothelium-dependent relaxations to an APJ receptor biased agonist, CMF-019 (10-9-10-5 M), which preferentially activates the G-protein-dependent pathway with minimal effect on GRK2, did not differ between SHR and WKY. In the presence of apelin (10-7 M), endothelium-dependent relaxation to acetylcholine (ACh) (10-9-10-5 M) was impaired in SHR coronary arteries (pD2 = 6.82 ± 0.10 vs 6.52 ± 0.18, and Emax = 81 ± 8 vs 60 ± 10% relaxation, in the absence and presence of apelin, respectively; n=5; p<0.05); however, apelin had no effect on ACh-induced relaxation of WKY coronary arteries. By contrast, ACh-induced relaxation of SHR and WKY coronary arterial rings was unaffected by CMF-019 (10-7 M). Moreover, the GRK2 inhibitor, CMPD 101 (3 × 10-5 M), partially restored the relaxation response to apelin in SHR coronary arteries (pD2 = 6.24 ± 0.30; Emax = 42 ± 13% relaxation; n=3). Relaxations in response to the NO-donor, DEA NONOate (10-9-10-4 M), were not altered by apelin (10-7 M) treatment in either WKY or SHR coronary arteries. Taken together, the data indicate that apelin failed to cause relaxation in hypertensive coronary arteries, likely due to impaired production or release of NO from endothelial cells rather than interfering with the action of NO on coronary smooth muscle cells. In SHR coronary arteries, apelin instead actively inhibited relaxation to another endothelium-dependent vasodilator; i.e. ACh. That the effects of the G-protein biased agonist, CMF-019, did not differ between WKY and SHR coronary arteries is consistent with a role for GRK2 activation in the altered response to apelin in hypertensive arteries. APJ receptor signaling via the GRK2 pathway may contribute to both the loss of relaxation to apelin itself, as well as the ability of apelin to inhibit endothelium-dependent relaxation to ACh in coronary arteries from SHR.

Mineralocorticoid receptor blockade with finerenone improves heart function and exercise capacity in ovariectomized mice.

AimsIn post‐menopausal women, incidence of heart failure with preserved ejection fraction is higher than in men. Hormonal replacement therapies did not demonstrate benefits. We tested whether the non‐steroidal mineralocorticoid receptor antagonist finerenone limits the progression of heart failure in ovariectomized (OVX) mice with metabolic disorders.Methods and resultsOvariectomy was performed in 4‐month‐old mice, treated or not at 7 months old for 1 month with finerenone (Fine) 1 mg/kg/day. Left ventricular (LV) cardiac and coronary endothelial functions were assessed by echocardiography, catheterization, and myography. Blood pressure was measured by plethysmography. Insulin and glucose tolerance tests were performed. Exercise capacity and spontaneous activity were measured on treadmill and in combined indirect calorimetric cages equipped with voluntary running wheel. OVX mice presented LV diastolic dysfunction without modification of ejection fraction compared with controls (CTL), whereas finerenone improved LV filling pressure (LV end‐diastolic pressure, mmHg: CTL 3.48 ± 0.41, OVX 6.17 ± 0.30**, OVX + Fine 3.65 ± 0.55†, **P < 0.01 vs. CTL, † P < 0.05 vs. OVX) and compliance (LV end‐diastolic pressure–volume relation, mmHg/RVU: CTL 1.65 ± 0.42, OVX 4.77 ± 0.37***, OVX + Fine 2.87 ± 0.26††, ***P < 0.001 vs. CTL, †† P < 0.01 vs. OVX). Acetylcholine‐induced endothelial‐dependent relaxation of coronary arteries was impaired in ovariectomized mice and improved by finerenone (relaxation, %: CTL 86 ± 8, OVX 38 ± 3**, OVX + Fine 83 ± 7††, **P < 0.01 vs. CTL, †† P < 0.01 vs. OVX). Finerenone improved decreased ATP production by subsarcolemmal mitochondria after ovariectomy. Weight gain, increased blood pressure, and decreased insulin and glucose tolerance in OVX mice were improved by finerenone. The exercise capacity at race was diminished in untreated OVX mice only. Spontaneous activity measurements in ovariectomized mice showed decreased horizontal movements, reduced time spent in a running wheel, and reduced VO2 and VCO2, all parameters improved by finerenone.ConclusionsFinerenone improved cardiovascular dysfunction and exercise capacity after ovariectomy‐induced LV diastolic dysfunction with preserved ejection fraction.

Transient heart rate reduction improves acute decompensated heart failure-induced left ventricular and coronary dysfunction.

AimsAcute decompensated heart failure (ADHF), a live‐threatening complication of heart failure (HF), associates a further decrease of the already by HF‐impaired cardiac function with an increase in heart rate. We evaluated, using a new model of ADHF, whether heart rate reduction (HRR) opposes the acute decompensation‐related aggravation of cardiovascular dysfunction.Methods and resultsCardiac output (echocardiography), cardiac tissue perfusion (magnetic resonance imaging), pulmonary wet weight, and in vitro coronary artery relaxation (Mulvany) were assessed 1 and 14 days after acute decompensation induced by salt‐loading (1.8 g/kg, PO) in rats with well‐established HF due to coronary ligation. HRR was induced by administration of the If current inhibitor S38844, 12 mg/kg PO twice daily for 2.5 days initiated 12 h or 6 days after salt‐loading (early or delayed treatment, respectively). After 24 h, salt‐loading resulted in acute decompensation, characterized by a reduction in cardiac output (HF: 130 ± 5 mL/min, ADHF: 105 ± 8 mL/min; P < 0.01), associated with a decreased myocardial perfusion (HF: 6.41 ± 0.53 mL/min/g, ADHF: 4.20 ± 0.11 mL/min/g; P < 0.01), a slight increase in pulmonary weight (HF: 1.68 ± 0.09 g, ADHF: 1.81 ± 0.15 g), and impaired coronary relaxation (HF: 55 ± 1% of pre‐contraction at acetylcholine 4.5 10−5 M, ADHF: 27 ± 7 %; P < 0.01). Fourteen days after salt‐loading, cardiac output only partially recovered (117 ± 5 mL/min; P < 0.05), while myocardial tissue perfusion (4.51 ± 0.44 mL/min; P < 0.01) and coronary relaxation (28 ± 4%; P < 0.01) remained impaired, but pulmonary weight further increased (2.06 ± 0.15 g, P < 0.05). Compared with untreated ADHF, HRR induced by S38844 improved cardiac output (125 ± 1 mL/min; P < 0.05), myocardial tissue perfusion (6.46 ± 0.42 mL/min/g; P < 0.01), and coronary relaxation (79 ± 2%; P < 0.01) as soon as 12 h after S38844 administration. These effects persisted beyond S38844 administration, illustrated by the improvements in cardiac output (130 ± 6 mL/min; P < 0.05), myocardial tissue perfusion (6.38 ± 0.48 mL/min/g; P < 0.01), and coronary relaxation (71 ± 4%; P < 0.01) at Day 14. S38844 did not modify pulmonary weight at Day 1 (1.78 ± 0.04 g) but tended to decrease pulmonary weight at Day 14 (1.80 ± 0.18 g). While delayed HRR induced by S38844 never improved cardiac function, early HRR rendered less prone to a second acute decompensation.ConclusionsIn a model mimicking human ADHF, early, but not delayed, transient HRR induced by the If current inhibitor S38844 opposes acute decompensation by preventing the decompensated‐related aggravation of cardiovascular dysfunction as well as the development of pulmonary congestion, and these protective effects persist beyond the transient treatment. Whether early transient HRR induced by If current inhibitors or other bradycardic agents, i.e. beta‐blockers, exerts beneficial effects in human ADHF warrants further investigation.

Hawthorn berry (crataegus songarica) causes endothelium-dependent relaxation of the porcine coronary artery: Role of Estrogen receptors

BACKGROUND: Fruits of Crataegus songarica are commonly used for the treatment of vascular insufficiency and heart problems. OBJECTIVE: Our aim was to determine the effect of C. songarica on vascular tone and to determine the mechanisms underlying the vasorelaxant properties. METHODS: Extracts of C. songarica were tested for vasodilator activity of porcine coronary artery after pre-contraction with the thromboxane mimetic U46619 in the presence or absence of inhibitors of intracellular signaling cascades. Reactive oxygen species were assessed by dihydroethidine staining and the level of eNOS and AKT phosphorylation was measured by immunohistochemical staining. RESULTS: Extracts of C. songarica berries produced endothelium dependent vasorelaxation, with most significant effect induced by aqueous fraction (AS-CS). This vasorelaxant effect of AS-CS was reduced by inhibition of nitric oxide pathways and inhibition of potassium channels. Inhibition of phosphatidylinositol 3- kinase and Src tyrosine kinase, as well as scavenging of reactive oxygen species, produced an attenuation of the relaxation response. Estrogen receptor antagonists (tamoxifen and ICI 182,782) reduced the AS-CS mediated vasorelaxation. AS-CS also stimulated the endothelial formation of ROS and phosphorylation of Akt and eNOS. CONCLUSION: The data indicated that C. songarica produces an endothelium-dependent vasorelaxation, which is partly dependent upon estrogen receptors, and sensitive to inhibition of ROS/Src/PI3K/NO pathways.

Endothelial Function in Coronary Arteries from Pneumonia-Infected Pigs

This study investigated whether pneumonia is accompanied with endothelial dysfunction in coronary arteries. The lungs and hearts of normal and Mycoplasma hyopneumoniae-infected pigs (6-month-old females and castrated males) were obtained from a local abattoir, and left anterior descending coronary arteries were isolated from the hearts. Lymphocyte infiltration was observed in the lung tissue of pneumonia-infected pigs (PP). However, the histopathological images of coronary arteries in PP were not different from the images in normal pigs (NP). In organ chamber experiments, endothelium-dependent relaxation of coronary arteries by bradykinin (0.1 nM–0.1 μM) and A23187 (1 nM–1 μM) were identical between PP and NP; the relaxant responses were not different even in the presence of L-NAME (100 μM, nitric oxide synthase inhibitor) alone or in combination with indomethacin (10 μM, cyclooxygenase inhibitor). In addition, the relaxant responses to nitric oxide [sodium nitroprusside (1 nM–10 μM)], hydrogen peroxide (10 μM–1 mM), and 11, 12-epoxyeicosatrienoic acids (30 nM–3 μM), all of which function as endothelium-derived relaxing factors in pig coronary arteries, were also identical between PP and NP. These findings suggest that mycoplasma pneumonia in pigs is not a risk factor for endothelial dysfunction in coronary arteries.