Doxorubicin alters G-protein coupled receptor-mediated vasocontraction in rat coronary arteries.

Doxorubicin (Doxo)-associated cardio-and vasotoxicity has been recognised as a serious complication of cancer chemotherapy. The purpose of this novel paper was to determine the effect of Doxo on G-protein coupled receptor (GPCR)-mediated vasocontraction located on vascular smooth muscle cells. Rat left anterior descending artery segments were incubated for 24 h with 0.5 µM Doxo. The vasocontractile responses by activation of endothelin receptor type A (ETA) and type B (ETB), serotonin receptor 1B (5-HT1B) and thromboxane A2 prostanoid receptor (TP) were investigated by a sensitive myography using specific agonists, while the specificity of the GPCR agonists was verified by applying selective antagonists (i.e. ETA and ETB agonist = 10- 14-10- 7.5 M endothelin-1 (ET-1); ETA antagonist = 10 µM BQ123; ETB agonists = 10- 14-10- 7.5 M sarafotoxin 6c (S6c) and ET-1; ETB antagonist = 0.1 µM BQ788; 5-HT1B agonist = 10- 12-10- 5.5 M 5-carboxamidotryptamine (5-CT); 5-HT1B antagonist = 1 µM GR55562; TP agonist = 10- 12-10- 6.5 M U46619; TP antagonist = 1 µM Seratrodast). Our results show that 0.5 µM Doxo incubation of LAD segments leads to an increased VSMC vasocontraction through the ETB, 5-HT1B and TP GPCRs, with a 2.2-fold increase in ETB-mediated vasocontraction at 10- 10.5 M S6c, a 2.0-fold increase in 5-HT1B-mediated vasocontraction at 10- 5.5 M 5-CT, and a 1.3-fold increase in TP-mediated vasocontraction at 10- 6.5 M U46619. Further studies unravelling the involvement of intracellular GPCR signalling pathways will broaden our understanding of the Doxo-induced vasotoxicity, and thus pave the way to mitigate the adverse effects by potential implementation of adjunct therapy options.

Cardiovascular implications and physical activity in middle-aged and older adults with a history of COVID-19 (CV COVID): a protocol for a randomised controlled trial.

BACKGROUND: The clinical manifestation of COVID-19 is associated with infection and inflammation of the lungs, but there is evidence to suggest that COVID-19 may also affect the structure and function of the cardiovascular system. At present, it is not fully understood to what extent COVID-19 impacts cardiovascular function in the short- and long-term following infection. The aim of the present study is twofold: (i) to define the effect of COVID-19 on cardiovascular function (i.e. arterial stiffness, cardiac systolic and diastolic function) in otherwise healthy individuals and (ii) to evaluate the effect of a home-based physical activity intervention on cardiovascular function in people with a history of COVID-19. METHODS: This prospective, single-centre, observational study will recruit 120 COVID-19-vaccinated adult participants aged between 50 and 85 years, i.e. 80 with a history of COVID-19 and 40 healthy controls without a history of COVID-19. All participants will undergo baseline assessments including 12-lead electrocardiography, heart rate variability, arterial stiffness, rest and stress echocardiography with speckle tracking imaging, spirometry, maximal cardiopulmonary exercise testing, 7-day physical activity and sleep measures and quality of life questionnaires. Blood samples will be collected to assess the microRNA expression profiles, cardiac and inflammatory biomarkers, i.e. cardiac troponin T; N-terminal pro B-type natriuretic peptide; tumour necrosis factor alpha; interleukins 1, 6 and 10; C-reactive protein; D-dimer; and vascular endothelial growth factors. Following baseline assessments, COVID-19 participants will be randomised 1:1 into a 12-week home-based physical activity intervention aiming to increase their daily number of steps by 2000 from baseline. The primary outcome is change in left ventricular global longitudinal strain. Secondary outcomes are arterial stiffness, systolic and diastolic function of the heart, functional capacity, lung function, sleep measures, quality of life and well-being (depression, anxiety, stress and sleep efficiency). DISCUSSION: The study will provide insights into the cardiovascular implications of COVID-19 and their malleability with a home-based physical activity intervention. TRIAL REGISTRATION: ClinicalTrials.gov NCT05492552. Registered on 7 April 2022.

Profiling the Biomechanical Responses to Workload on the Human Myocyte to Explore the Concept of Myocardial Fatigue and Reversibility: Rationale and Design of the POWER Heart Failure Study.

It remains unclear why some patients develop heart failure without evidence of structural damage. One theory relates to impaired myocardial energetics and ventricular-arterial decoupling as the heart works against adverse mechanical load. In this original study, we propose the novel concept of myocardial fatigue to capture this phenomenon and aim to investigate this using human cardiomyocytes subjected to a modern work-loop contractility model that closely mimics in vivo cardiac cycles. This proof-of-concept study (NCT04899635) will use human myocardial tissue samples from patients undergoing cardiac surgery to develop a reproducible protocol to isolate robust calcium-tolerant cardiomyocytes. Thereafter, work-loop contractility experiments will be performed over a range of preload, afterload and cycle frequency as a function of time to elicit any reversible reduction in contractile performance (i.e. fatigue). This will provide novel insight into mechanisms behind heart failure and myocardial recovery and serve as a valuable research platform in translational cardiovascular research.

Metformin Protects Against Sunitinib-induced Cardiotoxicity: Investigating the Role of AMPK.

ABSTRACT: Sunitinib is associated with cardiotoxicity through inhibition of AMP-protein kinase (AMPK) signaling. By contrast, the common antidiabetic agent metformin has demonstrated cardioprotection through indirect AMPK activation. In this study, we investigate the effects of metformin during sunitinib-induced cytotoxicity. Left ventricular developed pressure, coronary flow, heart rate, and infarct size were measured in Langendorff-perfused rat hearts treated with 1 µM sunitinib ±50 µM metformin ±1 µM human equilibrative nucleoside transporter inhibitor S-(4-Nitrobenzyl)-6-thioinosine (NBTI). Western blot analysis was performed for p-AMPKα levels. Primary isolated cardiac myocytes from the left ventricular tissue were used to measure live cell population levels. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to assess adjunctive treatment of and metformin in human hepatoma G2 and promyelocytic leukemia (HL-60) cells treated with 0.1-100 µM sunitinib ±50 µM metformin. In the perfused hearts, coadministration of metformin attenuated the sunitinib-induced changes to left ventricular developed pressure, infarct size, and cardiac myocyte population. Western blot analysis revealed a significant decrease in p-AMPKα during sunitinib treatment, which was attenuated after coadministration with metformin. All metformin-induced effects were attenuated, and NBTI was coadministered. The MTT assay demonstrated an increase in the EC50 value during coadministration of metformin with sunitinib compared with sunitinib monotherapy in hepatoma G2 and HL-60 cell lines, demonstrating the impact and complexity of metformin coadministration and the possible role of AMPK signaling. This study highlights the novel cardioprotective properties of metformin and AMPK activation during sunitinib-induced cardiotoxicity when administered together in the Langendorff heart model.

Anti-cancer Therapy Leads to Increased Cardiovascular Susceptibility to COVID-19.

Anti-cancer treatment regimens can lead to both acute- and long-term myocardial injury due to off-target effects. Besides, cancer patients and survivors are severely immunocompromised due to the harsh effect of anti-cancer therapy targeting the bone marrow cells. Cancer patients and survivors can therefore be potentially extremely clinically vulnerable and at risk from infectious diseases. The recent global outbreak of the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its infection called coronavirus disease 2019 (COVID-19) has rapidly become a worldwide health emergency, and on March 11, 2020, COVID-19 was declared a global pandemic by the World Health Organization (WHO). A high fatality rate has been reported in COVID-19 patients suffering from underlying cardiovascular diseases. This highlights the critical and crucial aspect of monitoring cancer patients and survivors for potential cardiovascular complications during this unprecedented health crisis involving the progressive worldwide spread of COVID-19. COVID-19 is primarily a respiratory disease; however, COVID-19 has shown cardiac injury symptoms similar to the cardiotoxicity associated with anti-cancer therapy, including arrhythmia, myocardial injury and infarction, and heart failure. Due to the significant prevalence of micro- and macro-emboli and damaged vessels, clinicians worldwide have begun to consider whether COVID-19 may in fact be as much a vascular disease as a respiratory disease. However, the underlying mechanisms and pathways facilitating the COVID-19-induced cardiac injury in cancer and non-cancer patients remain unclear. Investigations into whether COVID-19 cardiac injury and anti-cancer drug-induced cardiac injury in cancer patients and survivors might synergistically increase the cardiovascular complications and comorbidity risk through a "two-hit" model are needed. Identification of cardiac injury mechanisms and pathways associated with COVID-19 development overlapping with anti-cancer therapy could help clinicians to allow a more optimized prognosis and treatment of cancer survivors suffering from COVID-19. The following review will focus on summarizing the harmful cardiovascular risk of COVID-19 in cancer patients and survivors treated with an anti-cancer drug. This review will improve the knowledge of COVID-19 impact in the field of cardio-oncology and potentially improve the outcome of patients.

Ageing alters the severity of Sunitinib-induced cardiotoxicity: Investigating the mitogen activated kinase kinase 7 pathway association.

Anti-cancer drug Sunitinib is linked to adverse cardiovascular events, which have shown to involve mitogen activated kinase kinase 7 (MKK7) pathway. Sunitinib-induced cardiotoxicity in 3, 12 and 24 months old male Sprague-Dawley rats and MKK7 expression and activation was investigated using the Langendorff perfused heart model followed by Western blot analysis. Cardiac function and infarct size were measured during/after 125 min of Sunitinib treatment. Left ventricular cardiac samples were analysed by qRT-PCR for expression of MKK7 mRNA and cardiac injury associated microRNAs. Infarct size was increased in all Sunitinib treated age groups. Haemodynamic alterations were observed following Sunitinib administration. Left ventricular developed pressure (LVDP) was decreased in all age groups, while heart rate (HR) was decreased in 3 and 12 months groups. Sunitinib treatment decreased the expression of miR-27a in all age groups, while miR-133a and miR-133b levels were increased in 3 months and decreased in 24 months groups. MKK7 mRNA and p-MKK7 levels were decreased in the 3 months group after Sunitinib treatment. MKK7 mRNA level was increased in 24 months group and p-MKK7 levels were increased in 12 months group following Sunitinib treatment. This study highlights the importance and impact of ageing and anti-cancer therapy-induced cardiotoxicity.

The 10th Biennial Hatter Cardiovascular Institute workshop: cellular protection-evaluating new directions in the setting of myocardial infarction, ischaemic stroke, and cardio-oncology.

Due to its poor capacity for regeneration, the heart is particularly sensitive to the loss of contractile cardiomyocytes. The onslaught of damage caused by ischaemia and reperfusion, occurring during an acute myocardial infarction and the subsequent reperfusion therapy, can wipe out upwards of a billion cardiomyocytes. A similar program of cell death can cause the irreversible loss of neurons in ischaemic stroke. Similar pathways of lethal cell injury can contribute to other pathologies such as left ventricular dysfunction and heart failure caused by cancer therapy. Consequently, strategies designed to protect the heart from lethal cell injury have the potential to be applicable across all three pathologies. The investigators meeting at the 10th Hatter Cardiovascular Institute workshop examined the parallels between ST-segment elevation myocardial infarction (STEMI), ischaemic stroke, and other pathologies that cause the loss of cardiomyocytes including cancer therapeutic cardiotoxicity. They examined the prospects for protection by remote ischaemic conditioning (RIC) in each scenario, and evaluated impasses and novel opportunities for cellular protection, with the future landscape for RIC in the clinical setting to be determined by the outcome of the large ERIC-PPCI/CONDI2 study. It was agreed that the way forward must include measures to improve experimental methodologies, such that they better reflect the clinical scenario and to judiciously select combinations of therapies targeting specific pathways of cellular death and injury.

Involvement of mitogen activated kinase kinase 7 intracellular signalling pathway in Sunitinib-induced cardiotoxicity.

The tyrosine kinase inhibitor Sunitinib is used to treat cancer and is linked to severe adverse cardiovascular events. Mitogen activated kinase kinase 7 (MKK7) is involved in the development of cardiac injury and is a component of the c-Jun N-terminal kinase (JNK) signal transduction pathway. Apoptosis signal-regulating kinase 1 (ASK1) is the upstream activator of MKK7 and is specifically inhibited by 2,7-dihydro-2,7-dioxo-3H-naphtho[1,2,3-de]quinoline-1-carboxylic acid ethyl ester (NQDI-1). This study investigates the role of ASK1, MKK7 and JNK during Sunitinib-induced cardiotoxicity. Infarct size were measured in isolated male Sprague-Dawley rat Langendorff perfused hearts treated for 125 min with Sunitinib in the presence and absence of NQDI-1. Left ventricular cardiac tissue samples were analysed by qRT-PCR for MKK7 mRNA expression and cardiotoxicity associated microRNAs (miR-1, miR-27a, miR-133a and miR-133b) or Western blot analysis to measure ASK1/MKK7/JNK phosphorylation. Administration of Sunitinib (1 µM) during Langendorff perfusion resulted in increased infarct size, increased miR-133a expression, and decreased phosphorylation of the ASK1/MKK7/JNK pathway compared to control. Co-administration of NQDI-1 (2.5 µM) attenuated the increased Sunitinib-induced infarct size, reversed miR-133a expression and restored phosphorylated levels of ASK1/MKK7/JNK. These findings suggest that the ASK1/MKK7/JNK intracellular signalling pathway is important in Sunitinib-induced cardiotoxicity. The anti-cancer properties of Sunitinib were also assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay. Sunitinib significantly decreased the cell viability of human acute myeloid leukemia 60 cell line (HL60). The combination of Sunitinib (1 nM-10 µM) with NQDI-1 (2.5 µM) enhanced the cancer-fighting properties of Sunitinib. Investigations into the ASK1/MKK7/JNK transduction pathway could lead to development of cardioprotective adjunct therapy, which could prevent Sunitinib-induced cardiac injury.

Attenuation of Sunitinib-induced cardiotoxicity through the A3 adenosine receptor activation.

Sunitinib is an anti-cancer tyrosine kinase inhibitor associated with severe cardiotoxic adverse effects. Using rat Langendorff heart model and human acute myeloid leukaemia 60 (HL60) cell line we detected the involvement of protein kinase C (PKC) α during Sunitinib-induced cardiotoxicity and the effect of Sunitinib on cancer progression. The cardioprotective and anti-cancer properties of the A3 adenosine receptor agonist 2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) were investigated. The cardiac effect of Sunitinib (1µM) and IB-MECA (1nM) treatment was measured through haemodynamic and infarct size assessment. The cytotoxic effect of Sunitinib (0.1 - 10µM) and IB-MECA (10nM - 10µM) on HL60 cells was assessed using the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay technique. Myocardial injury associated microRNAs (miR-1, miR-27a, miR-133a and miR-133b) and cancer associated microRNAs (miR-15a, miR-16-1 and miR-155) were profiled by qRT-PCR in the cardiac tissue and HL60 cells, while phosphorylated PKCα levels were measured by Western Blot analysis. Sunitinib treatment increased infarct size and decreased left ventricular developed pressure and heart rate. Co-treatment of IB-MECA reversed the myocardial injury produced by Sunitinib administration. IB-MECA did not jeopardize the anti-cancer effect of Sunitinib in HL60 cells. The expression signature of the specific microRNAs in cardiac tissue and HL60 cells showed an altered expression profile when treated with Sunitinib and IB-MECA. pPKCα levels were increased by Sunitinib treatment in cardiac tissue and HL60 cells and co-administration of IB-MECA attenuated this increase in the cardiac tissue. This study reveals that A3 adenosine receptor activation by IB-MECA attenuates Sunitinib-induced cardiotoxicity through the involvement of PKCα.

Investigation into the cardiotoxic effects of doxorubicin on contractile function and the protection afforded by cyclosporin A using the work-loop assay.

Doxorubicin is known to cause cardiotoxicity through multiple routes including the build-up of reactive oxygen species and disruption of the calcium homeostasis in cardiac myocytes, but the effect of drug treatment on the associated biomechanics of cardiac injury remains unclear. Detecting and understanding the adverse effects of drugs on cardiac contractility is becoming a priority in non-clinical safety pharmacology assessment. The work-loop technique enables the assessment of force-length work-loop contractions, which mimic those of the pressure-volume work-loops experienced by the heart in vivo. During this study we evaluated whether the work-loop technique could potentially provide improved insight into the biomechanics associated with drug-induced cardiac dysfunction. In order to do this we investigated the cardiotoxic effects of doxorubicin and characterised the protection afforded by the co-administration of cyclosporin A (CsA). This study provides detailed biomechanical in vitro insight into the cardiac dysfunction associated with Doxorubicin treatment, including reduction in peak force, force during shortening and power output. These effects were significantly abrogated in doxorubicin-CsA co-treatment studies. Closely mimicking the in vivo pressure-volume muscle mechanics, this assay provides a quick and easy technique to gain a better understanding of the detailed biomechanics of drug-induced cardiac dysfunction.

Caspase inhibition via A3 adenosine receptors: a new cardioprotective mechanism against myocardial infarction.

PURPOSE: 2-CL-IB-MECA, (A3 adenosine receptor agonist)(A3AR) mediated cardioprotection is well documented although the associated intracellular signalling pathways remain unclear. Here we demonstrate a role of the pro-survival signalling pathways MEK1/2-ERK1/2 and PI3K/AKT and their effect on modifying Caspase-3 activity in A3AR mediated cardioprotection. METHODS: Isolated perfused rat hearts or primary adult rat cardiac myocytes were subjected to ischaemia/hypoxia and reperfusion/reoxygenation, respectively. 2-CL-IB-MECA (1 nM) was administered at the onset of reperfusion/reoxygenation in the presence and absence of either the PI3K inhibitor Wortmannin (5 nM) or MEK1/2 inhibitor UO126 (10 µM). Heart tissues were harvested for assessment of p-ERK1/2(Thr202/Tyr204) or p-AKT (Ser-473) status or underwent infarct size assessment. Cardiac myocytes underwent flow-cytometric analysis for apoptosis, necrosis, cleaved-caspase 3/p-BAD (Ser-112 and Ser-136) activity post-reoxygenation. RESULTS: 2-CL-IB-MECA significantly reduced infarct size compared to non-treated controls, where co-administration with either of the kinase inhibitors abolished the infarct sparing effects. Administration of 2-CL-IB-MECA at reperfusion significantly upregulated the status of p-ERK1/2 and p-AKT compared to time matched controls in a UO126 and Wortmannin sensitive manner respectively. 2-CL-IB-MECA when administered throughout reoxygenation significantly reduced apoptosis, necrosis, cleaved-caspase 3 activity and increased p-BAD (Ser-112) and p-BAD (Ser-136) activity in myocytes subjected to hypoxia/reoxygenation injury. The cytoprotective effect was abolished by co-administration with the kinase inhibitors Wortmannin and/or UO126. CONCLUSIONS: We have described the molecular mechanisms associated with A3AR mediated cardioprotection indicating a role for the pro-survival signalling pathways that decrease caspase-3 activity. These observations provide novel insight into the pharmacological effects of A3ARs in ameliorating myocardial ischaemia/reperfusion injury.

Molecular basis of cancer-therapy-induced cardiotoxicity: introducing microRNA biomarkers for early assessment of subclinical myocardial injury.

Development of reliable biomarkers for early clinical assessment of drug-induced cardiotoxicity could allow the detection of subclinical cardiac injury risk in vulnerable patients before irreversible damage occurs. Currently, it is difficult to predict who will develop drug-induced cardiotoxicity owing to lack of sensitivity and/or specificity of currently used diagnostics. miRNAs are mRNA regulators and they are currently being extensively profiled for use as biomarkers due to their specific tissue and disease expression signature profiles. Identification of cardiotoxicity-specific miRNA biomarkers could provide clinicians with a valuable tool to allow prognosis of patients at risk of cardiovascular injury, alteration of a treatment regime or the introduction of an adjunct therapy in order to increase the long-term survival rate of patients treated with cardiotoxic drugs.

Attenuation of doxorubicin-induced cardiotoxicity by mdivi-1: a mitochondrial division/mitophagy inhibitor.

Doxorubicin is one of the most effective anti-cancer agents. However, its use is associated with adverse cardiac effects, including cardiomyopathy and progressive heart failure. Given the multiple beneficial effects of the mitochondrial division inhibitor (mdivi-1) in a variety of pathological conditions including heart failure and ischaemia and reperfusion injury, we investigated the effects of mdivi-1 on doxorubicin-induced cardiac dysfunction in naïve and stressed conditions using Langendorff perfused heart models and a model of oxidative stress was used to assess the effects of drug treatments on the mitochondrial depolarisation and hypercontracture of cardiac myocytes. Western blot analysis was used to measure the levels of p-Akt and p-Erk 1/2 and flow cytometry analysis was used to measure the levels p-Drp1 and p-p53 upon drug treatment. The HL60 leukaemia cell line was used to evaluate the effects of pharmacological inhibition of mitochondrial division on the cytotoxicity of doxorubicin in a cancer cell line. Doxorubicin caused a significant impairment of cardiac function and increased the infarct size to risk ratio in both naïve conditions and during ischaemia/reperfusion injury. Interestingly, co-treatment of doxorubicin with mdivi-1 attenuated these detrimental effects of doxorubicin. Doxorubicin also caused a reduction in the time taken to depolarisation and hypercontracture of cardiac myocytes, which were reversed with mdivi-1. Finally, doxorubicin caused a significant elevation in the levels of signalling proteins p-Akt, p-Erk 1/2, p-Drp1 and p-p53. Co-incubation of mdivi-1 with doxorubicin did not reduce the cytotoxicity of doxorubicin against HL-60 cells. These data suggest that the inhibition of mitochondrial fission protects the heart against doxorubicin-induced cardiac injury and identify mitochondrial fission as a new therapeutic target in ameliorating doxorubicin-induced cardiotoxicity without affecting its anti-cancer properties.

Protection from myocardial stunning by ischaemia and hypoxia with the adenosine A3 receptor agonist, IB-MECA.

Guinea pig isolated working hearts were exposed to 30-min ischaemia by reducing coronary flow to 10%, followed by reperfusion. Aortic output fell to 4.5+/-4.5% of the pre-ischaemic value at reperfusion, recovering to 48.2+/-14.6% at 20-min post-reperfusion; the index of myocardial stunning. IB-MECA (N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide, 3 x 10(-7) M), infused from 10 min into ischaemia, did not affect recovery of aortic output 20 min after reperfusion (41.9+/-1.9%). IB-MECA infused at reperfusion, however, significantly protected against stunning, aortic output recovering to 79.6+/-3.9% at 20-min post-reperfusion. Hypoxic gassing (5% CO(2) in nitrogen, 30 min) of guinea pig isolated paced left atria and papillary muscles reduced the developed tension, recovering to 75% 5 min after re-oxygenation. This myocardial stunning was unaffected by IB-MECA (3 x 10(-7) M) added 10 min into hypoxia. IB-MECA added at reoxygenation significantly improved recovery, which was prevented by the adenosine A(3) receptor antagonist, 1-propyl-3-(3-iodo-4-aminobenzyl)-8-(4-oxyacetate)phenylxanthine (I-ABOPX, 1 x 10(-5) M). Thus, stimulation of adenosine A(3) receptors at reperfusion/reoxygenation in guinea pig cardiac preparations protects against myocardial stunning.

The cardioprotective and mitochondrial depolarising properties of exogenous nitric oxide in mouse heart.

OBJECTIVE: Nitric oxide (NO) is reported to be both protective and detrimental in models of myocardial ischaemia/reperfusion injury, which may be concentration dependent. Our objective was to characterise this dichotomy using the nitric oxide donor, S-nitroso N-acetyl penicillamine (SNAP) in isolated perfused mouse heart and isolated mouse cardiac mitochondria. METHODS: To determine the effect of nitric oxide concentration on myocardial viability, isolated mouse hearts were subjected to 35 min global ischaemia and 30 min reperfusion in the presence of SNAP (0.02-20 microM). To determine whether NO mediated protection was via opening of the putative mitochondrial K(ATP) channel and/or free radical synthesis, SNAP perfused hearts were also treated with the mitochondrial K(ATP) channel blocker, 5-hydroxy decanoate (5-HD) and the free-radical scavenger, N-(2-mercaptopropionyl)-glycine (MPG). This data was correlated with mitochondrial membrane potential (Delta Psi(m)), measured with the potentiometric dye, tetra-methyl rhodium methyl ester (TMRM), in isolated mitochondria,by flow cytometry. RESULTS: SNAP dose-dependently attenuated infarct size, with maximal protection observed at 2 microM (17+/-4% versus controls 32+/-3%, P<0.01). At greater concentrations however, protection was lost with infarct sizes tending towards control at 20 microM (29+/-3%). These results were paralleled by changes in Delta Psi(m) in the isolated mitochondria: Delta Psi(m) depolarisation peaking with 1 microM SNAP (26+/-4% shift in TMRM fluorescence, P<0.01); at greater concentrations, this relationship was lost. The mitochondrial K(ATP) channel blocker, 5-HD, resulted in both abrogation of SNAP infarct size reduction and concomitant loss of Delta Psi(m) depolarisation in the mitochondria. MPG however did not influence the cardioprotective properties of SNAP. CONCLUSION: We demonstrate that nitric oxide can mediate cardioprotection in a dose-dependent fashion by an effect that may be related to Delta Psi(m). Both cardioprotection and Delta Psi(m) changes are sensitive to 5-HD and the cardioprotection appears independent of free-radical synthesis.

Adenosine A(3) receptor activation protects the myocardium from reperfusion/reoxygenation injury.

Ischemia-reperfusion induces both necrotic and apoptotic cell death. The ability of adenosine to attenuate reperfusion-induced injury (RI) and the role played by adenosine receptors are unclear. We therefore studied the role of the A(3) receptor (A(3)R) in ameliorating RI using the specific A(3)R agonist 1-[2-chloro-6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxi-N-methyl-b-D-ribofuranuronamide (2-Cl-IB-MECA). Isolated rat hearts and cardiomyocytes were subjected to ischemia or simulated ischemia, followed by reperfusion/reoxygenation. The end points were percent infarction/risk zone and annexin-V (apoptosis) and/or propidium iodide positivity (necrosis), respectively. In isolated hearts, 2-Cl-IB-MECA significantly limited infarct size (44.2 +/- 2.7% in control vs. 21.9 +/- 2.4% at 1 nM and 35.8 +/- 3.3% at 0.1 nM, P < 0.05). In isolated myocytes, apoptosis and necrosis were significantly reduced compared with controls (5.7 +/- 2.6% vs. 17.1 +/- 1.3% and 13.7 +/- 2.0% vs. 23.1 +/- 1.5%, respectively, P < 0.0001). In both models, the beneficial effects were abrogated using the A(3)R antagonist MRS-1191. The involvement of A(2a) receptor activation was also examined. This is the first study to demonstrate that A(3)R activation at reperfusion limits myocardial injury in the isolated rat heart and improves survival in isolated myocytes, possibly by antiapoptotic and antinecrotic mechanisms.

Inhibiting mitochondrial permeability transition pore opening: a new paradigm for myocardial preconditioning?

OBJECTIVE: We propose that ischemic preconditioning (IPC) and mitochondrial K(ATP) channel activation protect the myocardium by inhibiting mitochondrial permeability transition pore (MPTP) opening at reperfusion. METHODS: Isolated rat hearts were subjected to 35 min ischemia/120 min reperfusion and assigned to the following groups: (1) control; (2) IPC of 2x5 min each of preceding global ischemia; (3,4,5) 0.2 micromol/l cyclosporin A (CsA, which inhibits MPTP opening), 5 micromol/l FK506 (which inhibits the phosphatase calcineurin without inhibiting MPTP opening), or 20 micromol/l atractyloside (Atr, a MPTP opener) given at reperfusion; (6,7) pre-treatment with 30 micromol/l diazoxide (Diaz, a mitochondrial K(ATP) channel opener) or 200 nmol/l 2 chloro-N(6)-cyclopentyl-adenosine (CCPA, an adenosine A1 receptor agonist); (8) IPC+Atr; (9) Diaz+Atr; (10) CCPA+Atr. The effect of mitochondrial K(ATP) channel activation on calcium-induced MPTP opening in isolated calcein-loaded mitochondria was also assessed. RESULTS: IPC, CsA when given at reperfusion, and pre-treatment with diazoxide or CCPA all limited infarct size (19.9+/-2.6% in IPC; 24.6+/-1.9% in CsA, 18.0+/-1.7% in Diaz, 20.4+/-3.3% in CCPA vs. 44.7+/-2.0% in control, P<0.0001). Opening the MPTP with atractyloside at reperfusion abolished this cardio-protective effect (47.7+/-1.8% in IPC+Atr, 42.3+/-3.2% in Diaz+Atr, 51.2+/-1.6% in CCPA+Atr). Atractyloside and FK506, given at reperfusion, did not influence infarct size (45.7+/-2.1% in Atr and 43.1+/-3.6% in FK506 vs. 44.7+/-2.0% in control, P=NS). Diazoxide (30 micromol/l) was shown to reduce calcium-induced MPTP opening by 52.5+/-8.0% in calcein-loaded mitochondria. 5-Hydroxydecanoic acid (100 micromol/l) was able to abolish the cardio-protective effects of both diazoxide and IPC. CONCLUSION: One interpretation of these data is that IPC and mitochondrial K(ATP) channel activation may protect the myocardium by inhibiting MPTP opening at reperfusion.

Effects of adenosine receptor agonists on guinea-pig isolated working hearts and the role of endothelium and NO.

The hypothesis that the coronary vasodilator effects of adenosine receptor agonists are independent of the vascular endothelium or mediators derived therefrom was examined in guinea-pig isolated working hearts. Adenosine receptor agonists, 5'-(N-ethylcarboxamido)-adenosine (NECA; two-fold selective for A2 over A1 receptors), 2-[p-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS21680; A2A selective), N6-cyclopentyl-adenosine (CPA; A1 selective) and N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA; A3 selective), were infused (3 x 10(-7) M) after endothelium removal by passing oxygen through the coronary circulation. In spontaneously beating hearts, CGS21680 and NECA increased, while CPA decreased, coronary flow. NECA and CPA reduced heart rate, left ventricular pressure and aortic output. The nitric oxide synthase (NOS) inhibitor, N(G)-nitro-L-arginine (L-NOARG; 3 x 10(-5) M) abolished the vasodilatation by NECA but not CGS21680, indicating that nitric oxide (NO) of a non-endothelial source mediated the NECA response. Coronary vasodilatation by CGS21680 was inhibited bythe A2A receptor antagonist, 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo [2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM241385). Indometacin (10(-6) M) attenuated the coronary vasodilatation to CGS21680, suggesting a partial role for cyclooxygenase products. IB-MECA had no effect, indicating no A3 receptor involvement. In paced working hearts, the responses were similar except CPA had no effect on coronary flow or aortic output and CGS21680 increased left ventricular pressure and the maximum rate of ventricular pressure rise. This study has demonstrated functionally effective removal of the endothelium by a novel method of passing oxygen through the coronary vasculature. A coronary vasodilator action of adenosine receptor agonists mediated via A2A receptors is endothelium- and NO-independent, but partially involves cyclooxygenase products.