Tyler H. Rork

Adenosine A2A receptor activation reduces infarct size in the isolated, perfused mouse heart by inhibiting resident cardiac mast cell degranulation

Mast cells are found in the heart and contribute to reperfusion injury following myocardial ischemia. Since the activation of A2A adenosine receptors (A2AARs) inhibits reperfusion injury, we hypothesized that ATL146e (a selective A2AAR agonist) might protect hearts in part by reducing cardiac mast cell degranulation. Hearts were isolated from five groups of congenic mice: A2AAR+/+ mice, A2AAR(-/-) mice, mast cell-deficient (Kit(W-sh/W-sh)) mice, and chimeric mice prepared by transplanting bone marrow from A2AAR(-/-) or A2AAR+/+ mice to radiation-ablated A2AAR+/+ mice. Six weeks after bone marrow transplantation, cardiac mast cells were repopulated with >90% donor cells. In isolated, perfused hearts subjected to ischemia-reperfusion injury, ATL146e or CGS-21680 (100 nmol/l) decreased infarct size (IS; percent area at risk) from 38 +/- 2% to 24 +/- 2% and 22 +/- 2% in ATL146e- and CGS-21680-treated hearts, respectively (P < 0.05) and significantly reduced mast cell degranulation, measured as tryptase release into reperfusion buffer. These changes were absent in A2AAR(-/-) hearts and in hearts from chimeric mice with A2AAR(-/-) bone marrow. Vehicle-treated Kit(W-sh/W-sh) mice had lower IS (11 +/- 3%) than WT mice, and ATL146e had no significant protective effect (16 +/- 3%). These data suggest that in ex vivo, buffer-perfused hearts, mast cell degranulation contributes to ischemia-reperfusion injury. In addition, our data suggest that A2AAR activation is cardioprotective in the isolated heart, at least in part by attenuating resident mast cell degranulation.

Acetaminophen in the Post-ischemia Reperfused Myocardium

Acetaminophen was administered acutely at the onset of reperfusion after 20 min of low-flow, global myocardial ischemia in isolated, perfused guinea pig hearts (Langendorff) to evaluate its influence in the postischemia, reperfused myocardium. Similarly prepared hearts were treated with vehicle or with uric acid (another phenol for comparison). Functionally, acetaminophen-treated hearts (0.35 mM) achieved significantly greater recovery during reperfusion. For example, left ventricular developed pressures at 40 min reperfusion were 38 ± 3, 27 ± 3, and 20 ± 2 in the presence of acetaminophen (P < 0.05, relative to the other two groups), vehicle, and uric acid, respectively. Coronary perfusion pressures and calculated coronary vascular resistances, in the acetaminophen-treated hearts, were significantly lower at the same time (e.g., coronary perfusion pressures in the three groups, respectively, were 40 ± 2 [P < 0.05], 51 ± 3, and 65 ± 12 mm Hg). Under baseline, control conditions, creatine kinase ranged from 12–15 units/liter in the three groups. It increased to 35–40 units/liter (P < 0.05) during ischemia but was significantly reduced by acetaminophen during reperfusion (e.g., 5.3 ± 0.8 units/liter at 40 min). Oxidant-mediated chemiluminescence in all three treatment groups during baseline conditions and ischemia was similar (i.e., approximately 1.5–2.0 min for peak luminescence to reach its half maximal value). It took significantly more time during reperfusion for the oxidation of luminol in the presence of acetaminophen (>20 min, P < 0.05) than in its absence (3–8 min in uric acid- and vehicle-treated hearts). These results suggest that administration of acetaminophen (0.35 mM), at the onset of reperfusion, provides anti-oxidant–mediated cardioprotection in the postischemia, reperfused myocardium.

Chronically Administered Acetaminophen and the Ischemia/Reperfused Myocardium

Male and female Hartley strain guinea pigs weighing 280 ± 10 g were given acetaminophen-treated water ad libitum for 10 days. Sham-treated control animals were given similar quantities of untreated tap water (vehicle-treated control group). On Day 10, hearts were extracted, instrumented, and exposed to an ischemia (low-flow, 20 min)/reperfusion protocol. Our objective was to compare and contrast ventricular function, coronary circulation, and selected biochemical and histological indices in the two treatment groups. Left ventricular developed pressure in the early minutes of reperfusion was significantly greater in the presence of acetaminophen, e.g., at 1 min, 40 ± 4 vs 21 ± 3 mmHg (P < 0.05). Coronary perfusion pressure was significantly less from 3 to 40 min of reperfusion in the presence of acetaminophen. Creatine kinase release in vehicle-treated hearts rose from 42 ± 14 (baseline) to 78 ± 25 units/liter by the end of ischemia. Corresponding values in acetaminophen-treated hearts were 36 ± 8 and 44 ± 14 units/liter. Acetaminophen significantly (P < 0.05) attenuated release of creatine kinase. Chemiluminescence, an indicator of the in vitro production of peroxynitrite via the in vivo release of superoxide and nitric oxide, was also significantly attenuated by acetaminophen. Electron microscopy indicated a well-preserved myofibrillar ultrastructure in the postischemic myocardium of acetaminophen-treated hearts relative to vehicle-treated hearts (e.g., few signs of contraction bands, little or no evidence of swollen mitochondria, and well-defined light and dark bands in sarcomeres with acetaminophen; opposite with vehicle). We conclude that chronic administration of acetaminophen provides cardioprotection to the postischemic, reperfused rodent myocardium.

Antiarrhythmic Properties of Acetaminophen in the Dog

Mongrel dogs bred for research and weighing 25 ± 3 kg were used to test the hypothesis that acetaminophen has antiar-rhythmic properties. Only ventricular arrhythmias defined by the Lambeth Conventions were investigated. Dogs were exposed either to 60 mins of regional myocardial ischemia followed by 180 mins of reperfusion (n = 14) or were administered a high dose of ouabain (n = 14). Both groups of 14 dogs were further divided into vehicle and acetaminophen treatment groups (n = 7 in each). During selected 10-min intervals, we recorded the numbers of ventricular premature beats, ventricular salvos, ventricular bigeminy, ventricular tachycardia (nonsustained and sustained), and we recorded the heart rate, systemic arterial blood pressure, and left ventricular function. Neither heart rate nor the number of ventricular arrhythmias differed significantly under baseline conditions. Conversely, the combined average number of ventricular ectopic beats during ischemia and reperfusion was significantly less in the presence of acetaminophen (28 ± 4 vs. 6 ± 1; P < 0.05). Similarly, percent ectopy during reperfusion in vehicle- and acetaminophen-treated dogs was 1.4 ± 0.4 and 0.4 ± 0.2, respectively (P < 0.05). The number of all ventricular ectopic beats except ventricular salvos was also significantly reduced in the presence of acetaminophen. Similar results were obtained with ouabain. Our results reveal that systemic administration of a therapeutic dose of acetaminophen has previously unreported antiarrhythmic effects in the dog.

Acetaminophen in the Hypoxic and Reoxygenated Guinea Pig Myocardium

We investigated the effects of 0.35-mM acetaminophen and its vehicle on isolated, perfused guinea pig hearts made hypoxic and subsequently reoxygenated. Hearts were allowed 30 min postinstrumentation to reach baseline, steady-state values, and then were exposed to 6 min of hypoxia (5% O2, 5% CO2, balance N2) followed by 36 min of reoxygenation (95% O2, 5% CO2). We recorded hemodynamic, metabolic, and mechanical data in addition to assessing ultrastructure and the capacity of coronary venous effluent to reduce reactive oxygen species. We found that acetaminophen-treated hearts retained a greater fraction of mechanical function during hypoxia and reoxygenation. For example, the average percentage change from baseline of left ventricular developed pressure in acetaminophen- and vehicle-treated hearts at 6 min reoxygenation was 9 ± 2% and -8 ± 5% (P < 0.05), respectively. In addition, electron micrographs revealed greater preservation of myofibrillar ultrastructure in acetaminophen-treated hearts. Biochemical analyses revealed the potential of coronary effluent from acetaminophen-treated hearts to significantly neutralize peroxynitrite-dependent chemiluminescence in all recorded time periods. During early reoxygenation, the percentage inhibition of peroxynitrite-mediated chemiluminescence was 56 ± 10% in vehicle-treated hearts and 99 ± 1% in acetaminophen-treated hearts (P < 0.05). We conclude that acetaminophen has previously unreported cardioprotective properties in the nonischemic, hypoxic, and reoxygenated myocardium mediated through the reduction of reactive oxygen species.

An Old Drug with a New Purpose: Cardiovascular Actions of Acetaminophen (Paracetamol)

For over 50 years, acetaminophen (paracetamol) has been a staple in industrialized and non-industrialized countries for the treatment of pain and fever. Although its precise mechanisms of action are not known, the drug generates dose-dependent reduction in circulating prostaglandins, inhibits myeloperoxidase and the oxidation of lipoproteins, and appears to confer cardioprotection by blocking the effects of hydroxyl radical, peroxynitrite, and hydrogen peroxide. The drug might inhibit cyclooxygenase, although its ultimate target(s) is (are) still unclear. Sadly, since most investigations of acetaminophen have focused on its analgesic/antipyretic properties and hepatotoxicity, the effects of the drug on other mammalian organ systems, including the heart and circulation, have been ignored. Recently, work in our laboratory has shown acetaminophen to have a protective role in the injured mammalian myocardium. The cardioprotection was first observed in isolated, perfused guinea pig hearts subjected to ischemia-reperfusion injury. Hearts pretreated with acetaminophen recovered greater ventricular function and exhibited improved myofibrillar ultrastructure when compared to vehicle-treated hearts. More recent in vitro investigations have suggested protective roles for acetaminophen in barbiturate-induced arrhythmogenesis and myocardial hypoxia-reoxygenation injury. We have also extended our work to the in vivo arena. There, we found that acetaminophen reduced infarct size in dogs exposed to 60 minutes regional myocardial ischemia and 180 minutes reperfusion. We invite and encourage readers of this review to repeat/duplicate our experiments. Such work is needed to either challenge or support our findings. Further, more clinically-relevant work depends on these basic and related translational experiments.

New Perspectives on Acetaminophen

Acetaminophen (paracetamol) continues to be one of the leading, international over-the-counter analgesics. Unlike the COX-2 inhibitors, no adverse cardiovascular effects have been associated with acetaminophen usage in safe, therapeutic dosages. There are few rigorous physiological investigations of its actions/mechanisms of action. Several investigations, in recent years, have questioned its potential activity in the mammalian cardiovascular system. Our laboratory has found some positive salutary effects in myocardial ischemia/reperfusion injury and during myocardial infarction. In addition, another laboratory found concomitant cardioprotective effects during the acute phase of myocardial infarction. They report similar effects of the ability of acetaminophen to reduce the severity of myocardial infarction and ultimately result in improved mortality rates. They also postulate that this attenuation in myocardial damage is mediated through antioxidant means. Others have not found similar results but have reported no detrimental cardiovascular actions of acetaminophen in experimental animals. The inconsistency in results probably reflects multiple differences in experimental approaches, including use of different species, different experimental preparations, different drug concentrations, and different routes/modes of administering/preparing the drug. The current review focuses on this recent research and tries to provide new perspectives for future investigations. In particular, we have identified new cellular/subcellular targets at which acetaminophen might be acting. These include matrix metalloproteinases and the mitochondrial permeability transition pore. Future work will help identify additional tissue/organ actions of acetaminophen and its as-yet-unknown mechanisms of action.