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Background: Fatigue is one of the most common acute complications of radiation therapy (RT). It can have a serious impact on a patient’s quality of life and ability to engage in treatment, and has been associated with lower recurrence-free and overall survival in patients with breast cancer. The pathogenesis of radiation-induced fatigue remains elusive, and factors implicated include biological (low hemoglobin and elevated pro-inflammatory cytokines and chemokines) and psychological (pain, depression and anxiety). Although it is a common clinical issue, optimal treatment strategies are lacking, and a better understanding of the mechanisms involved is needed to devise effective interventions. The purpose of this exploratory, non-randomized, prospective study was to examine a number of possible biological and psychological factors influencing fatigue in patients undergoing breast irradiation for early stage breast cancer. Methods: All subjects were assessed at five time points: immediately before RT, mid-point of RT, end of RT, and 6 months and 1 year after completion of RT. Clinical evaluation of skin toxicity and cosmetic outcome and laboratory measures evaluating anemia and hepatic toxicity were performed. Laboratory markers of systemic inflammatory/stress response included salivary cortisol, CRP and cytokines. Caspase-1 and caspase-3, novel markers of apoptosis, were also collected. Fatigue, distress, depression, anxiety, sleep, energy level and pain were assessed at each time point using validated measures. A two-sample student t-test or a non-parametric Wilcoxon rank sum test was used to identify significant associations between fatigued and non-fatigued subjects at each time point. Results: Fifty-three subjects completed the study. Subjects were predominantly white and non-Hispanic, middle to upper-middle class, with a mean age of 59. Fatigued subjects were more likely than non-fatigued subjects to have a history of anxiety and/or depression. Across all subjects, fatigue increased during treatment and returned to or near baseline by 6 months after treatment. Fatigue was significantly associated with overall distress, energy level, and some measures of physical and functional well-being during treatment; these associations were not present at baseline and resolved by 6 months. Fatigued subjects were more likely to have increases in depression scores by the end of treatment, although mean depression scores did not reach clinical significance. Fatigue was significantly associated with breast-specific pain by the end of treatment, and this resolved by 6 months. Fatigue was significantly associated with some measures of sleep at baseline, and this persisted during treatment and resolved by 6 months to 1 year. Fatigue was not associated with measures of skin toxicity and cosmetic outcome and laboratory measures of anemia, hepatic toxicity, salivary cortisol, caspase-1, caspase-3, and IL-10; most other cytokines were undetectable. Conclusions: This was a comprehensive, longitudinal study evaluating the association of biological and psychological factors with the development of fatigue in patients undergoing breast irradiation. As expected, in patients experiencing heightened fatigue during breast irradiation, fatigue was greatest at the end of treatment and returned to or near baseline by 6 months after treatment. Important associations with the development of fatigue included physical and functional parameters, and of particular significance were patients with a prior history of mental health diagnoses. In contrast to previous studies, we did not find an increase in pro-inflammatory or in novel biomarkers including caspase-1 and caspase-3. Identification of and interventions directed at those patients at risk could impact positively on the experience associated with fatigue and radiation. Citation Format: Edward Perry, Sudhanshu Mulay, Jayesh Kamath, Robert Dowsett, Rong Wu, Xiaoyan Wang, Matthew Greenwood, Jacob Neuwirth, James Grady, Bruce Liang, Susan Tannenbaum. Factors influencing fatigue in breast cancer patients undergoing breast irradiation [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS9-29.

Background Anthracycline induced cardiomyopathy is a major cause of mortality and morbidity among pediatric cancer survivors. It has been postulated that oxidative stress induction and inflammation may play a role in the pathogenesis of this process. Accordingly, the present study performed an assessment of biomarker profiles and functional imaging parameters focused upon potential early determinants of anthracycline induced cardiomyopathy. Methods Patients (10–22 years) were prospectively enrolled between January 2013 and November 2014. Thirteen subjects completed the study and underwent serial cardiac magnetic resonance imaging and plasma biomarker profiling performed 24–48 h after the first anthracycline dose and at set dose intervals. In addition, we collected plasma samples from 62 healthy controls to examine normal plasma biomarker profiles. Results Left ventricular ejection fraction (LVEF) decreased from 64.3 ± 6.2 at the first visit to 57.5 ± 3.3 (p = 0.004) 1 year after chemotherapy. A decline in longitudinal strain magnitude occurred at lower cumulative doses. A differential inflammatory/matrix signature emerged in anthracycline induced cardiomyopathy patients compared to normal including increased interleukin-8 and MMP levels. With longer periods of anthracycline dosing, MMP-7, a marker of macrophage proteolytic activation, increased by 165 ± 54% whereas interleukin-10 an anti-inflammatory marker decreased by 75 ± 13% (both p

Introduction Acute ischemic injury leads to severe neuronal loss. One of the key mechanisms responsible for this effect is inflammation, which is characterized by the activation of myeloid cells, including resident microglia and infiltrating monocytes/macrophages. P2X4 receptors (P2X4Rs) present on these immune cells modulate the inflammatory response. For example, excessive release of adenosine triphosphate during acute ischemic stroke triggers stimulation of P2X4Rs, leading to myeloid cell activation and proliferation and further exacerbating post-ischemic inflammation. In contrast, during recovery P2X4Rs activation on microglia leads to the release of brain-derived neurotrophic factor (BDNF), which alleviate depression, maintain synaptic plasticity and hasten post-stroke behavioral recovery. Therefore, we hypothesized that deletion of the P2X4R specifically from myeloid cells would have differential effects on acute versus chronic recovery following stroke. Methods We subjected global or myeloid-specific (MS) P2X4R knock-out (KO) mice and wild-type littermates of both sexes to right middle cerebral artery occlusion (60 min). We performed histological, behavioral (sensorimotor and depressive), and biochemical (quantitative PCR and flow cytometry) analyses to determine the acute (three days after occlusion) and chronic (30 days after occlusion) effects of receptor deletion. Results Global P2X4R deletion led to reduced infarct size in both sexes. In MS P2X4R KO mice, only females showed reduced infarct size, an effect that did not change with ovariectomy. MS P2X4R KO mice of both sexes showed swift recovery from sensorimotor deficits during acute recovery but exhibited a more pronounced post-stroke depressive behavior phenotype that was independent of infarct size. Quantitative PCR analysis of whole cell lysate as well as flow-sorted myeloid cells from the perilesional cortex showed increased cellular interleukin 1 beta (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-α) mRNA levels but reduced plasma levels of these cytokines in MS P2X4R KO mice after stroke. The expression levels of BDNF and other depression-associated genes were reduced in MS P2X4R KO mice after stroke. Conclusions P2X4R deletion protects against stroke acutely but predisposes to depression-like behavior chronically after stroke. Thus, a time-sensitive approach should be considered when targeting P2X4Rs after stroke.

Background— Heart failure (HF), despite continuing progress, remains a leading cause of mortality and morbidity. P2X4 receptors (P2X4R) have emerged as potentially important molecules in regulating cardiac function and as potential targets for HF therapy. Transgenic P2X4R overexpression can protect against HF, but this does not explain the role of native cardiac P2X4R. Our goal is to define the physiological role of endogenous cardiac myocyte P2X4R under basal conditions and during HF induced by myocardial infarction or pressure overload. Methods and Results— Mice established with conditional cardiac-specific P2X4R knockout were subjected to left anterior descending coronary artery ligation–induced postinfarct or transverse aorta constriction–induced pressure overload HF. Knockout cardiac myocytes did not show P2X4R by immunoblotting or by any response to the P2X4R-specific allosteric enhancer ivermectin. Knockout hearts showed normal basal cardiac function but depressed contractile performance in postinfarct and pressure overload models of HF by in vivo echocardiography and ex vivo isolated working heart parameters. P2X4R coimmunoprecipitated and colocalized with nitric oxide synthase 3 (eNOS) in wild-type cardiac myocytes. Mice with cardiac-specific P2X4R overexpression had increased S-nitrosylation, cyclic GMP, NO formation, and were protected from postinfarct and pressure overload HF. Inhibitor of eNOS, L-N 5 -(1-iminoethyl)ornithine hydrochloride, blocked the salutary effect of cardiac P2X4R overexpression in postinfarct and pressure overload HF as did eNOS knockout. Conclusions— This study establishes a new protective role for endogenous cardiac myocyte P2X4R in HF and is the first to demonstrate a physical interaction between the myocyte receptor and eNOS, a mediator of HF protection.

Aims To determine the role of CD13 as an adhesion molecule in trafficking of inflammatory cells to the site of injury in vivo and its function in wound healing following myocardial infarction induced by permanent coronary artery occlusion. Methods and results Seven days post-permanent ligation, hearts from CD13 knockout (CD13KO) mice showed significant reductions in cardiac function, suggesting impaired healing in the absence of CD13. Mechanistically, CD13KO infarcts showed an increase in small, endothelial-lined luminal structures, but no increase in perfusion, arguing against an angiogenic defect in the absence of CD13. Cardiac myocytes of CD13KO mice showed normal basal contractile function, eliminating myocyte dysfunction as a mechanism of adverse remodelling. Conversely, immunohistochemical and flow cytometric analysis of CD13KO infarcts demonstrated a dramatic 65% reduction in infiltrating haematopoietic cells, including monocytes, macrophages, dendritic, and T cells, suggesting a critical role for CD13 adhesion in inflammatory trafficking. Accordingly, CD13KO infarcts also contained fewer myofibroblasts, consistent with attenuation of fibroblast differentiation resulting from the reduced inflammation, leading to adverse remodelling. Conclusion In the ischaemic heart, while compensatory mechanisms apparently relieve potential angiogenic defects, CD13 is essential for proper trafficking of the inflammatory cells necessary to prime and sustain the reparative response, thus promoting optimal post-infarction healing.

We have previously demonstrated that in response to traumatic injury in skeletal muscle, there is a dysregulation of the matrix metalloproteases (MMPs) and their inhibitors (TIMPs), a response hypothesized to interfere with proper skeletal muscle regeneration. Moreover, we have shown that pharmacological activation of the adenosine A3 receptor by Cl-IBMECA in skeletal muscle can protect against ischemia-reperfusion and eccentric exercise injury. However, the mechanism by which Cl-IBMECA protects muscle tissue is poorly defined. This study evaluated the effects of Cl-IBMECA on MMP/TIMP expression in skeletal muscle and tested the hypothesis that adenosine A3 receptor-stimulated protection of skeletal muscle following traumatic injury is associated with a blunting of MMPs involved in inflammatory processes and collagen degradation, and an increase in MMPs associated with extracellular matrix remodeling. Sixty C57BL/6J male mice were injected with Cl-IBMECA ( n = 30) or a vehicle ( n = 30), and Evans blue dye. Injury was induced by applying a cold steel probe (−79°C) to the tibialis anterior (TA) muscle for 10 s. TA muscles from uninjured and injured legs were collected 3, 10, and 24 h postinjury for analysis of muscle injury and MMP/TIMP mRNA and protein levels. Twenty-four hours postinjury, 56.8% of the fibers were damaged in vehicle-treated mice vs. 35.4% in Cl-IBMECA-treated mice ( P = 0.02). Cl-IBMECA treatment reduced membrane type 1 (MT1)-MMP, MMP-3, MMP-9, and TIMP-1 mRNA expression 2- to 20-fold compared with vehicle-treated mice ( P < 0.05). Cl-IBMECA decreased protein levels of latent/shed MT1-MMP 23–2,000%, respectively, 3–10 h postinjury. In Cl-IBMECA-treated mice, latent MMP-2 was decreased 20% 3 h postinjury, active MMP-3 was decreased 64% 3 h postinjury, and latent/active MMP-9 was decreased 417,631% 3 h postinjury and 20% 10 h postinjury. Protein levels of active MMP-2 and latent MMP-3 were increased 25% and 74% 3 h postinjury, respectively. The present study elucidates a new protective role of adenosine A3 receptor stimulation in posttraumatic skeletal muscle injury.

Introduction Anthracycline chemotherapy (AC) can be associated with a progressive decline in left ventricular ejection fraction (LVEF), but the contributory mechanisms remain unclear. While changes in microRNAs (miRs), a key translational control point, have been identified in adult cardiovascular disease, whether and to what degree a shift in miR profiles occurs in pediatric AC patients has not been well studied. The goal of this study was to examine miR profiles (unbiased array) in pediatric AC patients compared to age matched referent normal patients. Hypothesis We hypothesize that pediatric AC patients will express a unique miR profile at initiation and completion of therapy and will be related to LVEF. Methods Serum was collected in pediatric patients (n=12, 13-22 years old) with newly diagnosed malignancy requiring AC within 24-48 hours following initiation of therapy (30-60 mg/m2) and approximately one year after completing maximal therapy. A custom microarray of 84 miRs associated with cardiovascular disease pathways was utilized (RT-qPCR) and indexed to referent normal profiles (n=17, 13-17 years old). LVEF was computed by cardiac MRI. Results LVEF fell from AC initiation at one year following AC (64.28% ± 1.78% vs 57.53% ± 0.95% respectively, p=0.004). Of the 84 miRs profiled, significant shifts in 17 miRs occurred relative to referent normal (p ≤ 0.05) where a nearly equivalent number were upregulated and downregulated (41 vs 59%, respectively). Moreover, the functional domain of miRs associated with myocardial differentiation and development fell by over 3-fold at completion of AC (Figure, p ≤ 0.05). Lastly, miRs-143-3p, -499a-5p, and -142-3p were associated with the fall in LVEF (p ≤ 0.05). Conclusions This study demonstrated for the first time that dynamic changes in miR expression occurs in pediatric cancer patients following AC. These findings suggest that miRs are a potential strategy for early identification of patients most susceptible to LV dysfunction following AC exposure, as well as potential targets for protective therapies.

Liang BT. Adenosine A3 receptor stimulation induces protection of skeletal muscle from eccentric exercise-mediated injury. Am J Physiol Regul Integr Comp Physiol 299: R259‐R267, 2010. First published April 28, 2010; doi:10.1152/ajpregu.00060.2010.—Effective therapy to reduce skeletal muscle injury associated with severe or eccentric exercise is needed. The purpose of this study was to determine whether adenosine receptor stimulation can mediate protection from eccentric exercise-induced muscle injury. Downhill treadmill exercise (15°) was used to induce eccentric exercise-mediated skeletal muscle injury. Experiments were conducted in both normal wild-type (WT) mice and also in -sarcoglycan knockout dystrophic mice, animals that show an exaggerated muscle damage with the stress of exercise. In the vehicle-treated WT animals, eccentric exercise increased serum creatine kinase (CK) greater than 3-fold to 358.9 62.7 U/l (SE). This increase was totally abolished by stimulation of the A3 receptor. In the dystrophic -sarcoglycan-null mice, eccentric exercise caused CK levels to reach 55,124 5,558 U/l. A3 receptor stimulation in these animals reduced the CK response by nearly 50%. In the dystrophic mice at rest, 10% of the fibers were found to be damaged, as indicated by Evans blue dye staining. While this percentage was doubled after exercise, A3 receptor stimulation eliminated this increase. Neither the A1 receptor agonist 2-chloro-N 6 -cyclopentyladenosine (0.05 mg/kg) nor the A2A receptor agonist 2-p-(2carboxyethyl)phenethylamino-5=-N-ethylcarboxamidoadenosine (0.07 mg/kg) protected skeletal muscle from eccentric exercise injury in WT or dystrophic mice. The protective effect of adenosine A3 receptor stimulation was absent in mice, in which genes for phospholipase C 2/3 (PLC2/3) and -sarcoglycan were deleted. The present study elucidates a new protective role of the A3 receptor and PLC2/3 and points to a potentially effective therapeutic strategy for eccentric exerciseinduced skeletal muscle injury.

Evidence is accumulating to support a potentially important role for purinergic (P2X) receptors in heart failure (HF). We tested the hypothesis that a hydrolysis-resistant nucleotide analog with agonist activity at myocardial P2X receptors (P2XRs) improves the systolic HF phenotype in mouse and dog models. We developed a hydrolysis-resistant adenosine monophosphate derivative, (1'S,2R,3S,4'R,5'S)-4-(6-amino-2-chloro-9H-purin-9-yl)-1-[phosphoryloxymethyl] bicycle[3.1.0]hexane-2,3-diol) (MRS2339), with agonist activity at native cardiac P2XRs. Chronic MRS2339 infusion in postinfarct and calsequestrin (CSQ) mice with HF resulted in higher rates of pressure change (+dP/dt), left ventricle (LV)-developed pressure, and cardiac output in an in vitro working heart model. Heart function in vivo, as determined by echocardiography-derived fractional shortening, was also improved in MRS2339-infused mice. The beneficial effect of MRS2339 was dose-dependent and was identical to that produced by cardiac myocyte-specific overexpression of the P2X(4) receptor. The HF improvement was associated with the preservation of LV wall thickness in both systole and diastole in postinfarct and CSQ mice. In dogs with pacing-induced HF, MRS2339 infusion reduced left ventricular end-diastolic pressure, improved arterial oxygenation, and increased +dP/dt. MRS2339 treatment also decreased LV chamber size in mice and dogs with HF. In murine and canine models of systolic HF, in vivo administration of a P2X nucleotide agonist improved contractile function and cardiac performance. These actions were associated with preserved LV wall thickness and decreased LV remodeling. The data are consistent with a role of cardiac P2XRs in mediating the beneficial effect of this agonist.

Although adenosine exerts cardio-and vasculoprotective effects, the roles and signaling mechanisms of different adenosine receptors in mediating skeletal muscle protection are not well understood. We used a mouse hindlimb ischemia-reperfusion model to delineate the function of three adenosine receptor subtypes. Adenosine A3receptor-selective agonist 2-chloro- N6-(3-iodobenzyl)adenosine-5′- N-methyluronamide (Cl-IBMECA; 0.07 mg/kg ip) reduced skeletal muscle injury with a significant decrease in both Evans blue dye staining (5.4 ± 2.6%, n = 8 mice vs. vehicle-treated 28 ± 6%, n = 7 mice, P < 0.05) and serum creatine kinase level (1,840 ± 910 U/l, n = 13 vs. vehicle-treated 12,600 ± 3,300 U/l, n = 14, P < 0.05), an effect that was selectively blocked by an A3receptor antagonist 3-ethyl-5-benzyl-2-methyl-6-phenyl-4-phenylethynyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (MRS-1191; 0.05 mg/kg). The adenosine A1receptor agonist 2-chloro- N6-cyclopentyladenosine (CCPA; 0.05 mg/kg) also exerted a cytoprotective effect, which was selectively blocked by the A1antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 0.2 mg/kg). The adenosine A2Areceptor agonist 2- p-(2-carboxyethyl)phenethylamino-5′- N-ethylcarboxamidoadenosine (CGS-21680; 0.07 mg/kg)-induced decrease in skeletal muscle injury was selectively blocked by the A2Aantagonist 2-(2-furanyl)-7-[3-(4-methoxyphenyl)propyl]-7H-pyrazolo[4,3-e] [1,2,4]triazolo[1,5-C]pyrimidin-5-amine (SCH-442416; 0.017 mg/kg). The protection induced by the A3receptor was abrogated in phospholipase C-β2/β3 null mice, but the protection mediated by the A1or A2Areceptor remained unaffected in these animals. The adenosine A3receptor is a novel cytoprotective receptor that signals selectively via phospholipase C-β and represents a new target for ameliorating skeletal muscle injury.

The P2X4 purinergic receptor (P2X4R) is a ligand-gated ion channel. Its activation by extracellular ATP results in Ca2+ influx. Transgenic cardiac overexpression of the human P2X4 receptor showed an in vitro phenotype of enhanced basal contractility. The objective here was to determine the in vivo cardiac physiological role of this receptor. Specifically, we tested the hypothesis that this receptor plays an important role in modulating heart failure progression. Transgenic cardiac overexpression of canine calsequestrin (CSQ) showed hypertrophy, heart failure, and premature death. Crossing the P2X4R mouse with the CSQ mouse more than doubled the lifespan (182 ± 91 days for the binary CSQ/P2X4R mouse, n = 35) of the CSQ mouse (71.3 ± 25.4 days, n = 50, P < 0.0001). The prolonged survival in the binary CSQ/P2X4R mouse was associated with an improved left ventricular weight-to-body weight ratio and a restored β-adrenergic responsiveness. The beneficial phenotype of the binary mouse was not associated with any downregulation of the CSQ level but correlated with improved left ventricular developed pressure and ±dP/d t. The enhanced cardiac performance was manifested in young binary animals and persisted in older animals. The increased contractility likely underlies the survival benefit from P2X4 receptor overexpression. An increased expression or activation of this receptor may represent a new approach in the therapy of heart failure.

SPECIFIC AIMThe aim of the study was to elucidate the mechanism of protection from myocardial ischemia induced by the adenosine A3 receptor.PRINCIPAL FINDINGSThe adenosine A3 receptor mediates anti-ischemic function in the heart and appears to signal selectively via RhoA and phospholipase D (PLD) to exert its protective effect, different from the cardioprotective mechanism of the adenosine A1 receptor. While a direct RhoA-PLD1 interaction is important in stimulating PLD1 activity, the physiological role of this direct interaction in the intact cell—the cardiac myocyte—is unclear. In an established cardiac myocyte model of preconditioning using cultured chick embryo heart cells, overexpression of the RhoA-noninteracting PLD1 mutant I870R inhibited A3 agonist-induced PLD activation and selectively blocked A3 agonist but not A1 agonist-induced cardioprotection. Atrial cardiac myocytes were rendered null for native adenosine receptors by treatment with irreversible A1 antagonist m-DITC-XAC and selectively tra...

Clinical disorders associated with increased serotonin [5-hydroxytryptamine (5-HT)] levels, such as carcinoid syndrome, and the use of serotonin agonists, such as fenfluoramine have been associated with a valvulopathy characterized by hyperplastic valvular and endocardial lesions with increased extracellular matrix. Furthermore, 5-HT has been demonstrated to up-regulate transforming growth factor (TGF)-β in mesangial cells via G-protein signal transduction. We investigated the hypothesis that increased exposure of heart valve interstitial cells to 5-HT may result in increased TGF-β1 expression and activity because of serotonin receptor-mediated signal transduction with activation of Gαq, and subsequently up-regulation of phospholipase C. Thus, in the present study we performed a clinical-pathological investigation of retrieved carcinoid and normal valve cusps using immunohistochemical techniques to detect the presence of TGF-β1 and other proteins associated with TGF-β expression, including TGF-β receptors I and II, latent TGF-β-associated peptide (LAP), and α-smooth muscle actin. Carcinoid valve cusps demonstrated the unusual finding of widespread smooth muscle actin involving the interstitial cells in the periphery of carcinoid nodules; these same cells were also positive for LAP. Normal valve cusps were only focally positive for smooth muscle actin and LAP. In sheep aortic valve interstitial cell cultures 5-HT induced TGF-β1 mRNA production and increased TGF-β1 activity. 5-HT also increased collagen biosynthesis at the dosages studied. Furthermore, TGF-β1 added to SAVIC cultures increased the production of sulfated glycan and hyaluronic acid. In addition, overexpression of Gαq using an adenoviral expression vector for a constitutively active Gαq mutant (Q209L-Gαq) resulted in increased phospholipase C activity as well as up-regulation of TGF-β expression and activity. These results strongly support the view that G-protein-related signal transduction is involved in 5-HT up-regulation of TGF-β1. In conclusion, 5-HT-associated valve disease may be, in part, because of TGF-β1 mechanisms.

P2X4 receptors (P2X4Rs) are ligand-gated ion channels capable of conducting cations such as Na+. Endogenous cardiac P2X4R can mediate ATP-activated current in adult murine cardiomyocytes. In the present study, we tested the hypothesis that cardiac P2X receptors can induce Na+ entry and modulate Na+ handling. We further determined whether P2X receptor-induced stimulation of the Na+/Ca2+ exchanger (NCX) has a role in modulating the cardiac contractile state. Changes in Na+-K+-ATPase current ( Ip) and NCX current ( INCX) after agonist stimulation were measured in ventricular myocytes of P2X4 transgenic mice using whole cell patch-clamp techniques. The agonist 2-methylthio-ATP (2-meSATP) increased peak Ip from a basal level of 0.52 ± 0.02 to 0.58 ± 0.03 pA/pF. 2-meSATP also increased the Ca2+ entry mode of INCX (0.55 ± 0.09 pA/pF under control conditions vs. 0.82 ± 0.14 pA/pF with 2-meSATP) at a membrane potential of +50 mV. 2-meSATP shifted the reversal potential of INCX from −14 ± 2.3 to −25 ± 4.1 mV, causing an estimated intracellular Na+ concentration increase of 1.28 ± 0.42 mM. These experimental results were closely mimicked by mathematical simulations based on previously established models. KB-R7943 or a structurally different agent preferentially opposing the Ca2+ entry mode of NCX, YM-244769, could inhibit the 2-meSATP-induced increase in cell shortening in transgenic myocytes. Thus, the Ca2+ entry mode of INCX participates in P2X agonist-stimulated contractions. In ventricular myocytes from wild-type mice, the P2X agonist could increase INCX, and KB-R7943 was able to inhibit the contractile effect of endogenous P2X4Rs, indicating a physiological role of these receptors in wild-type cells. The data demonstrate a novel Na+ entry pathway through ligand-gated P2X4Rs in cardiomyocytes.

The P2X4 receptor is a newly identified receptor expressed in the heart cell. Its function was elucidated with cardiac transgenic (TG) expression of the receptor by using the myocardium-specific a-myosin heavy chain promoter. The presence of the transgene was determined by polymerase chain reaction by using primers specific to the receptor and the vector linker region, by Southern blotting of the genomic DNA, and by immunoblotting and immunohistochemistry of both isolated cardiac myocytes and intact hearts. In intact heart study, the P2X4 receptor TG mouse exhibited significantly elevated basal cardiac contractility with greater rates of contraction and relaxation, left ventricular developed pressure, and cardiac output compared with nontransgenic (NTG) animals but showed no evidence of hypertrophy or heart failure. The TG heart also showed a greater increase of cardiac contractility in response to the P2X receptor agonist 2-methylthioATP, consistent with overexpression of a functional P2X4 receptor with consequent increase in the receptor-mediated response. In isolated cardiac cell study, the TG heart cell showed a similar level of basal contraction amplitude as the NTG heart cell while exhibiting a threefold greater increase in contractility during stimulation by 2-methylthioATP. Thus, an increased responsiveness of the overexpressed P2X4 receptor to endogenous ATP is responsible for the enhanced basal cardiac performance in the intact TG heart. The sustained enhanced contractile function with no associated heart pathology in the P2X4 receptor TG mouse suggests a novel physiologic role of the P2X4 receptor, that of stimulating the cardiac contractility.

Activation of P2 purinergic receptors exerts a potent positive inotropic effect in the cardiac myocyte. However, it is unknown whether its activation can also cause an increased contractility in intact heart. With the use of isolated rat and mouse hearts, the objective of the present study was to investigate the effect of P2 receptor agonist on the function of the intact heart. In both Langendorff rat hearts and working rat and mouse heart models, the P2X receptor agonist 2-methylthio-ATP (2-meSATP) caused dose-dependent increases in left ventricular developed pressure, rate of contraction, and rate of relaxation. The extent of P2X receptor agonist-stimulated increase in contractility was significantly less than that stimulated by the β-adrenergic agonist isoproterenol. However, the increase in contractility occurred without a significant effect on the basal heart rate, in contrast to that caused by isoproterenol. In isolated rat ventricular myocytes, both ATP and the P2X receptor agonist 2-meSATP stimulated large increases in the myocyte contractile amplitude (107 ± 13% and 99 ± 9%, n = 17 cells from 5 rats and n = 19 cells from 6 rats, respectively). 2-meSATP caused only a slight increase in phospholipase C activity and could stimulate myocyte contractility in the presence of phospholipase C inhibitor U-73122, consistent with the role of a phospholipase C-independent P2X receptor in mediating the positive inotropic effect of 2-meSATP. The data provide evidence for a potentially important physiological role of the cardiac P2X receptor and for the concept that agonist at this receptor may be beneficial for the treatment of cardiac dysfunction.

The objective of the present study was to investigate the role of δ1-opioid receptors in mediating cardioprotection in isolated chick cardiac myocytes and to investigate whether protein kinase C and mitochondrial ATP-sensitive K+(KATP) channels act downstream of the δ1-opioid receptor in mediating this beneficial effect. A 5-min preexposure to the selective δ1-opioid receptor agonist (−)-TAN-67 (1 μM) resulted in less myocyte injury during the subsequent prolonged ischemia compared with untreated myocytes. 7-Benzylidenenaltrexone, a selective δ1-opioid receptor antagonist, completely blocked the cardioprotective effect of (−)-TAN-67. Naltriben methanesulfonate, a selective δ2-opioid receptor antagonist, had only a slight inhibitory effect on (−)-TAN-67-mediated cardioprotection. Nor-binaltorphimine dihydrochloride, a κ-opioid receptor antagonist, did not affect (−)-TAN-67-mediated cardioprotection. The protein kinase C inhibitor chelerythrine and the KATP channel inhibitors glibenclamide, a nonselective KATP antagonist, and 5-hydroxydecanoic acid, a mitochondrial selective KATPantagonist, reversed the cardioprotective effect of (−)-TAN-67. These results suggest that the δ1-opioid receptor is present on cardiac myocytes and mediates a potent cardioprotective effect via protein kinase C and the mitochondrial KATP channel.

Adenosine released during cardiac ischemia exerts a potent, protective effect in the heart. A newly recognized adenosine receptor, the A 3 subtype, is expressed on the cardiac ventricular cell, and its activation protects the ventricular heart cell against injury during a subsequent exposure to ischemia. A cultured chicken ventricular myocyte model was used to investigate the cardioprotective role of a novel adenosine A 3 receptor. The protection mediated by prior activation of A 3 receptors exhibits a significantly longer duration than that produced by activation of the adenosine A 1 receptor. Prior exposure of the myocytes to brief ischemia also protected them against injury sustained during a subsequent exposure to prolonged ischemia. The adenosine A 3 receptor-selective antagonist 3-ethyl 5-benzyl-2-methyl-6-phenyl-4-phenylethynyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (MRS1191) caused a biphasic inhibition of the protective effect of the brief ischemia. The concomitant presence of the A 1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) converted the MRS1191-induced dose inhibition curve to a monophasic one. The combined presence of both antagonists abolished the protective effect induced by the brief ischemia. Thus, activation of both A 1 and A 3 receptors is required to mediate the cardioprotective effect of the brief ischemia. Cardiac atrial cells lack native A 3 receptors and exhibit a shorter duration of cardioprotection than do ventricular cells. Transfection of atrial cells with cDNA encoding the human adenosine A 3 receptor causes a sustained A 3 agonist-mediated cardioprotection. The study indicates that cardiac adenosine A 3 receptor mediates a sustained cardioprotective function and represents a new cardiac therapeutic target.

Although ATP, acting through a P2 purinoceptor, can stimulate a pronounced positive inotropic effect in cardiac ventricular myocytes, the receptor-effector mechanism that underlies this stimulatory cardiac action is not well understood. The objectives of the present study were to develop the cultured chick embryo ventricular myocytes as a novel model for the cardiac P2 purinoceptor and to determine the mechanism underlying its positive inotropic effect. ATP caused an 89 ± 8.9% ( n = 14 cells) increase in the myocyte contractility, with an efficacy and potency order of ATP > ADP > AMP ≫ adenosine. 2-Methylthio-ATP (2-MeS-ATP) but not α,β-methylene-ATP was able to stimulate myocyte contractility, with a maximal increase of 54 ± 2.6% ( n = 11 cells). Although UTP potently stimulates phosphoinositide hydrolysis, it had an only modest positive inotropic effect (27 ± 7% maximal increase; n = 8 cells). In contrast to previous suggestions, the 2-MeS-ATP-stimulated positive inotropic response does not require the action of phospholipase C (PLC), such as that of the inositol phosphates; the UTP effect on contractility appears to be mediated via the 2-MeS-ATP-sensitive P2 receptor. The PLC inhibitor U-73122 had no effect on the 2-MeS-ATP-stimulated increase in contractility, providing further evidence against a role for PLC in the inotropic effect of 2-MeS-ATP. An adenosine 3′,5′-cyclic monophosphate-independent Ca2+entry-stimulating mechanism appears to underlie a direct coupling of the receptor to stimulation of the myocyte contractility. This new PLC- and adenosine 3′,5′-cyclic monophosphate-independent positive inotropic mechanism represents a target for developing novel positive inotropic therapeutics.

The possible cardioprotective roles of adenosine A1 and A3 receptors were investigated in a cardiac myocyte model of injury. The adenosine A3 receptor is a novel cardiac receptor capable of mediating potentially important cardioprotective functions. Prolonged hypoxia with glucose deprivation was used to simulate ischemia and to induce injury in cardiac ventricular myocytes cultured from chick embryos 14 days in ovo. When present during the prolonged hypoxia, the adenosine A3 agonists N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA) and 2-chloro-N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (CI-IB-MECA) caused a dose-dependent reduction in the extent of hypoxia-induced injury as manifested by a decrease in the amount of creatine kinase released and the percentage of myocytes killed. The adenosine A1 agonists 2-chloro-N6-cyclopentyladenosine (CCPA), N6-cyclohexyladenosine, and adenosine amine congener were also able to cause a decrease in the extent of myocyte injury. The A1 receptor-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine blocked the cardioprotective effect of the A1 but not of the A3 agonists. Conversely, the selective A3 antagonists MRS-1191 and MRS-1097 blocked the protection induced by CI-IB-MECA but had minimal effect on that caused by CCPA. Thus the cardioprotective effects of A1 and A3 agonists were mediated by their respective receptors. This study defines a novel cardioprotective function of the cardiac A3 receptor and provides conclusive evidence that activation of both A1 and A3 receptors during hypoxia can attenuate myocyte injury.

Background— More than 50% of >270 000 childhood cancer survivors in the United States have been treated with anthracyclines and are therefore at risk of developing cardiotoxicity. Cardiac magnetic resonance (CMR) has demonstrated utility to detect diffuse interstitial fibrosis and changes in regional myocardial function. We hypothesized that CMR would identify occult cardiotoxicity characterized by structural and functional myocardial abnormalities in a cohort of asymptomatic pediatric cancer survivors with normal global systolic function. Methods and Results— Forty-six long-term childhood cancer survivors with a cumulative anthracycline dose ≥200 mg/m 2 and normal systolic function were studied 2.5 to 26.9 years after anthracycline exposure. Subjects underwent transthoracic echocardiography, CMR with routine cine acquisition, tissue characterization, and left ventricular strain analysis using a modified 16-segment model. Extracellular volume was measured in 27 subjects, all of whom were late gadolinium enhancement negative. End-systolic fiber stress was elevated in 45 of 46 subjects. Low average circumferential strain magnitude ( ε cc ) −14.9±1.4; P ε ll ) −13.5±1.9; P P =0.01). Higher mean extracellular volume was observed in female subjects (0.34 versus 0.22; P =0.01). Conclusions— Asymptomatic postchemotherapy pediatric patients have abnormal myocardial characteristics and strain parameters by CMR despite normal global cardiac function by standard transthoracic echocardiography and CMR measures.

Both adenosine receptor and ATP-sensitive K (KATP) channel mediate the protective effect of ischemic preconditioning in the intact heart. The objective of the present study was to determine the role of adenosine receptor and KATP channel as well as their interaction in simulating and mediating preconditioning of the cardiac myocyte. Cardiac ventricular myocytes cultured from chick embryos 14 days in ovo were developed as a myocyte model of preconditioning. Myocytes were preconditioned by exposing them to 5-min hypoxia, termed preconditioning hypoxia, before a second 90-min hypoxia. Preconditioning resulted in a 64 +/- 3% decrease in the amount of creatine kinase released and a 66 +/- 2% reduction in the percentage of myocytes (+/-SE, n = 11) killed. Glibenclamide or 5-hydroxydecanoic acid (5-HD), when present during the preconditioning hypoxia, blocked the preconditioning effect. Prior exposure of the myocytes to pinacidil also led to a decrease in the injury sustained during the 90-min hypoxia. The protective effect of pinacidil was blocked by glibenclamide or 5-HD, suggesting that KATP channel activation can mimic as well as mediate preconditioning. Adenosine receptor antagonist 8-sulfophenyltheophylline (8-SPT) blocked the protective effect of preconditioning hypoxia. Adenosine or the A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA) can replace preconditioning hypoxia and mimic preconditioning; this effect was fully antagonized by 8-SPT, glibenclamide, or 5-HD. Adenosine A1-receptor activation caused a glibenclamide-sensitive inhibition of the basal 45Ca influx and basal myocyte contractile amplitude, consistent with coupling of A1 receptor to stimulation of KATP channel in the myocytes. The data provide direct evidence that myocyte KATP channel is the effector downstream from adenosine A1 receptor in mediating the direct preconditioning of cardiac myocytes.

The objectives of this study were to investigate the mechanism underlying the adenosine A2a receptor (A2aR)-mediated positive inotropic response and to define its contractile function using chick embryo ventricular cells as a model. Activation of the A2aR caused a marked stimulation of calcium entry and cell contractility, which were blocked by verapamil or nifedipine. The effects elicited by maximal concentrations of the A2aR agonist 2-[4-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenos ine and the beta-adrenergic agonist isoproterenol were additive, indicating that the two receptors do not share a common stimulatory mechanism. The cAMP antagonist (Rp)-adenosine cyclic 3':5'-monophosphorothioate was ineffective in inhibiting the A2aR-mediated stimulation of contractility or the L-type calcium channel, while it completely abolished the isoproterenol effects. Activation of the A2aR had no effect on Na+/Ca2+ exchange or inositol 1,4,5-trisphosphate accumulation. Blocking of the A2aR resulted in unopposed A1 receptor-mediated inhibitory effects and led to an inhibition of basal contractility and an enhanced anti-adrenergic effect by A1 agonist. The adenosine A2a receptor mediates a new cyclic AMP-independent mechanism and a new contractile function in the cardiac cell.

The expression and function of stimulatory adenosine A2 receptor on the cardiac myocyte is not well defined. The objective of the present study is to characterize the A2a receptor in adult rat cardiac ventricular myocytes. After selection of an optimal lot of collagenase for myocyte isolation and for consistent measurement of adenosine-mediated responses, the A1-receptor pathway was inactivated by pertussis toxin and by the A1-selective antagonist 1,3-dipropyl-8-cyclopentylxanthine. Effects of the adenosine agonist and antagonist or cardiac myocyte contractile amplitude and on adenosine 3',5'-cyclic monophosphate (cAMP) levels were determined. The A2a-receptor-selective agonist 2-[p-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoade nos ine (CGS-21680) caused a pronounced stimulation of myocyte contractile amplitude and an increase in the cAMP level, as did the nonselective agonists 5'-(N-ethylcarboxamido) adenosine (NECA) and adenosine. The A2a-receptor-selective antagonist 8-(3-chlorostyryl)caffeine blocked the NECA- and adenosine-induced positive inotropic response. Probing of myocyte RNA with a rat A2a-receptor cDNA demonstrated a 2.6-kb mRNA, corresponding to that encoding the A2a receptor. Together, data from contractile, cAMP, and RNA studies indicate that A2a receptors are expressed and are functionally coupled to stimulation of cAMP accumulation and cardiac contractility in adult rat ventricular myocytes.

Background— Vascular endothelial growth factor receptor-1 (VEGFR-1/Flt-1) is a potential therapeutic target for cardiovascular diseases, but its role in angiogenesis remains controversial. Whereas germline Vegfr-1 −/− embryos die of abnormal vascular development in association with excessive endothelial differentiation, mice lacking only the kinase domain appear healthy. Methods and Results— We performed Cre- lox P–mediated knockout to abrogate the expression of all known VEGFR-1 functional domains in neonatal and adult mice and analyzed developmental, pathophysiological, and molecular consequences. VEGFR-1 deficiency promoted tip cell formation and endothelial cell proliferation and facilitated angiogenesis of blood vessels that matured and perfused properly. Vascular permeability was normal at the basal level but elevated in response to high doses of exogenous VEGF-A. In the postinfarct ischemic cardiomyopathy model, VEGFR-1 deficiency supported robust angiogenesis and protected against myocardial infarction. VEGFR-1 knockout led to abundant accumulation of VEGFR-2 at the protein level, increased VEGFR-2 tyrosine phosphorylation transiently, and enhanced serine phosphorylation of Akt and ERK. Interestingly, increased angiogenesis, tip cell formation, vascular permeability, VEGFR-2 accumulation, and Akt phosphorylation could be partially rescued or suppressed by one or more of the following manipulations, including injection of the VEGFR-2 selective inhibitor SU1498, anti-VEGF-A, or introduction of Vegfr-2 +/− heterozygosity into Vegfr-1 somatic knockout mice. Conclusions— Upregulation of VEGFR-2 abundance at the protein level contributes in part to increased angiogenesis in VEGFR-1–deficient mice.

Binding of urokinase-type plasminogen activator (u-PA) to specific receptors (u-PAR) on the surface of endothelial cells contributes to the regulation of plasmin-dependent processes such as fibrinolysis and angiogenesis. We studied the effect of raising intracellular levels of cyclic AMP (cAMP) and/or activating protein kinase C on the expression of u-PAR in cultured human umbilical vein endothelial cells (HUVEC). Incubation of HUVEC with forskolin stimulated a time- and concentration-dependent increase in the expression of u-PAR, measured both by an increase in the specific binding of radiolabeled single-chain u-PA (scu-PA) and by increased binding of anti-u-PAR antibodies. Maximal increase in u-PAR expression (81 +/- 11% above control, n = 11) was not associated with a change in receptor affinity for scu-PA when HUVEC were incubated for 20 hours at 37 degrees C with 50 microM forskolin. Receptor induction by forskolin was inhibited when HUVEC were preincubated with deoxyadenosine monophosphate (DAM), an inhibitor of adenylyl cyclase. A similar increase in receptor expression (128 +/- 27% above control, n = 3) was induced by the cAMP analogue 8-bromoadenosine 3':5'-cyclic monophosphate (50 mM). Forskolin induced an approximately twofold increase in the expression of a single approximately 1.4-kb u-PAR messenger RNA (mRNA) transcript within 2 hours. Phorbol myristate acetate (PMA) also stimulated a time- and concentration-dependent increase in specific scu-PA binding. The maximal increase in u-PAR expression (254 +/- 27% above control, n = 11) was observed when HUVEC were preincubated with 10 nM PMA for 20 hours. Induction of u-PAR by PMA was inhibited when HUVEC were preincubated with either cycloheximide or H7 but was unaffected by DAM. u-PAR induced by PMA showed a reduced affinity for scu-PA (Kd, 14 +/- 2 nM versus 3.6 +/- 0.6 nM, p < 0.001; n = 8). PMA stimulation for 20 hours resulted in a sixfold increase in a single approximately 1.4-kb u-PAR mRNA transcript, with increased levels detectable within 30 minutes. Coincubation of HUVEC with optimal concentrations of forskolin and PMA for 20 hours produced a fully additive increase in u-PAR expression at both the mRNA and protein levels. These data suggest that both cAMP-dependent and protein kinase C-dependent protein kinase pathways may independently regulate u-PAR expression in human endothelial cells.

Objective: The aim was to characterise the process and the mechanisms of sensitisation of the atrial myocyte to adenosine receptor agonist. Methods: The ability of adenosine A1 receptor to mediate inhibition of adenylyl cyclase activity and myocyte contractility was determined in atrial myocytes cultured from 14 d chick embryos. Under conditions in which the myocytes were sensitised to the effects of A1 agonist, changes in the levels of adenosine A1 receptor and pertussis toxin sensitive G proteins were determined and correlated with alterations in the adenylyl cyclase activity and contractile responses of the myocyte to the A1 agonist. Results: Removal of adenosine from the culture medium with adenosine deaminase resulted in an enhanced ability of the adenosine A1 receptor agonist R-N6-(2-phenylisopropyl)-adenosine to exert a direct, negative inotropic effect and to inhibit isoprenaline stimulated adenylyl cyclase activity. The increase in the extent of maximum inhibition of adenylyl cyclase activity and of myocyte contractility was 215(30), n=5, and 90(10)%, n=14, respectively. Binding of the antagonist radioligand [3H]-8-cyclopentyl-1,3-dipropylxanthine in membranes from myocytes pre-exposed to adenosine deaminase showed a 70% increase in the adenosine A1 receptor density and a 54% increase in the proportion of the high affinity adenosine A1 receptor: control 33(5)%, n=5, versus sensitised 49(3)%, n=5, p

Rationale: Cardiac fibrosis contributes to pathogenesis of atrial fibrillation (AF), which is the most commonly sustained arrhythmia and a major cause of morbidity and mortality. Although it has been suggested that Ca 2+ signals are involved in fibrosis promotion, the molecular basis of Ca 2+ signaling mechanisms and how Ca 2+ signals contribute to fibrogenesis remain unknown. Objective: To determine the molecular mechanisms of Ca 2+ -permeable channel(s) in human atrial fibroblasts, and to investigate how Ca 2+ signals contribute to fibrogenesis in human AF. Methods and Results: We demonstrate that the transient receptor potential (TRP) melastatin related 7 (TRPM7) is the molecular basis of the major Ca 2+ -permeable channel in human atrial fibroblasts. Endogenous TRPM7 currents in atrial fibroblasts resemble the biophysical and pharmacological properties of heterologous expressed TRPM7. Knocking down TRPM7 by small hairpin RNA largely eliminates TRPM7 current and Ca 2+ influx in atrial fibroblasts. More importantly, atrial fibroblasts from AF patients show a striking upregulation of both TRPM7 currents and Ca 2+ influx and are more prone to myofibroblast differentiation, presumably attributable to the enhanced expression of TRPM7. TRPM7 small hairpin RNA markedly reduced basal AF fibroblast differentiation. Transforming growth factor (TGF)-β1, the major stimulator of atrial fibrosis, requires TRPM7-mediated Ca 2+ signal for its effect on fibroblast proliferation and differentiation. Furthermore, TGF-β1–induced differentiation of cultured human atrial fibroblasts is well correlated with an increase of TRPM7 expression induced by TGF-β1. Conclusions: Our results establish that TRPM7 is the major Ca 2+ -permeable channel in human atrial fibroblasts and likely plays an essential role in TGF-β1–elicited fibrogenesis in human AF.

Ventricular and atrial myocytes cultured from chick embryos 14 days in ovo were used as model systems to study cardiac adenosine receptors. In membranes of ventricular cultures, blocking of the A1-adenosine receptor pathway by the A1-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) or by pertussis toxin treatment of the myocyte resulted in a significant adenosine agonist-mediated stimulation of the adenylate cyclase activity. The maximal increases in adenylate cyclase activity caused by the equipotent or the A2-adenosine receptor-selective agonists (from 52.1 +/- 3% to 63 +/- 10% [mean +/- SEM]) were significantly greater than those caused by the A1-selective agonists (from 11 +/- 5% to 34.6 +/- 7%) (p less than 0.01, by t test, n = 4-8). However, in membranes of atrial myocytes, when A1-subtype had been blocked, the various adenosine agonists had no effect on the adenylate cyclase activity. Whether the stimulatory adenylate cyclase-coupled adenosine receptor is also capable of stimulating contractility in the intact ventricular myocyte was next investigated. In ventricular but not in atrial cells, the various adenosine agonists caused an increase in the contractile amplitude in the presence of DPCPX or in myocytes preexposed to pertussis toxin. The increase in contraction amplitude caused by each agonist was expressed as percent of maximum (maximum is the increase in contractility caused by 2.4 mM calcium). In the pertussis toxin-treated myocyte, the maximal increases caused by the equipotent or A2-agonists (NECA, MECA, CV-1808, and CGS21680, from 49.6 +/- 3% to 52.5 +/- 6%, n = 8-12) were significantly greater than those elicited by the A1-agonists (2-CADO, S-PIA, R-PIA, and DCCA, from 12 +/- 4% to 37 +/- 3%, n = 8) (p less than 0.05, by t test). These data demonstrated that a stimulatory adenosine receptor, likely the A2-adenosine receptor, was present on the ventricular but not the atrial myocytes and was linked directly to a stimulation of the cardiac contractility. The functional effects mediated by the A1-subtype became manifested in the presence of isoproterenol, as evidence by an inhibition of the isoproterenol-stimulated increases in adenylate cyclase activity and in cardiac contractility by adenosine agonists. Thus, both subtypes of adenosine receptors, each mediating opposing responses, were present on the ventricular myocytes, whereas only the A1-subtype was found in the atria. The presence of a stimulatory functional A2-adenosine receptor may help explain the absence of a direct negative inotropic response to adenosine in the ventricle.

Binary cardiac transgenic (Tg) overexpression of P2X4 receptors (P2X4R) improved the survival of the cardiomyopathic calsequestrin (CSQ) mice. Here we studied the mechanism of rescue using binary P2X4R/CSQ Tg and CSQ Tg mice as models. Cellular and intact heart properties were determined by simultaneous sarcomere shortening (SS) and Ca2+ transients in vitro and echocardiography in vivo. Similar to a delay in death, binary mice exhibited a slowed heart failure progression with a greater left ventricular (LV) fractional shortening (FS) and thickness and a concomitant lesser degree of LV dilatation in both systole and diastole at 8 or 12 wk. By 16 wk, binary hearts showed similarly depressed FS and thinned out LV and equal enlargement of LV as did 12-wk-old CSQ hearts. Binary cardiac myocytes showed higher peak basal cell shortening (CS) and SS as well as greater basal rates of shortening and relaxation than did the CSQ myocytes at either 8 or 12 wk. Similar data were obtained in comparing the Ca2+ transient. At 16 wk, binary myocytes were like the 12-wk-old CSQ myocytes with equally depressed CS, SS, and Ca2+ transient. CSQ myocytes were longer than myocytes from wild-type and binary mice at 12 wk of age. At 16 wk, the binary myocyte length increased to that of the 12-wk-old CSQ myocyte, parallel to LV dilatation. The data suggest a unique mechanism, which involves a reversal of cardiac myocyte dysfunction and a delay in heart failure progression. It represents an example of targeting the abnormal failing myocyte in treating heart failure.

Evidence is accumulating to support the presence of P2X purinergic receptors in the heart. However, the biological role of this receptor remains to be defined. The objectives here were to determine the role of cardiac P2X receptors in modulating the progression of post-myocardial infarction ischemic heart failure and to investigate the underlying mechanism. The P2X4 receptor (P2X4R) is an important subunit of native cardiac P2X receptors, and the cardiac-specific transgenic overexpression of P2X4R (Tg) was developed as a model. Left anterior descending artery ligation resulted in similar infarct size between Tg and wild-type (WT) mice ( P > 0.1). However, Tg mice showed an enhanced cardiac contractile performance at 7 days, 1 mo, and 2 mo after infarction and an increased survival at 1 and 2 mo after infarction ( P < 0.01). The enhanced intact heart function was manifested by a greater global left ventricular developed pressure and rate of contraction of left ventricular pressure in vitro and by a significantly increased fractional shortening and systolic thickening in the noninfarcted region in vivo ( P < 0.05). The salutary effects on the ischemic heart failure phenotype were seen in both sexes and were not the result of any difference in infarct size in Tg versus WT hearts. An enhanced contractile function of the noninfarcted area in the Tg heart was likely an important rescuing mechanism. The cardiac P2X receptor is a novel target to treat post-myocardial infarction ischemic heart failure.

Cardiac P2X purinergic receptors can mediate an increase in myocyte contractility and a potentially important role in the heart. The P2X4 receptor (P2X4R) is an important subunit of native cardiac P2X receptors. With transgenic mice with cardiac-specific overexpression of P2X4R (Tg) used as a model, the objectives here were to characterize the P2X receptor-mediated cellular contractile and Ca2+ transient effects and to determine the mechanism underlying the receptor-induced increase in myocyte contractility. In response to the agonist 2-methylthioATP (2-meSATP), Tg myocytes showed an increased intracellular Ca2+ transient, as defined by fura 2 fluorescence ratio, and an enhanced contraction shortening that were unaccompanied by cAMP accumulation or L-type Ca2+ channel activation. The increased Ca2+ transient was not associated with any alteration in action potential duration, resting membrane potential, or diastolic fluorescence ratio or rates of rise and decline of the Ca2+ transient. Simultaneous Ca2+ transient and contraction measurements did not show any agonist-mediated change in myofilament Ca2+ sensitivity. However, activation of the overexpressed P2X4 receptor caused an enhanced SR Ca2+ loading, as evidenced by a 2-meSATP-evoked increase in the caffeine-induced inward current and Ca2+ transient. Similar data were obtained in wild-type mouse ventricular myocytes. Thus an increased SR Ca2+ content, occurring in the absence of cAMP accumulation or L-type Ca2+ channel activation, is the principal mechanism by which cardiac P2X receptor mediates a stimulatory effect on cardiac myocyte contractility.

96 Background: We are conducting an exploratory, prospective study to investigate factors associated with radiation-induced fatigue in women with early breast cancer undergoing radiation therapy (RT) for breast conservation. Our hypothesis is that fatigue associated with adjuvant RT is related to tissue damage by apoptosis and inflammation, and to baseline psychological profiles. Methods: All subjects were assessed immediately before RT (T1), mid-point of RT (T2), end of RT (T3), 6 months (T4) and 1 year (T5) after completing RT. Clinical evaluations of skin toxicity, laboratory measures, fatigue, distress, depression, anxiety, sleep, energy and pain were assessed using validated measures. Results: Thirty-one subjects (target 50) have been enrolled to date; 23 have completed T3 and are presented here. 35% had a history of depression and 17% are currently depressed; 22% had past and current anxiety. BCTOS breast pain, tenderness and sensitivity increased during RT and were consistent with functional pain scores. No clinically relevant changes in energy or distress were seen. Despite a notable prevalence of depression and anxiety at baseline, there were no relevant changes in depression or anxiety. Most IL-1β, IL-4, IL-6 and IL-10 levels were undetectable. 39% of subjects showed increases in fatigue and were 14 years younger than non-fatigued subjects. Fatigued subjects had more frequent past (44%) and current (33%) depression than non-fatigued subjects (29% and 7%, respectively), and woke up tired more frequently (44% versus 0%). Current depression, but not fatigue, was associated with decreased a.m. cortisol levels during RT. 56% of fatigued subjects had an RTOG Acute Skin Toxicity score > 1 and 78% had BCTOS breast-specific pain subscales scores ≥ 3 during RT vs. 21% and 14% of non-fatigued subjects, respectively. Serum caspase-1 (inflammation) and caspase-3 (apoptosis), hs-CRP and TNF-α were increased but not associated with fatigue. Conclusions: Our preliminary findings suggest that radiation-associated fatigue is complex and, as hypothesized, is dependent on a patient’s psychological profile in the setting of skin toxicity and insomnia. If this data holds at study completion, it may guide clinical interventions.

P2X receptors, activated by extracellular ATP, may be important in regulating cardiac function. The objective of the present study was to characterize the electrophysiologic actions of P2X4 receptors in cardiac myocytes and to determine whether they are involved in mediating the effect of extracellular ATP. Membrane currents under voltage clamp were determined in myocytes from both wild-type (WT) and P2X4 receptor-overexpressing transgenic (TG) mice. The P2X agonist 2-meSATP induced an inward current at -100 mV that was greater in magnitude (2-fold) in TG than in WT ventricular cells. In the presence of the P2X4 receptor-selective allosteric enhancer ivermectin (3 microM), the 2-meSATP-stimulated current increased significantly in both WT and TG ventricular cells, consistent with an important role of P2X4 receptors in mediating the ATP current not only in TG but also WT myocytes. That the current in both WT and TG cells showed similar voltage-dependence and reverse potential (approximately 0 mV) further suggests a role for this receptor in the normal electrophysiological action of ATP in WT murine cardiac myocytes. The P2X antagonist suramin was only able to block partially the 2-meSATP-stimulated current in WT cells, implying that both P2X4 receptor and another yet-to-be-identified P2X receptor mediate this current.

Background The survival rate of breast cancer patients has increased due to improvements in cancer treatment. However, many survivors develop irreversible or reversible cardiotoxicity associated with anthracycline or trastuzumab therapy, respectively. To detect cardiac damage, the currently accepted method is to measure left ventricular ejection fraction (LVEF) by echocardiography, which lacks the sensitivity to predict early cardiac dysfunction. Early identification of cardiotoxicity is essential to cancer survivors, as development of cardiomyopathy carries a worse outcome independent of cancer prognosis. Currently, there are no accepted guidelines for the early detection of myocardial injury. The use of cardiac biomarkers and more sensitive echocardiographic techniques have expanded options for monitoring, but have yet to reach a consensus. Hence, our study will evaluate the potential predictive value of novel cardiac biomarkers and advanced echocardiographic and cardiac magnetic resonance imaging (CMR) techniques to detect subclinical myocardial damage. Our findings may be applicable for monitoring new antineoplastic agents during food and drug administration (FDA) clinical trials. Trial Design Prospective cohort study with internal control of 20 patients newly diagnosed with breast cancer. The trial will assess endpoints at baseline, 2 weeks after initiation of therapy, and 2 weeks and 6 months after chemotherapy completion. 1. Primary Endpoint a. Decline in left ventricular ejection fraction assessed by CMR and 3D-echo not detected by conventional methods b. Presence of either myocardial fibrosis or edema detected by CMR c. Changes in myocardial deformation detected by echo or CMR strain d. Increase in cardiac biomarkers (Serum caspase-3 p17 peptide, Troponin I, B-type natriuretic peptide) and possible correlation with imaging parameters 2. Secondary Endpoint a. Development New York Heart Association class 1 to 4 symptoms b. Decrease in LVEF of ≥5% to ≤50% with or without symptoms Eligibility Criteria Inclusion Criteria: 1. Newly diagnosed stage I, II, or III breast cancer 2. Age between 18 and 75 years old. 3. Treatment with trastuzumab or anthracycline-based chemotherapy Exclusion Criteria: 1. History of cardiovascular disease 2. Pacemaker 3. History of mediastinal radiotherapy 4. Creatinine clearance 5. Serum bilirubin >2.0 mg/dl, ALT and AST > 100 U/1) 6. Hypertension, uncontrolled >140/90 7. LVEF 8. Claustrophobia Specific Aims 1. Detect early myocardial injury. 2. Evaluate early predictors of left ventricular dysfunction. 3. Evaluate timing of monitoring during or post treatment Statistical Method This is a pilot study and 20 patients are required to reach statistical significance with 85% power. All values will be analyzed as mean±SD or n (%). Categorical indicators will be analyzed using nonparametric statistics such as Cochran's Q. Changes in imaging and biomarker parameters will be assessed using analysis of variance, while correlation between the two will be assessed using mixed models appropriate for binary outcome. Significance will be accepted at p ≤0.05 for all tests. Present accrual and target accrual Nine subjects are enrolled with a goal of 20. Contact for people interested in trial: 1. Dr. Erick Avelar, eavelar@uchc.edu 2. Dr. Susan Tannenbaum, stannenbaum@uchc.edu Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr OT1-02-12.

To the Editor: Infarct size is a major determinant of mortality after acute myocardial infarction (AMI) ([1][1]). Although timely reperfusion of the ischemic myocardium in patients with ST-segment elevation myocardial infarction (STEMI) is the most effective way to limit infarct size, reperfusion

Background Fatigue, a common complication of radiation therapy, can impact patients’ quality of life and ability to engage in their cancer treatments. The pathogenesis of radiation-induced fatigue is unclear. This lack of clear understanding has hindered the development of treatment strategies to overcome this morbidity. The purpose of our exploratory, non-randomized, prospective study is to investigate the factors associated with development of radiation-induced fatigue in women with early breast cancer. Our study is based on the hypotheses that adjuvant radiation therapy given to women with breast cancer for local disease control causes tissue damage by apoptosis and inflammation. This leads to increased levels of inflammatory cytokines and biomarkers of apoptosis. This inflammatory cascade differentially induces fatigue in patients depending on their baseline psychological profile. Trial design Existing patient flow in the radiation oncology division will be utilized to screen eligible subjects. All subjects will assessed at five different time points, beginning immediately before radiation therapy through 1 year after completion of therapy. Detailed clinical evaluation of skin toxicity (RTOG acute toxicity criteria) and laboratory parameters assessing anemia, cardiac and hepatic toxicity will be performed. Comprehensive psychological assessment will be done using questionnaires for fatigue, distress, depression, sleep, quality of life and pain. Buccal mucosa swabs will be obtained for genetic analysis in future studies. Selection criteria Inclusion: Women with breast cancer undergoing radiation therapy for breast conservation Exclusion: Prior non-breast malignancy with active treatment (radiation or chemotherapy) within 1 year of enrolment on the study Specific aims 1. To measure the severity of radiation-induced tissue damage 2. To measure circulating markers of apoptosis (caspase 3 p17) and inflammation 3. To establish a baseline psychological profile for each subject prior to start of radiation and determine temporal changes during the course of radiation therapy. 4. To determine the correlation between goals 1, 2 and 3. 5. To propose interventions for reducing radiation-induced fatigue which will be evaluated in a future study. Statistical methods The association between fatigue and severity of tissue toxicity (Yes vs. No) will be assessed using chi-square tests (eg, row mean score test). ANOVA will be used to compare mean visual analogue scales (VAS) and psychological questionnaire scores across the levels of the tissue toxicity scale. Mean log cytokine levels will be compared across levels of the dermatitis scale using ANOVA. The association between cytokines and fatigue will be assessed using correlation (eg, VAS) and two-group t-tests (eg, Yes vs. No) depending on which fatigue outcome is used. Two statistical models will be used to assess fatigue and its relationship to dermatitis, cytokine level, and their interactive effects using logistic regression (for dichotomous) and OLS regression (for VAS) approaches. Present accrual: 0; Study initiation planned for July 1, 2013 Target accrual : 100. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr OT2-1-01.

Adenosine released during cardiac ischemia exerts a marked protective effect in the heart that is mediated by the A1 and A3 subtypes of adenosine receptors. The signaling pathways activated by these adenosine receptors have now been characterized in a chick embryo ventricular myocyte culture model of cardioprotection against ischemia. Selective A1 and A3 receptor agonists were shown to activate phospholipases C and D, respectively, to achieve their distinct cardioprotective effects. The specificity of the A3 receptor–phospholipase D interaction was also demonstrated in chick embryo atrial myocytes (which do not express endogenous A3 receptors) that had been transfected with a vector encoding the human A3 receptor. Activation of both endogenous A1 and A3 receptors in ventricular myocytes resulted in a protective response greater than that induced by stimulation of either receptor alone. Agonists that activate both adenosine A1 and A3 receptors may thus prove beneficial for the treatment of myocardial ischemia.—Parsons, M., Young, L., Lee, J. E., Jacobson, K. A., Liang, B. T. Distinct cardioprotective effects of adenosine mediated by differential coupling of receptor subtypes to phospholipases C and D.

Background —The cardioprotective effect of preconditioning can be exerted within 1 to 2 hours after initial ischemia, termed classical or early preconditioning, or can reappear 24 hours later as second window or late preconditioning. The objective of this study was to study the interaction between late and early preconditioning and to determine the potential underlying mechanism. Methods and Results —Adenosine receptor agonists and a K ATP channel opener were used to achieve early preconditioning, and Monophosphoryl lipid A (MLA) was used to induce late preconditioning. Cultured chick ventricular myocytes were used as a myocyte model of simulated ischemia and preconditioning. Prior treatment of the myocyte with MLA caused a dose-dependent decrease in the ischemia-induced myocyte injury 24 hours later, consistent with a late preconditioning effect. L-NMMA, glibenclamide, or 5-hydroxydecanoic acid administered during the ischemia blocked the MLA effect. Twenty four hours after MLA treatment, a 5-minute exposure to ischemia, adenosine, adenosine A 1 agonist CCPA, or A 3 agonist resulted in less myocyte injury during the subsequent prolonged ischemia, as compared with cells pretreated with the vehicle and subsequently exposed to the same early preconditioning stimuli. In addition to its ability to enhance the early preconditioning effect by A 1 and A 3 agonists, MLA pretreatment also increased the phorbol ester- and pinacidil-mediated early preconditioning effect. Conclusions —This study defined a novel interaction in which the cardioprotective effect of early preconditioning is additive to that of late preconditioning and raised the possibility that both agents can be used as combined therapy in the treatment of ischemic heart disease.

Adenosine is an important mediator of the endogenous defense against ischemia-induced injury in the heart. Adenosine can achieve cardioprotection by mediating the effect of ischemic preconditioning and by protecting against myocyte injury when it is present during the infarct-producing ischemia. A novel adenosine A3 receptor can mediate this protective function. One approach to achieve cardioprotection is to enhance myocardial sensitivity to the endogenous adenosine by increasing the number of adenosine receptors instead of administering an adenosine receptor agonist. The objective of the present study was to investigate whether genetic manipulation of the cardiac myocyte, achieved by gene transfer and overexpression of the human A3 receptor cDNA, renders the myocytes resistant to the deleterious effect of ischemia. Prolonged hypoxia with glucose deprivation, causing myocyte injury and adenosine release, was used to simulate ischemia in cultured chick embryo ventricular myocytes. During simulated ischemia, cultured myocytes with enhanced expression of the human A3 receptor and showed significantly higher ATP content, fewer cells killed, and less creatine kinase released into the medium than either control or mock-transfected myocytes. Also, increased expression of the A3 receptor caused an enhanced cardioprotective effect by the preconditioning ischemia. Overexpressing the adenosine A1 receptor also led to increased protection against ischemia-induced myocyte injury as well as an enhanced preconditioning effect. Thus, increasing the receptor level improves the myocyte sensitivity to the endogenous adenosine, which in turn causes all of the cardioprotective effects found for exogenously administered adenosine agonists. The study provides the first proof for the new concept that an increased expression of the human A3 receptor in the cardiac myocyte can be an important cardioprotective therapeutic approach.

Prior activation of protein kinase C (PKC) can precondition the cardiac cell against injury during subsequent ischaemia. By using cultured chick ventricular cell model for simulated ischaemia and preconditioning, the present study investigated the biochemical mechanism underlying the PKC-mediated preconditioning. A 5 min exposure to PMA enhanced the ability of pinacidil to mediate cardioprotection during a subsequent 90 min period of ischaemia, which is consistent with a sustained activation of the KATP channel initiated by PKC. The brief prior exposure to PMA was also associated with an enhanced ability of the adenosine A1 or A3 receptor agonist 2-chloro-N6-cyclopentyladenosine or N6-(3-iodobenzyl)adenosine-5´-N-methyluronamide to elicit a cardioprotective response during the subsequent ischaemia. In myocytes pretreated with PMA, the cardioprotection mediated by receptor agonist was blocked by the concomitant presence of KATP-channel antagonists glibenclamide or 5-hydroxydecanoic acid during the ischaemia. Thus the KATP channel acts downstream of the adenosine A1 and A3 receptors in mediating the protective effect due to prior PMA exposure. KATP channel activation is responsible for the adenosine receptor-mediated effect. PMA treatment had no effect on other A1 or A3 receptor-mediated effects such as the inhibition of adenylate cyclase, ruling out a direct stimulation of the receptor or G-protein by PMA. The present results indicate that prior stimulation of PKC causes a sustained KATP channel activation, which in turn renders the myocyte more responsive to the protective action of adenosine A1 and A3 receptor agonists during the subsequent ischaemia.

Preface. I: Adenosine. 1. Molecular Biology and Pharmacology of Recombinant Human Adenosine Receptors J. Linden, K.A. Jacobson. 2. Cardiac Physiology of Adenosine J.G. Dobson Jr., R.A. Fenton. 3. Vascular Biology and Pharmacology of Adenosine Receptors P.L. Martin, R.A. Olsson. 4. Roles of Adenosine in Angiogenesis H.J. Granger, et al. 5. Adenosine KATP Channel and Cardioprotection in the Intact Heart D.W. Green, G.J. Grover. 6. Adenosine Receptor Subtypes and Cardioprotection in Cardiac Myocyte and Transgenic Models G.P. Matherne, et al. 7. Regulation of Adenosine Receptor Subtypes and Cardiac Dysfunction in Human Heart Failure B. Stein, et al. 8. Adenosine and Cardiac Arrhythmias D.J. Slotwiner, B.B. Lerman. 9. Adenosine and Cardiac Aging R.A. Fenton, et al. II: Nucleotides. 10. Molecular Biology of P2X Purinoceptors B.F. King. 11. The G Protein-Coupled P2 Receptors T.K. Harden. 12. P2 Purioceptors in Blood Vessels V. Ralevic. 13. P2 Purinoceptors and Cardiac Functions A. Rubino. 14. Vascular P2 Receptors and their Possible Role in Hypertension C. Kennedy. III: Interaction between P1 and P2 Receptors. 15. Neuromodulation in the Cardiovascular System C. Allgaier, P. Illes. 16. Adenosine Receptors, Blood Vessel and Inflammation G.W. Sullivan. 17. P2 Purinoceptors and Regulation of the Function of Platelets, Erythrocytes and Mast Cells F. di Virgilio, S. Falzoni. 18. Purinoreceptors and T Cell Function: Regulation of Lymphocyte Activation by Extracellular ATP and Adenosine M. Sitkovsky, et al. 19. Recent Advances in Cardiac Adenosine Metabolism J. Schrader, et al. 20. The Role of Peripheral Autonomic Neurons with P1- and/or P2 Purinoreceptors in Cardiac Regulation J.A. Armour. 21. Extracellular Metabolism of Nucleotides and Adenosine in the Cardiovascular System H. Zimmermann, J.D. Pearson. Index.