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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[degrees]) was used to induce eccentric exercise-mediated skeletal muscle injury. Experiments were conducted in both normal wild-type (WT) mice and also in [beta]-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 [+ or -] 62.7 U/l (SE). This increase was totally abolished by stimulation of the [A.sub.3] receptor. In the dystrophic [beta]-sarcoglycan-null mice, eccentric exercise caused CK levels to reach 55,124 [+ or -] 5,558 U/l. [A.sub.3] 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, [A.sub.3] receptor stimulation eliminated this increase. Neither the [A.sub.1] receptor agonist 2-chloro-[N.sup.6]-cyclopentyladenosine (0.05 mg/kg) nor the [A.sub.2A] receptor agonist 2-p-(2-carboxyethyl)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 [A.sub.3] receptor stimulation was absent in mice, in which genes for phospholipase C [beta]2/[beta]3 (PLC[beta]2/[beta]3) and [beta]-sarcoglycan were deleted. The present study elucidates a new protective role of the [A.sub.3] receptor and PLC[beta]2/[beta]3 and points to a potentially effective therapeutic strategy for eccentric exercise-induced skeletal muscle injury. [beta]-sarcoglycan; muscle force; creatine kinase; inflammation doi: 10.1152/ajpregu.00060.2010.

Binary cardiac transgenic (Tg) overexpression of P2[X.sub.4] receptors (P2[X.sub.4]R) improved the survival of the cardiomyopathic calsequestrin (CSQ) mice. Here we studied the mechanism of rescue using binary P2[X.sub.4]R/CSQ Tg and CSQ Tg mice as models. Cellular and intact heart properties were determined by simultaneous sarcomere shortening (SS) and [Ca.sup.2+] 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 [Ca.sup.2+] transient. At 16 wk, binary myocytes were like the 12-wk-old CSQ myocytes with equally depressed CS, SS, and [Ca.sup.2+] 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. contractility; heart failure; hypertrophy

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 [P2X.sub.4] receptor ([P2X.sub.4]R) is an important subunit of native cardiac P2X receptors, and the cardiac-specific transgenic overexpression of [P2X.sub.4]R (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. purines; contractility; infarction

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 A3 receptor-selective agonist 2-chloro-[N.sup.6]-(3-iodobenzyl)adenosine-5'-N-methyluronamide (CI-IB-MECA; 0.07 mg/kg ip) reduced skeletal muscle injury with a significant decrease in both Evans blue dye staining (5.4 [+ or -] 2.6%, n = 8 mice vs. vehicle-treated 28 [+ or -] 6%, n = 7 mice, P < 0.05) and serum creatine kinase level (1,840 [+ or -] 910 U/l, n = 13 vs. vehicle-treated 12,600 [+ or -] 3,300 U/l, n = 14, P < 0.05), an effect that was selectively blocked by an A3 receptor antagonist 3-ethyl-5-benzyl-2-methyl-6-phenyl-4-phenylethynyl-1,4-([+ or -]) -dihydropyridine-3,5-dicarboxylate (MRS-1191; 0.05 mg/kg). The adenosine [A.sub.1] receptor agonist 2-chloro-[N.sup.6]-cyclopentyladenosine (CCPA; 0.05 mg/kg) also exerted a cytoprotective effect, which was selectively blocked by the [A.sub.1] antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 0.2 mg/kg). The adenosine [A.sub.2A] receptor 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 [A.sub.2A] antagonist 2-(2-furanyl)-7- [3-(4-methoxyphenyl)propyl]-7Hpyrazolo[4,3-e] [1,2,4]triazolo[1,5-C]pydmid in-5-amine (SCH-442416; 0.017 rag/ kg). The protection induced by the [A.sub.3] receptor was abrogated in phospholipase C-[beta]2/[beta]3 null mice, but the protection mediated by the [A.sub.1] or [A.sub.2A] receptor remained unaffected in these animals. The adenosine [A.sub.3] receptor is a novel cytoprotective receptor that signals selectively via phospholipase C-[beta] and represents a new target for ameliorating skeletal muscle injury. wild-type mice; creatine kinase; phosolipase C

Cardiac P2X purinergic receptors can mediate an increase in myocyte contractility and a potentially important role in the heart. The P2[X.sub.4] receptor (P2[X.sub.4]R) is an important subunit of native cardiac P2X receptors. With transgenic mice with cardiac-specific overexpression of P2[X.sub.4]R (Tg) used as a model, the objectives here were to characterize the P2X receptor-mediated cellular contractile and [Ca.sup.2+] 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 [Ca.sup.2+] transient, as defined by fura 2 fluorescence ratio, and an enhanced contraction shortening that were unaccompanied by cAMP accumulation or L-type [Ca.sup.2+] channel activation. The increased [Ca.sup.2+] 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 [Ca.sup.2+] transient. Simultaneous [Ca.sup.2+] transient and contraction measurements did not show any agonist-mediated change in myofilament [Ca.sup.2+] sensitivity. However, activation of the overexpressed P2[X.sub.4] receptor caused an enhanced SR [Ca.sup.2+] loading, as evidenced by a 2-meSATP-evoked increase in the caffeine-induced inward current and [Ca.sup.2+] transient. Similar data were obtained in wild-type mouse ventricular myocytes. Thus an increased SR [Ca.sup.2+] content, occurring in the absence of cAMP accumulation or L-type [Ca.sup.2+] channel activation, is the principal mechanism by which cardiac P2X receptor mediates a stimulatory effect on cardiac myocyte contractility. purines; calcium; sarcoplasmic reticulum

P2X purinergic receptors, activated by extracellular ATP, mediate a number of cardiac cellular effects and may be important under pathophysiological conditions. The objective of the present study was to characterize the P2X receptor-mediated ionic current and determine its role in heart failure using the calsequestrin (CSQ) model of cardiomyopathy. Membrane currents under voltage clamp were determined in myocytes from both wild-type (WT) and CSQ mice. The P2X agonist 2-methylthio-ATP (2-meSATP) induced an inward current that was greater in magnitude in CSQ than in WT ventricular cells. The novel agonist, MRS-2339, an N-methanocarba derivative of 2-chloro-AMP relatively resistant to nucleotidase, induced a current in the CSQ myocyte similar to that by 2-meSATP. When administered via a miniosmotic pump (Alzet), it significantly increased longevity compared with vehicle-injected mice (log rank test, P = 0.02). The improvement in survival was associated with decreases in the heart weight-to-body weight ratio and in cardiac myocyte cross-sectional area [MRS-2339-treated mice: 281 [+ or -] 15.4 (SE) [micro][m.sup.2], n = 6 mice vs. vehicle-treated mice: 358 [+ or -] 27.8 [micro][m.sup.2], n = 6 mice, P < 0.05]. MRS-2339 had no vasodilator effect in mouse aorta ring preparations, indicating that its salutary effect in heart failure is not because of any vascular unloading. The cardiac P2X current is upregulated in the CSQ heart failure myocytes. Chronic administration of a nucleotidase-resistant agonist confers a beneficial effect in the CSQ model of heart failure, apparently via an activation of the cardiac P2X receptor. Cardiac P2X receptors represent a novel and potentially important therapeutic target for the treatment of heart failure. ion channels; membrane current; mouse; heart

The P2[X.sub.4] purinergic receptor (P2[X.sub.4]R) is a ligand-gated ion channel. Its activation by extracellular ATP results in [Ca.sub.2+] influx. Transgenic cardiac overexpression of the human P2[X.sub.4] 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 P2[X.sub.4]R mouse with the CSQ mouse more than doubled the lifespan (182 [+ or -] 91 days for the binary CSQ/P2[X.sub.4]R mouse, n = 35) of the CSQ mouse (71.3 [+ or -] 25.4 days, n = 50, P < 0.0001). The prolonged survival in the binary CSQ/P2[X.sub.4]R mouse was associated with an improved left ventricular weight-to-body weight ratio and a restored [beta]-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 [+ or -]dP/dt. The enhanced cardiac performance was manifested in young binary animals and persisted in older animals. The increased contractility likely underlies the survival benefit from P2[X.sub.4] receptor overexpression. An increased expression or activation of this receptor may represent a new approach in the therapy of heart failure. cardiac failure; contraction; isoproterenol; purines; adenine nucleotide

The mechanism governing the positive inotropic response of cardiac ventricular myocytes to P2 purinoceptor ATP stimulation was investigated through preparation of a myocyte model using cultured chick embryo ventricular myocytes. Results have shown that the novel cAMP and PLC-independent calcium entry pathway mediated the direct coupling of a P2 purinoceptor to stimulate myocyte contractility. It was suggested that the pathway occurred inside the myocardiac cell.

The role of adenosine receptor and KATP channel in protein kinase C (PKC)-mediated preconditioning of cardiac myocytes was analyzed using a myocyte model of preconditioning. Results of the study indicated that PKC serves as an effector downstream from the adenosine receptor. Furthermore, PKC activation can precondition the myocytes without necessarily using adenosine-receptor activation.

Researchers have investigated whether activation of adenosine receptors during prolonged hypoxia can attenuate the hypoxia-induced myocyte injury. They have also looked at the role of A1 and A3 receptors in mediating the adenosine-induced cardiac myocyte protection. This involved using prolonged hypoxia with glucose deprivation to bring about ischemia and to induce injury in cardiac ventricular myocytes obtained from chick embryos 14 days in ovo. It was found that the activation of both receptors can attenuate myocyte injury during prolonged hypoxia.

An investigation of the potassium (K) channel in cardiac myocytes from chick embryos has confirmed the results of previous studies indicating the presence of the K(sub ATP) channel in preconditioning of cardiac myocytes. First, in the presence of the K(sub ATP) channel blocker, glibenclamide, the effect preconditioning during preconditioning hypoxia was abolished. Second, the addition of another agonist, pinacidil, prior to prolonged hypoxia reduced the severity of hypoxia-induced injury. However, the results failed to provide direct evidence linking the adenosine receptor and K(sub ATP) channel in myocyte preconditioning.

P2 purinergic receptor activation enhances cardiac contractility in isolated rat and mouse hearts. Am J Physiol Heart Circ Physiol 281: H334-H341, 2001.--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 [Beta]-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 [+ or -] 13% and 99 [+ or -] 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. heart; drugs; ATP; purines; inotropy

Protection of cardiac myocytes via [[Delta].sub.1]-opioid receptors, protein kinase C, and mitochondrial [K.sub.ATP] channels. Am J Physiol Heart Circ Physiol 280: H377-H383, 2001.--The objective of the present study was to investigate the role of [[Delta].sub.1]-opioid receptors in mediating cardioprotection in isolated chick cardiac myocytes and to investigate whether protein kinase C and mitochondrial ATP-sensitive [K.sup.+] ([K.sub.ATP]) channels act downstream of the [[Delta].sub.1]-opioid receptor in mediating this beneficial effect. A 5-min preexposure to the selective [[Delta].sub.1]-opioid receptor agonist (-)-TAN-67 (1 [micro]M) resulted in less myocyte injury during the subsequent prolonged ischemia compared with untreated myocytes. 7-Benzylidenenaltrexone, a selective [[Delta].sub.1]-opioid receptor antagonist, completely blocked the cardioprotective effect of (-)-TAN-67. Naltriben methanesulfonate, a selective [[Delta].sub.2]-opioid receptor antagonist, had only a slight inhibitory effect on (-)-TAN-67-mediated cardioprotection. Nor-binaltorphimine dihydrochloride, a [Kappa]-opioid receptor antagonist, did not affect (-)-TAN-67-mediated cardioprotection. The protein kinase C inhibitor chelerythrine and the [K.sub.ATP] channel inhibitors glibenclamide, a nonselective [K.sub.ATP] antagonist, and 5-hydroxydecanoic acid, a mitochondrial selective [K.sub.ATP] antagonist, reversed the cardioprotective effect of (-)-TAN-67. These results suggest that the [[Delta].sub.1]-opioid receptor is present on cardiac myocytes and mediates a potent cardioprotective effect via protein kinase C and the mitochondrial [K.sub.ATP] channel. ATP-sensitive [K.sup.+] channels; ischemic preconditioning; cardioprotective effect; [Delta]-1 opioid receptor