Although interactions between superoxide (O 2•؊ ) and nitric oxide underlie many physiologic and pathophysiologic processes, regulation of this crosstalk at the enzymatic level is poorly understood. Here, we demonstrate that xanthine oxidoreductase (XOR), a prototypic superoxide O 2•؊ -producing enzyme, and neuronal nitric oxide synthase (NOS1) coimmunoprecipitate and colocalize in the sarcoplasmic reticulum of cardiac myocytes. Deficiency of NOS1 (but not endothelial NOS, NOS3) leads to profound increases in XOR-mediated O 2•؊ production, which in turn depresses myocardial excitation-contraction coupling in a manner reversible by XOR inhibition with allopurinol. These data demonstrate a unique interaction between a nitric oxide and an O 2•؊ -generating enzyme that accounts for crosstalk between these signaling pathways; these findings demonstrate a direct antioxidant mechanism for NOS1 and have pathophysiologic implications for the growing number of disease states in which increased XOR activity plays a role.

Platelets become activated during myocardial infarction (MI), but the direct contribution of activated platelets to myocardial reperfusion injury in vivo has yet to be reported. We tested the hypothesis that activated platelets contribute importantly to reperfusion injury during MI in mice. After 30 min of ischemia and 60 min of reperfusion, P-selectin knockout mice had a significantly smaller infarct size than that of wild-type mice (P < 0.05). Platelets were detected by P-selectin antibody in the previously ischemic region of wild-type mice as early as 2 min postreperfusion after 45 min, but not 20 min, of ischemia. The appearance of neutrophils in the heart was delayed when compared with platelets. Flow cytometry showed that the number of activated platelets more than doubled after 45 min of ischemia when compared with 20 min of ischemia or sham treatment (P < 0.05). Platelet-rich or platelet-poor plasma was then transfused from either sham-operated or infarcted mice after 45 and 10 min of ischemia-reperfusion to mice undergoing 20 and 60 min of ischemia-reperfusion. Infarct size was increased by threefold and platelet accumulation was remarkably enhanced in mice treated with wild-type, MI-activated platelet-rich plasma but not in mice receiving either platelet-poor plasma from wild types or MI-activated platelet-rich plasma from P-selectin knockout mice. In conclusion, circulating platelets become activated early during reperfusion and their activation depends on the duration of the preceding coronary occlusion and is proportional to the extent of myocardial injury. Activated platelets play an important role in the process of myocardial ischemia-reperfusion injury, and platelet-derived P-selectin is a critical mediator.

Histamine stimulation of swine carotid artery induces both contraction and actin polymerization. The importance of stimulus-induced actin polymerization is not known. Tyrosine phosphorylation of the scaffolding protein paxillin is thought to be an important regulator of actin polymerization. Noise temperature, hysteresivity, and phase angle are rheological measures of the fluidity of a tissue, i.e., whether the muscle is more a "Hookean solid" or a "Newtonian liquid." Y118 paxillin phosphorylation, crossbridge phosphorylation, actin polymerization, noise temperature, hysteresivity, phase angle, real stiffness, and stress were measured in intact swine carotid arteries that were depolarized with high K ϩ or stimulated with histamine. The initial rapid force development phase of high-K ϩ or histamine-induced contraction was associated with increased crossbridge phosphorylation but no significant change in Y118 paxillin phosphorylation, actin polymerization, noise temperature, hysteresivity, or phase angle. This suggests that the initial contraction was caused by the increase in crossbridge phosphorylation and did not alter the tissue's rheology. Only after full force development was there a significant increase in Y118 paxillin phosphorylation and actin polymerization associated with a significant decrease in noise temperature and hysteresivity. These data suggest that some part of the sustained contraction may depend on stimulated actin polymerization and/or a transition to a more "solid" rheology. Supporting this contention was the finding that an inhibitor of actin polymerization, latrunculin-A, reduced force while increasing noise temperature/hysteresivity. Further research is needed to determine whether Y118 paxillin phosphorylation, actin polymerization, and changes in rheology could have a role in arterial smooth muscle contraction. cytoskeleton; hysteresivity; latch hypothesis; vascular smooth muscle MAXIMAL STIMULATION of arterial smooth muscle typically produces a large increase in myoplasmic Ca 2ϩ , which activates myosin kinase. The activated myosin kinase phosphorylates crossbridges on Ser 19 of the myosin regulatory light chain (MRLC). The resulting large increase in crossbridge phosphorylation is felt to produce rapid force development (reviewed in Ref. 23). During the sustained phase of a maximal contraction, crossbridge phosphorylation typically falls to intermediate levels while force is maintained at peak levels, a phenomenon termed "latch" (8, 26). At first, it was thought that a second regulatory system was necessary to explain the latch phenomenon (6). However, dynamic crossbridge models suggest that accumulating attached dephosphorylated crossbridges (latchbridges) can explain many of the characteristics of the latch phenomenon, specifically, maintained force despite reduced crossbridge phosphorylation, shortening velocity, and ATP consumption (14,30,45). These models suggest that a second regulatory system is not necessary to explain the latch phenomenon.In other types of smooth muscle, ther...

Impaired leptin signalling in obesity is increasingly implicated in cardiovascular pathophysiology. To explore mechanisms for leptin activity in the heart, we hypothesized that physiological leptin signalling participates in maintaining cardiac β-adrenergic regulation of excitation-contraction coupling. We studied 10-week-old (before development of cardiac hypertrophy) leptin-deficient (ob/ob, n = 12) and C57Bl/6 (wild-type (WT), n = 15) mice at baseline and after recombinant leptin infusion (0.3 mg kg −1 day −1 for 28 days, n = 6 in each group). Ob/ob-isolated myocytes had attenuated sarcomere shortening and calcium transients ([Ca 2+ ] i ) versus WT (P < 0.01 for both) following stimulation of the β-receptor (with isoproterenol (isoprenaline)) or at the post-receptor level (with forskolin and dibutryl-cAMP). In addition, sarcoplasmic reticulum (SR) Ca 2+ stores were depressed. Leptin replenishment in ob/ob mice restored each of these abnormalities towards normal without affecting gross (wall thickness) or microscopic (cell size) measures of cardiac architecture. Immunoblots revealed alterations of several proteins involved in excitation-contraction coupling in the ob/ob mice, including decreased abundance of G s α-52 kDa, as well as alterations in the expression of Ca 2+ cycling proteins (increased SR Ca 2+ -ATPase, and depressed phosphorylated phospholamban). In addition, protein kinase A (PKA) activity in ob/ob mice was depressed at baseline and correctable towards the activity found in WT with leptin repletion, a finding that could account for impaired β-adrenergic responsiveness. Taken together, these data reveal a novel link between the leptin signalling pathway and normal cardiac function and suggest a mechanism by which leptin deficiency or resistance may lead to cardiac depression.

Tejani AD, Walsh MP, Rembold CM. Tissue length modulates "stimulated actin polymerization," force augmentation, and the rate of swine carotid arterial contraction. Am J Physiol Cell Physiol 301: C1470 -C1478, 2011. First published August 24, 2011 doi:10.1152/ajpcell.00149.2011.-"Stimulated actin polymerization" has been proposed to be involved in force augmentation, in which prior submaximal activation of vascular smooth muscle increases the force of a subsequent maximal contraction by ϳ15%. In this study, we altered stimulated actin polymerization by adjusting tissue length and then measured the effect on force augmentation. At optimal tissue length (1.0 L o), force augmentation was observed and was associated with increased prior stimulated actin polymerization, as evidenced by increased prior Y118 paxillin phosphorylation without changes in prior S3 cofilin or cross-bridge phosphorylation. Tissue length, per se, regulated Y118 paxillin, but not S3 cofilin, phosphorylation. At short tissue length (0.6 L o), force augmentation was observed and was associated with increased prior stimulated actin polymerization, as evidenced by reduced prior S3 cofilin phosphorylation without changes in Y118 paxillin or cross-bridge phosphorylation. At long tissue length (1.4 L o), force augmentation was not observed, and there were no prior changes in Y118 paxillin, S3 cofilin, or cross-bridge phosphorylation. There were no significant differences in the cross-bridge phosphorylation transients before and after the force augmentation protocol at all three lengths tested. Tissues contracted faster at longer tissue lengths; contractile rate correlated with prior Y118 paxillin phosphorylation. Total stress, per se, predicted Y118 paxillin phosphorylation. These data suggest that force augmentation is regulated by stimulated actin polymerization and that stimulated actin polymerization is regulated by total arterial stress. We suggest that K ϩ depolarization first leads to cross-bridge phosphorylation and contraction, and the contraction-induced increase in mechanical strain increases Y118 paxillin phosphorylation, leading to stimulated actin polymerization, which further increases force, i.e., force augmentation and, possibly, latch.