Ectonucleoside triphosphate diphosphohydrolase-1 (CD39) impacts TGF-ß1 responses: insights into cardiac fibrosis and function following myocardial infarction.

Extracellular purine nucleotides and nucleosides released from activated or injured cells influence multiple aspects of cardiac physiology and pathophysiology. Ectonucleoside triphosphate diphosphohydrolase-1 (ENTPD1; CD39) hydrolyzes released nucleotides and thereby regulates the magnitude and duration of purinergic signaling. However, the impact of CD39 activity on post-myocardial infarction (MI) remodeling is incompletely understood. We measured the levels and activity of ectonucleotidases in human left ventricular samples from control and ischemic cardiomyopathy (ICM) hearts and examined the impact of ablation of Cd39 expression on post-myocardial infarction remodeling in mice. We found that human CD39 levels and activity are significantly decreased in ICM hearts (n = 5) compared with control hearts (n = 5). In mice null for Cd39, cardiac function and remodeling are significantly compromised in Cd39-/- mice following myocardial infarction. Fibrotic markers including plasminogen activator inhibitor-1 (PAI-1) expression, fibrin deposition, α-smooth muscle actin (αSMA), and collagen expression are increased in Cd39-/- hearts. Importantly, we found that transforming growth factor ß1 (TGF-ß1) stimulates ATP release and induces Cd39 expression and activity on cardiac fibroblasts, constituting an autocrine regulatory pathway not previously appreciated. Absence of CD39 activity on cardiac fibroblasts exacerbates TGF-ß1 profibrotic responses. Treatment with exogenous ectonucleotidase rescues this profibrotic response in Cd39-/- fibroblasts. Together, these data demonstrate that CD39 has important interactions with TGF-ß1-stimulated autocrine purinergic signaling in cardiac fibroblasts and dictates outcomes of cardiac remodeling following myocardial infarction. Our results reveal that ENTPD1 (CD39) regulates TGF-ß1-mediated fibroblast activation and limits adverse cardiac remodeling following myocardial infarction.NEW & NOTEWORTHY We show that CD39 is a critical modulator of TGF-ß1-mediated fibroblast activation and cardiac remodeling following myocardial infarction via modulation of nucleotide signaling. TGF-ß1-induced CD39 expression generates a negative feedback loop that attenuates cardiac fibroblast activation. In the absence of CD39 activity, collagen deposition is increased, elastin expression is decreased, and diastolic dysfunction is worsened. Treatment with ecto-apyrase attenuates the TGF-ß1-induced profibrotic cardiac fibroblast phenotype, revealing a novel approach to combat post-myocardial infarction cardiac fibrosis.

Preclinical Development of a vWF Aptamer to Limit Thrombosis and Engender Arterial Recanalization of Occluded Vessels.

Endothelial surface and circulating glycoprotein von Willebrand factor (vWF) regulates platelet adhesion and is associated with thrombotic diseases, including ischemic stroke, myocardial infarction, and peripheral vascular disease. Thrombosis, as manifested in these diseases, is the leading cause of disability and death in the western world. Current parenteral antithrombotic and thrombolytic agents used to treat these conditions are limited by a short therapeutic window, irreversibility, and major risk of hemorrhage. To overcome these limitations, we developed a novel anti-vWF aptamer, called DTRI-031, that selectively binds and inhibits vWF-mediated platelet adhesion and arterial thrombosis while enabling rapid reversal of this antiplatelet activity by an antidote oligonucleotide (AO). Aptamer DTRI-031 exerts dose-dependent inhibition of platelet aggregation and thrombosis in whole blood and mice, respectively. Moreover, DTRI-031 can achieve potent vascular recanalization of platelet-rich thrombotic occlusions in murine and canine carotid arteries. Finally, DTRI-031 activity is rapidly (<5 min) and completely reversed by AO administration in a murine saphenous vein hemorrhage model, and murine toxicology studies indicate the aptamer is well tolerated. These findings suggest that targeting vWF with an antidote-controllable aptamer potentially represents an effective and safer treatment for thrombosis patients having platelet-rich arterial occlusions in the brain, heart, or periphery.

Role of the CD39/CD73 Purinergic Pathway in Modulating Arterial Thrombosis in Mice.

OBJECTIVE: Circulating blood cells and endothelial cells express ectonucleoside triphosphate diphosphohydrolase-1 (CD39) and ecto-5'-nucleotidase (CD73). CD39 hydrolyzes extracellular ATP or ADP to AMP. CD73 hydrolyzes AMP to adenosine. The goal of this study was to examine the interplay between CD39 and CD73 cascade in arterial thrombosis. APPROACH AND RESULTS: To determine how CD73 activity influences in vivo thrombosis, the time to ferric chloride-induced arterial thrombosis was measured in CD73-null mice. In response to 5% FeCl3, but not to 10% FeCl3, there was a significant decrease in the time to thrombosis in CD73-null mice compared with wild-type mice. In mice overexpressing CD39, ablation of CD73 did not inhibit the prolongation in the time to thrombosis conveyed by CD39 overexpression. However, the CD73 inhibitor α-ß-methylene-ADP nullified the prolongation in the time to thrombosis in human CD39 transgenic (hC39-Tg)/CD73-null mice. To determine whether hematopoietic-derived cells or endothelial cell CD39 activity regulates in vivo arterial thrombus, bone marrow transplant studies were conducted. FeCl3-induced arterial thrombosis in chimeric mice revealed a significant prolongation in the time to thrombosis in hCD39-Tg reconstituted wild-type mice, but not on wild-type reconstituted hCD39-Tg mice. Monocyte depletion with clodronate-loaded liposomes normalized the time to thrombosis in hCD39-Tg mice compared with hCD39-Tg mice treated with control liposomes, demonstrating that increased CD39 expression on monocytes protects against thrombosis. CONCLUSIONS: These data demonstrate that ablation of CD73 minimally effects in vivo thrombosis, but increased CD39 expression on hematopoietic-derived cells, especially monocytes, attenuates in vivo arterial thrombosis.

Ectonucleotide triphosphate diphosphohydrolase-1 (CD39) mediates resistance to occlusive arterial thrombus formation after vascular injury in mice.

Modulation of purinergic signaling, which is critical for vascular homeostasis and the response to vascular injury, is regulated by hydrolysis of proinflammatory ATP and/or ADP by ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD-1; CD39) to AMP, which then is hydrolyzed by ecto-5'-nucleotidase (CD73) to adenosine. We report here that compared with littermate controls (wild type), transgenic mice expressing human ENTPDase-1 were resistant to the formation of an occlusive thrombus after FeCl(3)-induced carotid artery injury. Treatment of mice with the nonhydrolyzable ADP analog, adenosine-5'-0-(2-thiodiphosphate) trilithium salt, Ado-5'-PP[S], negated the protection from thrombosis, consistent with a role for ADP in platelet recruitment and thrombus formation. ENTPD-1 expression decreased whole-blood aggregation after stimulation by ADP, an effect negated by adenosine-5'-0-(2-thiodiphosphate) trilithium salt, Ado-5'-PP[S] stimulation, and limited the ability to maintain the platelet fibrinogen receptor, glycoprotein α(IIb)/ß(3), in a fully activated state, which is critical for thrombus formation. In vivo treatment with a CD73 antagonist, a nonselective adenosine-receptor antagonist, or a selective A(2A) or A(2B) adenosine-receptor antagonist, negated the resistance to thrombosis in transgenic mice expressing human ENTPD-1, suggesting a role for adenosine generation and engagement of adenosine receptors in conferring in vivo resistance to occlusive thrombosis in this model. In summary, our findings identify ENTPDase-1 modulation of purinergic signaling as a key determinant of the formation of an occlusive thrombus after vascular injury.

A canine thromboembolic model of anterior circulation large vessel occlusion stroke.

Purpose: To develop a large animal preclinical model of thromboembolic stroke with stable, protracted large vessel occlusion (LVO) utilizing an autologous clot. Materials and methods: A reproducible canine model of large vessel occlusion stroke was established by endovascular placement of an autologous clot into the middle cerebral artery (MCA) of six adult hounds and confirmed using digital subtraction angiography (DSA). Infarct volume and evidence of hemorrhage were determined by magnetic resonance imaging (MRI) 7 h after occlusion and Thrombolysis in Cerebral Infarction scale (TICI) was assessed before and after clot placement and at 1, 6, 7, and 9 h after middle cerebral artery occlusion (MCAO). Heart rate (HR) and blood pressure (BP) were monitored continuously and invasively through an arterial sheath throughout the procedures and complete blood count and blood gas analysis completed at time of sacrifice. Histopathological findings at time of sacrifice were used to confirm stroke volume and hemorrhage. Results: MCAO with resulting TICI 0 flow was observed in all six animals, verified by serial DSA, and lack of collateral flow persisted for 9 h after clot placement until time of sacrifice. The mean infarct volume was 47.0 ± 6.7% of the ipsilateral hemisphere and no events of spontaneous recanalization or clot autolysis were observed. Conclusion: We demonstrate a thromboembolic canine model of MCAO that is both feasible and results in consistent infarct volumes to generate a clinically relevant LVO. This model is important to evaluate treatment of LVO in acute ischemic stroke (AIS) outside the established 4.5 h recombinant tissue plasminogen activator (rTPA) therapeutic window utilizing a prolonged occlusive thrombus.

Transgenic swine: expression of human CD39 protects against myocardial injury.

UNLABELLED: CD39 (ectonucleoside triphosphate diphosphohydrolase-1; ENTPD-1) rapidly hydrolyzes ATP and ADP to AMP; AMP is hydrolyzed by ecto-5'-nucleotidase (CD73) to adenosine, an anti-thrombotic and cardiovascular protective mediator. While expression of human CD39 in a murine model of myocardial ischemia/reperfusion (I/R) injury confers cardiac protection, the translational therapeutic potential of these findings requires further testing in a large animal model. To determine if transgenic expression of CD39 reduces infarct size in a swine model of myocardial ischemia/reperfusion injury, transgenic pigs expressing human CD39 (hCD39) were generated via somatic cell nuclear transfer and characterized. Expression of hC39 in cardiac tissue was confirmed by immunoblot and immunohistochemistry. Myocardial I/R injury was induced by intracoronary balloon inflation in the left anterior descending (LAD) artery for 60 min followed by 3 hours of reperfusion. The ischemic area was delineated by perfusion with 5% phthalo blue and the myocardial infarct size was determined by triphenyl tetrazolium chloride (TTC) staining. During ischemia, the rate-pressure product was significantly lower in control versus hCD39-Tg swine. Following reperfusion, compared to littermate control swine, hCD39-Tg animals displayed a significant reduction in infarct size (hCD39-Tg: 17.2 ± 4.3% vs. CONTROL: 44.7 ± 5.2%, P=0.0025). Our findings demonstrate for the first time that the findings in transgenic mouse models translate to large animal transgenic models and validate the potential to translate CD39 into the clinical arena to attenuate human myocardial ischemia/reperfusion injury.

Transgenic over expression of ectonucleotide triphosphate diphosphohydrolase-1 protects against murine myocardial ischemic injury.

Modulation of purinergic signaling is critical to myocardial homeostasis. Ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD-1; CD39) which converts the proinflammatory molecules ATP or ADP to AMP is a key regulator of purinergic modulation. However, the salutary effects of transgenic over expression of ENTPD-1 on myocardial response to ischemic injury have not been tested to date. Therefore we hypothesized that ENTPD-1 over expression affords myocardial protection from ischemia-reperfusion injury via specific cell signaling pathways. ENTPD-1 transgenic mice, which over express human ENTPDase-1, and wild-type (WT) littermates were subjected to either ex vivo or in vivo ischemia-reperfusion injury. Infarct size, inflammatory cell infiltrate and intracellular signaling molecule activation were evaluated. Infarct size was significantly reduced in ENTPD-1 versus WT hearts in both ex vivo and in vivo studies. Following ischemia-reperfusion injury, ENTPD-1 cardiac tissues demonstrated an increase in the phosphorylation of the cellular signaling molecule extracellular signal-regulated kinases 1/2 (ERK 1/2) and glycogen synthase kinase-3ß (GSK-3ß). Resistance to myocardial injury was abrogated by treatment with a non-selective adenosine receptor antagonist, 8-SPT or the more selective A(2B) adenosine receptor antagonist, MRS 1754, but not the A(1) selective antagonists, DPCPX. Additionally, treatment with the ERK 1/2 inhibitor PD98059 or the mitochondrial permeability transition pore opener, atractyloside, abrogated the cardiac protection provided by ENTPDase-1 expression. These results suggest that transgenic ENTPDase-1 expression preferentially conveys myocardial protection from ischemic injury via adenosine A(2B) receptor engagement and associated phosphorylation of the cellular protective signaling molecules, Akt, ERK 1/2 and GSK-3ß that prevents detrimental opening of the mitochondrial permeability transition pore.

Quantification of Cerebral Perfusion using Laser Speckle Imaging and Infarct Volume using MRI in a Pre-clinical Model of Posterior Circulation Stroke.

BACKGROUND: Basilar artery occlusion (BAO) is a subset of posterior circulation stroke that carries a mortality as high as 90%.  The current clinical standard to diagnose ischemic stroke include computerized tomography (CT), CT angiography and perfusion and magnetic resonance imaging (MRI). Large animal pre-clinical models to accurately reflect the clinical disease as well as methods to assess stroke burden and evaluate treatments are lacking. METHODS: We describe a canine model of large vessel occlusion (LVO) stroke in the posterior circulation, and developed a laser speckle imaging (LSI) protocol to monitor perfusion changes in real time.  We then utilized high b-value DWI (b=1800s/mm2) MRI to increase detection sensitivity. We also evaluated the ability of magnetic resonance angiography (MRA) to assess arterial occlusion and correlate with DSA. Finally, we verified infarct size from apparent diffusion coefficient (ADC) mapping with histology.  Results:  Administration of thromboembolism occluded the basilar artery as tracked by DSA (n=7).   LSI correlated with DSA, demonstrating a reduction in perfusion after stroke onset that persisted throughout the experiment, allowing us to monitor perfusion in real time.  DWI with an optimized b-value for dogs illustrated the stroke volume and allowed us to derive ADC and magnetic resonance angiography (MRA) images. The MRA performed at the end of the experiment correlated with DSA performed after occlusion. Finally, stroke burden on MRI correlated with histology. CONCLUSIONS: Our studies demonstrate real time perfusion imaging using LSI of a canine thromboembolic LVO model of posterior circulation stroke, which utilizes multimodal imaging important in the diagnosis and treatment of ischemic stroke.

Targeting of phospholamban by peroxynitrite decreases beta-adrenergic stimulation in cardiomyocytes.

AIMS: Peroxynitrite production increases during the pathogenesis of numerous cardiac disorders (e.g. heart failure). However, limited studies have investigated the mechanism through which peroxynitrite exerts anti-adrenergic effects. Thus, the purpose of this study is to investigate the contribution of phospholamban (PLB), a critical excitation-contraction coupling protein, to the peroxynitrite-induced dysfunction. METHODS AND RESULTS: Isolated myocytes from wild-type (WT, CF-1) and PLB knockout (PLB(-/-)) mice were stimulated at 1 Hz, and myocyte shortening and Ca(2+) transients were simultaneously recorded. PLB phosphorylation was measured via western blot. Myocytes were superfused with isoproterenol, a beta-adrenergic agonist, and SIN-1, a peroxynitrite donor. SIN-1 superfusion dramatically decreased isoproterenol-stimulated Ca(2+) transients and myocyte shortening in WT myocytes. These effects were inhibited upon addition of the peroxynitrite decomposition catalyst, FeTPPS. Surprisingly, SIN-1 had no functional effect on beta-adrenergic-stimulated PLB(-/-) myocytes. Western blot analyses revealed that SIN-1 significantly decreased isoproterenol-stimulated PLB(Ser16) phosphorylation. Experiments with the protein phosphatase inhibitor, okadaic acid, alleviated the SIN-1-induced functional effects and the decrease in PLB phosphorylation. CONCLUSIONS: The peroxynitrite donor SIN-1 decreases beta-adrenergic stimulation by reducing PLB(Ser16) phosphorylation via protein phosphatase activation. This peroxynitrite-induced decrease in PLB phosphorylation may be a key mechanism in the beta-adrenergic dysfunction observed in many cardiomyopathies.

Biphasic effect of SIN-1 is reliant upon cardiomyocyte contractile state.

Many studies have demonstrated a biphasic effect of peroxynitrite in the myocardium, but few studies have investigated this biphasic effect on beta-adrenergic responsiveness and its dependence on contractile state. We have previously shown that high 3-morpholinosydnonimine (SIN-1) (source of peroxynitrite, 200 micromol/L) produced significant anti-adrenergic effects during maximal beta-adrenergic stimulation in cardiomyocytes. In the current study, we hypothesize that the negative effects of high SIN-1 will be greatest during high contractile states, whereas the positive effects of low SIN-1 (10 micromol/L) will predominate during low contractility. Isolated murine cardiomyocytes were field stimulated at 1 Hz, and [Ca(2+)](i) transients and shortening were recorded. After submaximal isoproterenol (ISO) (beta-adrenergic agonist, 0.01 micromol/L) stimulation, 200 micromol/L SIN-1 induced two distinct phenomena. Cardiomyocytes undergoing a large response to ISO showed a significant reduction in contractility, whereas cardiomyocytes exhibiting a modest response to ISO showed a further increase in contractility. Additionally, 10 micromol/L SIN-1 always increased contractility during low ISO stimulation, but had no effect during maximal ISO (1 micromol/L) stimulation. SIN-1 at 10 micromol/L also increased basal contractility. Interestingly, SIN-1 produced a contractile effect under only one condition in phospholamban-knockout cardiomyocytes, providing a potential mechanism for the biphasic effect of peroxynitrite. These results provide clear evidence for a biphasic effect of peroxynitrite, with high peroxynitrite modulating high levels of beta-adrenergic responsiveness and low peroxynitrite regulating basal function and low levels of beta-adrenergic stimulation.

Ferric Chloride-induced Canine Carotid Artery Thrombosis: A Large Animal Model of Vascular Injury.

Occlusive arterial thrombosis leading to cerebral ischemic stroke and myocardial infarction contributes to ~13 million deaths every year globally. Here, we have translated a vascular injury model from a small animal into a large animal (canine), with slight modifications that can be used for pre-clinical screening of prophylactic and thrombolytic agents. In addition to the surgical methods, the modified protocol describes the step-by-step methods to assess carotid artery canalization by angiography, detailed instructions to process both the brain and carotid artery for histological analysis to verify carotid canalization and cerebral hemorrhage, and specific parameters to complete an assessment of downstream thromboembolic events by utilizing magnetic resonance imaging (MRI). In addition, specific procedural changes from the previously well-established small animal model necessary to translate into a large animal (canine) vascular injury are discussed.

Impact of cardiac-specific expression of CD39 on myocardial infarct size in mice.

AIMS: Prior work suggests that ischemic preconditioning increases the level of CD39 in the heart and contributes to cardiac protection. Therefore, we examined if targeted cardiac expression of CD39 protects against myocardial injury. MAIN METHODS: Mice with cardiac-specific expression of human CD39 (αMHC/hCD39-Tg) were generated, characterized and subjected to left coronary artery ischemia-reperfusion injury and infarct size at 24h following injury quantified. KEY FINDINGS: αMHC/hCD39-Tg mice have increased in cardiac ATPase and ADPase activity compared to WT littermates. The increased activity in αMHC/hCD39-mice was inhibited by the CD39 antagonist sodium polyoxotungstate (POM-1). Measurement of basal cardiac function by echocardiography revealed that αMHC/hCD39-Tg mice have a lower resting heart rate and increased stroke volume. In response to myocardial ischemia, systolic and diastolic function was better preserved in αMHC/hCD39-Tg compared to WT mice. Comparison of Tau also revealed preserved cardiac relaxation during ischemia in αMHC/hCD39-Tg hearts. Assessment of myocardial infarct size in response to 60min of ischemia and 24h of reperfusion demonstrated a significant reduction in infarct size in αMHC/hCD39-Tg hearts. Analysis of isolated cardiomyocytes revealed no basal difference in calcium transients between WT and αMHC/hCD39-Tg cardiomyocytes. However, in response to isoproterenol stimulation, there was a trend toward lower calcium transients in αMHC/hCD39 cardiomyocytes suggesting less calcium accumulation in response to metabolic stress. SIGNIFICANCE: Cardiac-specific expression of CD39 reduces myocardial dysfunction and infarct size following ischemia-reperfusion injury. Increasing nucleotidase expression in the heart may be a novel approach to protect the heart from ischemic injury.

Kcnj11 Ablation Is Associated With Increased Nitro-Oxidative Stress During Ischemia-Reperfusion Injury: Implications for Human Ischemic Cardiomyopathy.

BACKGROUND: Despite increased secondary cardiovascular events in patients with ischemic cardiomyopathy (ICM), the expression of innate cardiac protective molecules in the hearts of patients with ICM is incompletely characterized. Therefore, we used a nonbiased RNAseq approach to determine whether differences in cardiac protective molecules occur with ICM. METHODS AND RESULTS: RNAseq analysis of human control and ICM left ventricular samples demonstrated a significant decrease in KCNJ11 expression with ICM. KCNJ11 encodes the Kir6.2 subunit of the cardioprotective KATP channel. Using wild-type mice and kcnj11-deficient (kcnj11-null) mice, we examined the effect of kcnj11 expression on cardiac function during ischemia-reperfusion injury. Reactive oxygen species generation increased in kcnj11-null hearts above that found in wild-type mice hearts after ischemia-reperfusion injury. Continuous left ventricular pressure measurement during ischemia and reperfusion demonstrated a more compromised diastolic function in kcnj11-null compared with wild-type mice during reperfusion. Analysis of key calcium-regulating proteins revealed significant differences in kcnj11-null mice. Despite impaired relaxation, kcnj11-null hearts increased phospholamban Ser16 phosphorylation, a modification that results in the dissociation of phospholamban from sarcoendoplasmic reticulum Ca2+, thereby increasing sarcoendoplasmic reticulum Ca2+-mediated calcium reuptake. However, kcnj11-null mice also had increased 3-nitrotyrosine modification of the sarcoendoplasmic reticulum Ca2+-ATPase, a modification that irreversibly impairs sarcoendoplasmic reticulum Ca2+ function, thereby contributing to diastolic dysfunction. CONCLUSIONS: KCNJ11 expression is decreased in human ICM. Lack of kcnj11 expression increases peroxynitrite-mediated modification of the key calcium-handling protein sarcoendoplasmic reticulum Ca2+-ATPase after myocardial ischemia-reperfusion injury, contributing to impaired diastolic function. These data suggest a mechanism for ischemia-induced diastolic dysfunction in patients with ICM.

Effect of glutathione augmentation on lipid peroxidation after spinal cord injury.

Lipid peroxidation (LPO) is considered a major factor in damage spread after spinal cord injury (SCI). Therapies that limit LPO after SCI have demonstrated some utility in clinical trials, but more effective treatments are needed. In the present study the effects of augmenting SC levels of the endogenous antioxidant glutathione (GSH) on LPO after SCI were studied in a rat contusion injury model. A significant decrease in GSH occurred 1h after SCI which was paralleled by increases of 123% in malondialdehyde (MDA) and >500% in the 4-hydroxyalkenals (4-HA's), two LPO products. SC irrigation with gamma-glutamylcysteine (GC) preserved GSH and reduced 4-HA's below naive levels but had no effect on MDA. By 24 h after SCI, MDA returned to naive levels but 4-HA's were still elevated. Once again, GC treatment reduced 4-HA's. 4-HA's are much more reactive than MDA and are considered among the most toxic LPO products. These results suggest that (1) conditions after SCI may favor particular branches of the LPO pathway leading to differential LPO product levels, (2) MDA measurement is not by itself an adequate test for the presence or magnitude of LPO after SCI, (3) binding of GSH to 4-HA's may be an important mechanism by which the GSH system confers protection against LPO after SCI, and (4) SC GSH can be augmented after trauma by local irrigation with GC. These results also suggest that GSH augmentation may be an effective strategy for curtailment of LPO-mediated damage in acute phase SCI.

Endothelial nitric oxide synthase decreases beta-adrenergic responsiveness via inhibition of the L-type Ca2+ current.

Signaling via endothelial nitric oxide synthase (NOS3) limits the heart's response to beta-adrenergic (beta-AR) stimulation, which may be protective against arrhythmias. However, mechanistic data are limited. Therefore, we performed simultaneous measurements of action potential (AP, using patch clamp), Ca2+ transients (fluo 4), and myocyte shortening (edge detection). L-type Ca2+ current (ICa) was directly measured by the whole cell ruptured patch-clamp technique. Myocytes were isolated from wild-type (WT) and NOS3 knockout (NOS3-/-) mice. NOS3-/- myocytes exhibited a larger incidence of beta-AR (isoproterenol, 1 microM)-induced early afterdepolarizations (EADs) and spontaneous activity (defined as aftercontractions). We also examined ICa, a major trigger for EADs. NOS3-/- myocytes had a significantly larger beta-AR-stimulated increase in ICa compared with WT myocytes. In addition, NOS3-/- myocytes had a larger response to beta-AR stimulation compared with WT myocytes in Ca2+ transient amplitude, shortening amplitude, and AP duration (APD). We observed similar effects with specific NOS3 inhibition [L-N5-(1-iminoethyl)-ornithine (l-NIO), 10 microM] in WT myocytes as with NOS3 knockout. Specifically, l-NIO further increased isoproterenol-stimulated EADs and aftercontractions. l-NIO also further increased the isoproterenol-stimulated ICa, Ca2+ transient amplitude, shortening amplitude, and APD (all P < 0.05 vs isoproterenol alone). l-NIO had no effect in NOS3-/- myocytes. These results indicate that NOS3 signaling inhibits the beta-AR response by reducing ICa and protects against arrhythmias. This mechanism may play an important role in heart failure, where arrhythmias are increased and NOS3 expression is decreased.

Neuronal nitric oxide synthase signaling within cardiac myocytes targets phospholamban.

Studies have shown that neuronal nitric oxide synthase (nNOS, NOS1) knockout mice (NOS1-/-) have increased or decreased contractility, but consistently have found a slowed rate of intracellular Ca2+ ([Ca2+]i) decline and relengthening. Contraction and [Ca2+]i decline are determined by many factors, one of which is phospholamban (PLB). The purpose of this study is to determine the involvement of PLB in the NOS1-mediated effects. Force-frequency experiments were performed in trabeculae isolated from NOS1-/- and wild-type (WT) mice. We also simultaneously measured Ca2+ transients (Fluo-4) and cell shortening (edge detection) in myocytes isolated from WT, NOS1-/-, and PLB-/- mice. NOS1-/- trabeculae had a blunted force-frequency response and prolonged relaxation. We observed similar effects in myocytes with NOS1 knockout or specific NOS1 inhibition with S-methyl-l-thiocitrulline (SMLT) in WT myocytes (i.e., decreased Ca2+ transient and cell shortening amplitudes and prolonged decline of [Ca2+]i). Alternatively, NOS1 inhibition with SMLT in PLB-/- myocytes had no effect. Acute inhibition of NOS1 with SMLT in WT myocytes also decreased basal PLB serine16 phosphorylation. Furthermore, there was a decreased SR Ca2+ load with NOS1 knockout or inhibition, which is consistent with the negative contractile effects. Perfusion with FeTPPS (peroxynitrite decomposition catalyst) mimicked the effects of NOS1 knockout or inhibition. beta-Adrenergic stimulation restored the slowed [Ca2+]i decline in NOS1-/- myocytes, but a blunted contraction remained, suggesting additional protein target(s). In summary, NOS1 inhibition or knockout leads to decreased contraction and slowed [Ca2+]i decline, and this effect is absent in PLB-/- myocytes. Thus NOS1 signaling modulates PLB serine16 phosphorylation, in part, via peroxynitrite.