Mark T. Ziolo

Nitric oxide signaling and the regulation of myocardial function.

Nitric oxide, which is produced endogenously within cardiac myocytes by three distinct isoforms of nitric oxide synthase, is a key regulator of myocardial function. This review will focus on the regulation of myocardial function by each nitric oxide synthase isoform during health and disease, with a specific emphasis on the proposed end-targets and signaling pathways.

Regulation of myocyte contraction via neuronal nitric oxide synthase: role of ryanodine receptor S-nitrosylation

The sarcoplasmic reticulum (SR) Ca2+ release channel (ryanodine receptor, RyR2) has been proposed to be an end target of neuronal nitric oxide synthase (NOS1) signalling. The purpose of this study is to investigate the mechanism of NOS1 modulation of RyR2 activity and the corresponding effect on myocyte function. Myocytes were isolated from NOS1 knockout (NOS1−/−) and wild‐type mice. NOS1−/− myocytes displayed a decreased fractional SR Ca2+ release, NOS1 knockout also led to reduced RyR2 S‐nitrosylation levels. RyR2 channels from NOS1−/− hearts had decreased RyR2 open probability. Additionally, knockout of NOS1 led to a decrease in [3H]ryanodine binding, Ca2+ spark frequency (CaSpF) and a rightward shift in the SR Ca2+ leak/load relationship. Similar effects were observed with acute inhibition of NOS1. These data are indicative of decreased RyR2 activity in myocytes with NOS1 knockout or acute inhibition. Interestingly, the NO donor and nitrosylating agent SNAP reversed the depressed RyR2 open probability, the reduced CaSpF, and caused a leftward shift in the leak/load relationship in NOS1−/− myocytes. SNAP also normalized Ca2+ transient and cell shortening amplitudes and SR fractional release in myocytes with NOS1 knockout or acute inhibition. Furthermore, SNAP was able to normalize the RyR2 S‐nitrosylation levels. These data suggest that NOS1 signalling increases RyR2 activity via S‐nitrosylation, which contributes to the NOS1‐induced positive inotropic effect. Thus, RyR2 is an important end target of NOS1.

Targeted Overexpression of Sarcolipin in the Mouse Heart Decreases Sarcoplasmic Reticulum Calcium Transport and Cardiac Contractility

The role of sarcolipin (SLN) in cardiac physiology was critically evaluated by generating a transgenic (TG) mouse model in which the SLN to sarco(endoplasmic)reticulum (SR) Ca2+ ATPase (SERCA) ratio was increased in the ventricle. Overexpression of SLN decreases SR calcium transport function and results in decreased calcium transient amplitude and rate of relaxation. SLN TG hearts exhibit a significant decrease in rates of contraction and relaxation when assessed by ex vivo work-performing heart preparations. Similar results were also observed with muscle preparations and myocytes from SLN TG ventricles. Interestingly, the inhibitory effect of SLN was partially relieved upon high dose of isoproterenol treatment and stimulation at high frequency. Biochemical analyses show that an increase in SLN level does not affect PLB levels, monomer to pentamer ratio, or its phosphorylation status. No compensatory changes were seen in the expression of other calcium-handling proteins. These studies suggest that the SLN effect on SERCA pump is direct and is not mediated through increased monomerization of PLB or by a change in PLB phosphorylation status. We conclude that SLN is a novel regulator of SERCA pump activity, and its inhibitory effect can be reversed by β-adrenergic agonists.

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

Signaling via endothelial nitric oxide synthase (NOS3) limits the hearts 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.

Antagonistic activities of the immunomodulator and PP2A-activating drug FTY720 (Fingolimod, Gilenya) in Jak2-driven hematologic malignancies

FTY720 (Fingolimod, Gilenya) is a sphingosine analog used as an immunosuppressant in multiple sclerosis patients. FTY720 is also a potent protein phosphatase 2A (PP2A)-activating drug (PAD). PP2A is a tumor suppressor found inactivated in different types of cancer. We show here that PP2A is inactive in polycythemia vera (PV) and other myeloproliferative neoplasms characterized by the expression of the transforming Jak2(V617F) oncogene. PP2A inactivation occurs in a Jak2(V617F) dose/kinase-dependent manner through the PI-3Kγ-PKC-induced phosphorylation of the PP2A inhibitor SET. Genetic or PAD-mediated PP2A reactivation induces Jak2(V617F) inactivation/downregulation and impairs clonogenic potential of Jak2(V617F) cell lines and PV but not normal CD34(+) progenitors. Likewise, FTY720 decreases leukemic allelic burden, reduces splenomegaly, and significantly increases survival of Jak2(V617F) leukemic mice without adverse effects. Mechanistically, we show that in Jak2(V617F) cells, FTY720 antileukemic activity requires neither FTY720 phosphorylation (FTY720-P) nor SET dimerization or ceramide induction but depends on interaction with SET K209. Moreover, we show that Jak2(V617F) also utilizes an alternative sphingosine kinase-1-mediated pathway to inhibit PP2A and that FTY720-P, acting as a sphingosine-1-phosphate-receptor-1 agonist, elicits signals leading to the Jak2-PI-3Kγ-PKC-SET-mediated PP2A inhibition. Thus, PADs (eg, FTY720) represent suitable therapeutic alternatives for Jak2(V617F) MPNs.

The Real Estate of NOS Signaling Location, Location, Location

While sympathetic stimulation of the heart produces chronotropic, inotropic, and lusitropic effects, increased frequency alone causes a positive force-frequency relationship (FFR) and frequency-dependent acceleration of relaxation (FDAR).1 That is, contraction amplitude and relaxation rate are increased with increasing frequency in most species (including humans). The key mechanism involved in the positive FFR is increased sarcoplasmic reticulum (SR) Ca2+ load, due to increased Ca2+ influx and decreased Ca2+ efflux.1,2 Ca2+ influx increases due to more L-type Ca2+ current ( I Ca) per unit time, while Ca2+ efflux via Na+-Ca2+ exchange (NCX) decreases because the diastolic time is reduced and [Na+]i increases. Enhanced SR Ca2+-pump function causes FDAR and also augments SR Ca2+ loading. Various signaling pathways are involved (eg, CaMKII).3 In human heart failure, the FFR reverses (ie, from positive to negative) due to an inability of the SR to increase Ca2+ content.4 This negative FFR is a main contributor to the loss of contractile reserve in the failing heart. Of many pathways that can modify FFR, nitric oxide (NO) signaling is the topic addressed by Khan et al in this issue of Circulation Research .5 NO synthase (NOS) produces NO from l-arginine, and cardiac myocytes express all three NOS isoforms.6,7 NOS1 (nNOS) and NOS3 (eNOS) are constitutively expressed and produce low amounts of NO (regulated by [Ca2+-calmodulin]i levels). NOS2 (iNOS) …

Phosphodiesterase 5 restricts NOS3/Soluble guanylate cyclase signaling to L-type Ca2+ current in cardiac myocytes

Endothelial nitric oxide synthase (NOS3) regulates the functional response to beta-adrenergic (beta-AR) stimulation via modulation of the L-type Ca(2+) current (I(Ca)). However, the NOS3 signaling pathway modulating I(Ca) is unknown. This study investigated the contribution of soluble guanylate cyclase (sGC) and phosphodiesterase type 5 (PDE5), a cGMP-specific PDE, in the NOS3-mediated regulation of I(Ca). Myocytes were isolated from NOS3 knockout (NOS3(-/-)) and wildtype (WT) mice. We measured I(Ca) (whole-cell voltage-clamp), and simultaneously measured Ca(2+) transients (Fluo-4 AM) and cell shortening (edge detection). Zaprinast (selective inhibitor of PDE5), decreased beta-AR stimulated (isoproterenol, ISO)-I(Ca), and Ca(2+) transient and cell shortening amplitudes in WT myocytes. However, YC-1 (NO-independent activator of sGC) only reduced ISO-stimulated I(Ca), but not cardiac contraction. We further investigated the NOS3/sGC/PDE5 pathway in NOS3(-/-) myocytes. PDE5 is mislocalized in these myocytes and we observed dissimilar effects of PDE5 inhibition and sGC activation compared to WT. That is, zaprinast had no effect on ISO-stimulated I(Ca), or Ca(2+) transient and cell shortening amplitudes. Conversely, YC-1 significantly decreased both ISO-stimulated I(Ca), and cardiac contraction. Further confirming that PDE5 localizes NOS3/cGMP signaling to I(Ca); YC-1, in the presence of zaprinast, now significantly decreased ISO-stimulated Ca(2+) transient and cell shortening amplitudes in WT myocytes. The effects of YC-1 on I(Ca) and cardiac contraction were blocked by KT5823 (a selective inhibitor of the cGMP-dependent protein kinase, PKG). Our data suggests a novel physiological role for PDE5 in restricting the effects of NOS3/sGC/PKG signaling pathway to modulating beta-AR stimulated I(Ca), while limiting effects on cardiac contraction.

HNO Enhances SERCA2a activity and cardiomyocyte function by promoting redox-dependent phospholamban oligomerization

AIMS Nitroxyl (HNO) interacts with thiols to act as a redox-sensitive modulator of protein function. It enhances sarcoplasmic reticular Ca(2+) uptake and myofilament Ca(2+) sensitivity, improving cardiac contractility. This activity has led to clinical testing of HNO donors for heart failure. Here we tested whether HNO alters the inhibitory interaction between phospholamban (PLN) and the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a) in a redox-dependent manner, improving Ca(2+) handling in isolated myocytes/hearts. RESULTS Ventriculocytes, sarcoplasmic reticulum (SR) vesicles, and whole hearts were isolated from control (wildtype [WT]) or PLN knockout (pln(-/-)) mice. Compared to WT, pln(-/-) myocytes displayed enhanced resting sarcomere shortening, peak Ca(2+) transient, and blunted β-adrenergic responsiveness. HNO stimulated shortening, relaxation, and Ca(2+) transient in WT cardiomyocytes, and evoked positive inotropy/lusitropy in intact hearts. These changes were markedly blunted in pln(-/-) cells/hearts. HNO enhanced SR Ca(2+) uptake in WT but not pln(-/-) SR-vesicles. Spectroscopic studies in insect cell microsomes expressing SERCA2a±PLN showed that HNO increased Ca(2+)-dependent SERCA2a conformational flexibility but only when PLN was present. In cardiomyocytes, HNO achieved this effect by stabilizing PLN in an oligomeric disulfide bond-dependent configuration, decreasing the amount of free inhibitory monomeric PLN available. INNOVATION HNO-dependent redox changes in myocyte PLN oligomerization relieve PLN inhibition of SERCA2a. CONCLUSIONS PLN plays a central role in HNO-induced enhancement of SERCA2a activity, leading to increased inotropy/lusitropy in intact myocytes and hearts. PLN remains physically associated with SERCA2a; however, less monomeric PLN is available resulting in decreased inhibition of the enzyme. These findings offer new avenues to improve Ca(2+) handling in failing hearts.

Nitric Oxide-Dependent Activation of CaMKII Increases Diastolic Sarcoplasmic Reticulum Calcium Release in Cardiac Myocytes in Response to Adrenergic Stimulation

Spontaneous calcium waves in cardiac myocytes are caused by diastolic sarcoplasmic reticulum release (SR Ca2+ leak) through ryanodine receptors. Beta-adrenergic (β-AR) tone is known to increase this leak through the activation of Ca-calmodulin-dependent protein kinase (CaMKII) and the subsequent phosphorylation of the ryanodine receptor. When β-AR drive is chronic, as observed in heart failure, this CaMKII-dependent effect is exaggerated and becomes potentially arrhythmogenic. Recent evidence has indicated that CaMKII activation can be regulated by cellular oxidizing agents, such as reactive oxygen species. Here, we investigate how the cellular second messenger, nitric oxide, mediates CaMKII activity downstream of the adrenergic signaling cascade and promotes the generation of arrhythmogenic spontaneous Ca2+ waves in intact cardiomyocytes. Both SCaWs and SR Ca2+ leak were measured in intact rabbit and mouse ventricular myocytes loaded with the Ca-dependent fluorescent dye, fluo-4. CaMKII activity in vitro and immunoblotting for phosphorylated residues on CaMKII, nitric oxide synthase, and Akt were measured to confirm activity of these enzymes as part of the adrenergic cascade. We demonstrate that stimulation of the β-AR pathway by isoproterenol increased the CaMKII-dependent SR Ca2+ leak. This increased leak was prevented by inhibition of nitric oxide synthase 1 but not nitric oxide synthase 3. In ventricular myocytes isolated from wild-type mice, isoproterenol stimulation also increased the CaMKII-dependent leak. Critically, in myocytes isolated from nitric oxide synthase 1 knock-out mice this effect is ablated. We show that isoproterenol stimulation leads to an increase in nitric oxide production, and nitric oxide alone is sufficient to activate CaMKII and increase SR Ca2+ leak. Mechanistically, our data links Akt to nitric oxide synthase 1 activation downstream of β-AR stimulation. Collectively, this evidence supports the hypothesis that CaMKII is regulated by nitric oxide as part of the adrenergic cascade leading to arrhythmogenesis.