Deficient ryanodine receptor S -nitrosylation increases sarcoplasmic reticulum calcium leak and arrhythmogenesis in cardiomyocytes

Abstract

Altered Ca 2؉ homeostasis is a salient feature of heart disease, where the calcium release channel ryanodine receptor (RyR) plays a major role. Accumulating data support the notion that neuronal nitric oxide synthase (NOS1) regulates the cardiac RyR via Snitrosylation. We tested the hypothesis that NOS1 deficiency impairs RyR S-nitrosylation, leading to altered Ca 2؉ homeostasis. Diastolic Ca 2؉ levels are elevated in NOS1 ؊/؊ and NOS1/NOS3 ؊/؊ but not NOS3 ؊/؊ myocytes compared with wild-type (WT), suggesting diastolic Ca 2؉ leakage. Measured leak was increased in NOS1 ؊/؊ and NOS1/NOS3 ؊/؊ but not in NOS3 ؊/؊ myocytes compared with WT. Importantly, NOS1 ؊/؊ and NOS1/NOS3 ؊/؊ myocytes also exhibited spontaneous calcium waves. Whereas the stoichiometry and binding of FK-binding protein 12.6 to RyR and the degree of RyR phosphorylation were not altered in NOS1 ؊/؊ hearts, RyR2 S-nitrosylation was substantially decreased, and the level of thiol oxidation increased. Together, these findings demonstrate that NOS1 deficiency causes RyR2 hyponitrosylation, leading to diastolic Ca 2؉ leak and a proarrhythmic phenotype. NOS1 dysregulation may be a proximate cause of key phenotypes associated with heart disease.heart ͉ nitric oxide ͉ excitation-contraction coupling ͉ oxidative stress ͉ heart failure T he cardiac myocyte has emerged as a prototypic example of the manner in which nitric oxide (NO) signaling occurs in a spatially confined manner. Although neuronal (NOS1) and endothelial (NOS3) isoforms of nitric oxide synthase are located extremely close to one another within the cell on opposite sides of the dyad, they exert opposite effects on myocardial contractility (1). The mechanism(s) for this effect remains controversial. One explanation derived from in vitro observations is that NOS3 inhibits the sarcolemmal L-type calcium channel on the sarcolemmal aspect of the dyad, whereas NOS1 modulates ryanodine receptor (RyR) activity on the sarcoplasmic reticulum (SR) (1-3). Although this paradigm explains many facets of NO activity within the heart, other studies suggest that in the myocyte, NOS1 may bind to and/or regulate other ion channels or effectors, including the plasma membrane calcium/calmodulin-dependent calcium ATPase (4), sarcoplamic reticulum Ca 2ϩ -ATPase (SERCA) (5), and possibly phospholamban (PLB). In addition, there is support for the notion that this effect is mediated by a direct protein posttranslational modification; but again, this assertion is controversial (6).Another facet of NO cardiobiology has emerged that further motivates the importance of understanding the direct NOS effector molecules. In heart failure and/or other states of cardiac injury, NOS1 levels within the heart rise, and NOS1 effectively translocates from the SR to the plasma membrane (2,7,8). Because this phenomenon could have either deleterious effects or adaptive consequences, it is imperative to address definitively the physiologic role of NOS1 in the heart.To address these issues, we tested the hypothesis that the cardiac RyR i...

nNOS regulation of skeletal muscle fatigue and exercise performance

“…Although S-nitrosylation of RyR1 reduced the binding affinity of FKBP12 (calstabin1) for RyR1 in vitro, it did not prevent FKBP12 reassociation with the RyR1 complex in vivo (Aracena et al 2005;Bellinger et al 2008). Interestingly, both RyR2 hyponitrosylation resulting from nNOSμ deficiency and RyR1 hypernitrosylation resulting from excessive nNOS activity promote Ca 2+ leakage (Gonzalez et al 2007;Durham et al 2008).…”

“…A pool of nNOSμ is associated with the ryanodine receptor 1 Ca 2+ release channel (RyR1) at the sarcoplasmic reticulum (SR) Ca 2+ store where it can S-nitrosylate and regulate RyR1 channel activity (Xu et al 1999;Stamler and Meissner 2001;Salanova et al 2008;Li et al 2011a, Figure 1). nNOSμ association and regulation of the ryanodine receptor is also seen in the heart (Gonzalez et al 2007). nNOSβ associates with the subsarcolemmal Golgi complex (Percival et al 2010), suggesting the presence of a high concentration of nNOS-derived NO from both nNOSμ and nNOSβ just under the plasma membrane.…”

Targeting ryanodine receptors for anti-arrhythmic therapy

“…Increased S-nitrosylation of RyR2 has been associated with cardiac arrhythmias in a mouse model of Duchenne's muscular dystrophy, and inhibition with S107 (see above) was shown to normalize both RyR1 and RyR2 function and prevent arrhythmias [56,66] . In contrast, Gonzalez et al [17] demonstrated that decreased rather [63] .…”

“…McCauley MD et al Acta Pharmacologica Sinica npg nitrosylation, oxidation, and phosphorylation, which might also increase susceptibility to diastolic Ca 2+ release and arrhythmias [16][17][18][19][20] . Given that there are many excellent reviews on strategies to modify intracellular signaling to reduce RyR2 activation [21,22] , we will restrict the scope of this review mainly to pharmacological strategies to stabilize RyR2 directly to reduce arrhythmic potential.…”

Regulation of protein function by S-nitrosation and S-glutathionylation: processes and targets in cardiovascular pathophysiology

“…Conversely, the decreased S-nitrosation of RyR2 in hearts of nNOS knock-out mice has been suggested to expose its Cys residues to oxidation under oxidative stress, leading to Ca 2+ leakage from SR and arrhythmogenesis (Gonzalez et al, 2007).…”