Abstract
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.
Highlights
In the heart, increases in the inotropic, chronotropic, and lusitropic states are primarily brought about by the stimulation of b-adrenergic receptors (b-ARs) [1]
We find that 1) Inhibition of nitric oxide synthase (NOS) attenuates SR Ca2+ waves (SCaW) formation as a result of b-AR stimulation in isolated rabbit myocytes; 2) the increased SCaWs are associated with an increase in RyR-dependent diastolic sarcoplasmic reticulum (SR) Ca2+ release (SR Ca2+ leak) and this leak is dependent upon Akt-mediated NOS1 activity in cells from rabbit and NOS1 knockout (NOS12/2) mice; and 3) NO directly affects calmodulin-dependent protein kinase II (CaMKII) to sustain its activity leading to the increase in SR Ca2+ leak
We previously demonstrated that the CaMKII-dependent increased SR Ca2+ leak contributes to increased incidence of arrhythmogenic spontaneous SR Ca2+ waves (SCaW) in both healthy myocytes and those isolated from failing hearts [5,7]
Summary
Increases in the inotropic, chronotropic, and lusitropic states are primarily brought about by the stimulation of b-adrenergic receptors (b-ARs) [1] Upon their stimulation, signaling cascades are initiated within the myocyte that alter the way Ca2+ is handled and stored by the various proteins of the excitation-contraction coupling (ECC) machinery [2]. A new paradigm involving the regulation of ECC by reactive oxygen species (ROS) and reactive nitrogen species (RNS), such as nitric oxide (NO) and peroxynitrite (ONOO2), has emerged. Ranging from acute to long-term regulation, the ROS/RNS axis has been shown to play an important role in controlling Ca2+ handling during the fight or flight reaction and the chronic pathological condition of heart failure (HF) in both humans and animal models of heart disease [4]. The extent to which these effects are related to arrhythmogenesis as a cause of or as a response to heart disease is unknown
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