Spontaneous calcium waves in cardiac myocytes are caused by diastolic sarcoplasmic reticulum release (SR Ca(2+) 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 Ca(2+) waves in intact cardiomyocytes. Both SCaWs and SR Ca(2+) 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 Ca(2+) 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 Ca(2+) 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.