Caveolin3eNOS Interaction in Cardiac Myocytes

To explore the dual roles of eNOS caveolar targeting in cardiac myocytes (e. g., compartmentation to facilitate activation upon agonist stimulation and inhibition of the eNOS catalytic activity in basal conditions), we refer to the paradigm of the muscarinic cholinergic NO-mediated regulation of heart rate. eNOS activated by muscarinic cholinergic agonists contributes to the so-called accentuated antagonism - that is, the ability of muscarinic cholinergic stimulation to attenuate b-adrenergic signaling in various models [4]. In order to examine the impact of eNOS compartmentation in caveolae of cardiac myocytes, we first used neonatal myocytes isolated from eNOS-deficient mouse, that were transfected with cDNA constructs encoding either the wild-type eNOS or a myristoylation-deficient eNOS mutant [23] (Fig. 11.6). These knock-in experiments provided us with myocytes expressing eNOS protein in either the caveolae or in the cytosolic compartment. In myocytes expressing wild-type eNOS, a muscarinic cholinergic agonist dramatically reduced the spontaneous heart beat rate (in a cGMP-dependent manner), whereas in the myr-eNOS myocytes the agonist failed to exert its negative chronotropic effect (Fig. 11.6). The second arm of the caveolae regulation (i.e., that cav-eolin-3 exerts inhibitory effects on myocyte eNOS) was demonstrated by documenting the blockade of muscarinic cholinergic agonist-induced negative chronotropic effects in cardiac myocytes loaded with caveolin-3 scaffolding domain-derived peptides.

Interestingly, a large fraction of sarcolemmal m2 muscarinic Cholinergic receptor (mAchR) was found to be targeted to cardiac myocyte caveolae upon agonist stimulation [60], thereby reinforcing the role of compartmentation in regulating NO signaling in myocyte. In a further study, we examined the impact of this translocation on the mAchR internalization process and the consecutive alteration in downstream NO signaling [22]. It was found that mAChR stimulation led to the

Fig. 11.6 The key roles of caveolin-3 and caveolae in the muscarinic cholinergic (mAchR) regulation of heart rate. Top left: Under basal conditions, caveolin-3 maintains eNOS in its inactivated state and thereby limits the negative chronotropic effects of NO. Top right: The m2 mAchR receptor targeting to caveolae upon agonist stimulation leads to the activation of eNOS through a local increase in intracellular calcium (the sarcoplasmic reticulum is in close vicinity to caveolae) and the consecutive disruption of the caveolin-3-eNOS heterocomplex: the myocyte beating rate is slowed (see representation of heart-beating chart recording).

Fig. 11.6 The key roles of caveolin-3 and caveolae in the muscarinic cholinergic (mAchR) regulation of heart rate. Top left: Under basal conditions, caveolin-3 maintains eNOS in its inactivated state and thereby limits the negative chronotropic effects of NO. Top right: The m2 mAchR receptor targeting to caveolae upon agonist stimulation leads to the activation of eNOS through a local increase in intracellular calcium (the sarcoplasmic reticulum is in close vicinity to caveolae) and the consecutive disruption of the caveolin-3-eNOS heterocomplex: the myocyte beating rate is slowed (see representation of heart-beating chart recording).

Bottom: Proof of principle that eNOS com-partmentation in caveolae is critical for mAchR signaling in the heart [23]. Left: In cardiac myocytes expressing only recombinant myristoylation-deficient eNOS in the cy-tosol, the coupling between agonist-bound m2 mAchR receptor and the mutant eNOS is lost, and no effect on the myocyte beating rate can be observed. Right: When caveolin-3-derived peptides (corresponding to the CSD sequence) are introduced in myocytes, eNOS is inactivated by this excess inhibitory clamping, preventing activation of the m2mAchR signaling cascade and the associated negative chronotropism.

sequestration of mAchRs through caveolae fission through a dynamin-dependent GTP-driven process. Repeated stimulations of mAchRs led to a progressive increase in mAchR sequestration (via the detachment of caveolae from myocyte sar-colemma) and a concurrent stabilization of the inhibitory eNOS-caveolin complex. These findings suggested that caveolae fission may contribute to G-protein-cou-pled receptor desensitization and thereby terminate the (initially facilitated) NO signaling cascade.

2461 11 Caveolae and the Endothelial Nitric Oxide Synthase 11.8

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