The cardiac neuronal nitric-oxide synthase (nNOS) has been described as a
modulator of cardiac contractility. We have demonstrated previously that
isoform 4b of the sarcolemmal calcium pump (PMCA4b) binds to nNOS in the heart
and that this complex regulates β-adrenergic signal transmission
in
vivo. Here, we investigated whether the nNOS-PMCA4b complex serves as a
specific signaling modulator in the heart. PMCA4b transgenic mice (PMCA4b-TG)
showed a significant reduction in nNOS and total NOS activities as well as in
cGMP levels in the heart compared with their wild type (WT) littermates. In
contrast, PMCA4b-TG hearts showed an elevation in cAMP levels compared with
the WT. Adult cardiomyocytes isolated from PMCA4b-TG mice demonstrated a
3-fold increase in Ser
16 phospholamban (PLB) phosphorylation as
well as Ser
22 and Ser
23 cardiac troponin I (cTnI)
phosphorylation at base line compared with the WT. In addition, the relative
induction of PLB phosphorylation and cTnI phosphorylation following
isoproterenol treatment was severely reduced in PMCA4b-TG myocytes, explaining
the blunted physiological response to the β-adrenergic stimulation. In
keeping with the data from the transgenic animals, neonatal rat cardiomyocytes
overexpressing PMCA4b showed a significant reduction in nitric oxide and cGMP
levels. This was accompanied by an increase in cAMP levels, which led to an
increase in both PLB and cTnI phosphorylation at base line. Elevated cAMP
levels were likely due to the modulation of cardiac phosphodiesterase, which
determined the balance between cGMP and cAMP following PMCA4b overexpression.
In conclusion, these results showed that the nNOS-PMCA4b complex regulates
contractility via cAMP and phosphorylation of both PLB and cTnI.Neuronal nitric-oxide synthase
(nNOS)
5 is involved in
a number of key processes in cardiomyocytes including calcium cycling
(
1), the β-adrenergic
contractile response (
2,
3), post-infarct left
ventricular remodeling (
4), and
the regulation of redox equilibrium
(
5). Moreover, a polymorphism
in an nNOS-interacting protein, CAPON, has been found to form a quantitative
trait for the determination of the QT interval in humans
(
6), whereas a mutation in
α1-syntrophin (SNTA1), another interacting partner of nNOS, has been
associated with long QT syndrome
(
7). The signaling events
downstream of the nNOS-CAPON
(
8) and nNOS-SNTA1
(
7) complexes, which are
responsible for mediating cardiac repolarization and sodium current
respectively, have been elucidated. The nNOS-containing protein complex is
therefore of immediate relevance to human pathology.In recent years, we have shown that the sarcolemmal calcium pump, which
ejects calcium to the extracellular compartment (reviewed in Refs.
9 and
10), is an important molecule
involved in signal regulation and transmission in the heart
(
11). We have demonstrated
that isoform 4b of the sarcolemmal calcium pump (also known as PMCA4b for
plasma
membrane
calcium/calmodulin-dependent
ATPase
4b) modulates signaling through a tight molecular
interaction with nNOS, leading to the modulation of β-adrenergic
responsiveness in the heart
(
12). However, the events
following signaling through the PMCA4b-nNOS complex remain unknown.In myocardial cells, nNOS has been localized to the sarcolemma
(
13), sarcoplasmic reticulum
(
2), and mitochondria
(
14), and translocation
between compartments has been demonstrated
(
15). It has been speculated
that these various localizations provide specificity to NO signaling, but the
exact mechanisms have yet to be elucidated. In this study, we show a mechanism
by which one fraction of nNOS serves highly specific functions through binding
to PMCA4b. As PMCA4b is confined to the sarcolemma and is a calcium pump, it
is the first identified protein to fulfill these aggregate functions. 1) It
acts as an anchoring protein; 2) it regulates nNOS activity; and 3) it
modulates a process at the plasma membrane,
i.e. β-adrenergic
signaling.
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