Research Report 2000 - MDC

Intracellular Signaling
in the Myocardium
Ernst-Georg Krause
The action of the heart as a pump
depends on the coordination of
electrical, mechanical and metabolic
activity of its cells. The prime
regulator of these activities appears to
be the intracellular calcium
concentration which is subject to
nerval and/or humoral mechanisms.
These involve second-messenger
mediated phosphorylation of specific
proteins which are known to directly
influence calcium transport through
the sarcolemma and the vesicles of the
sarcoplasmic reticulum (SR) in
cardiac as well as smooth muscle
cells.
Phospholamban, a pentameric protein
complex composed of identical
subunits, is thought to be the key
phosphoprotein in regulating calciumpump
(SERCA) activity of SR
vesicles, thereby controlling the
relaxation process. In the
dephosphorylated state,
phospholamban acts as an inhibitor of
SERCA, but this inhibition is
abolished with increasing
phosphorylation. Interestingly, both
cAMP-dependent protein kinase
(PKA) and Ca 2+ /calmodulin-dependent
protein kinase (CaM Kinase)
specifically phosphorylate each
phospholamban monomer at serine 16
and threonine 17 residues,
respectively. The functional
significance of this dual-site
phosphorylation is not yet known.
In the failing human heart, there is
increasing evidence that inotropic
stimulation by cAMP-dependent, as
well as cAMP-independent,
mechanisms results in inadequate
diastolic relaxation with consequences
for pumping efficiency. These
alterations have been suggested to be
due to altered intracellular calcium
handling. Indeed, a reduced amount of
SERCA and an impairment in
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phospholamban phosphorylation may
lead to a slower diastolic calcium
decay. Thus, the function of the SR
calcium pump may be modulated at
two levels by I) changes in the
expression of SERCA, phospholamban
and their respective protein kinases
and II) alterations of the regulation of
calcium transport at the level of SR
vesicles.
The research group “Intracellular
Signalling in the Myocardium” is
engaged in basic research that is
mainly focused on molecular
mechanisms related to protein
phosphorylation and dephosphorylation
and to the short-term regulation of
contraction and relaxation of the
normal and diseased myocardium, as
well as long-term processes with
regard to Ca 2+ homeostasis, growth,
and differentiation of cardiac muscle
cells.
Intracellular signaling in the
myocardium
S. Bartel and P. Karczewski in
cooperation with R. H. G.
Schwinger, University of Köln;
M. Kuschel, P. Xiao, E. Lakatta,
NIH, Baltimore, USA; A. Kaumann,
The Babraham Institute,
Cambridge, U. K.; P. Molenaar,
University of Melbourne, Australia
We have established that the
phosphorylation of phospholamban by
PKA at serine-16 prevails over the
phosphorylation at threonine-17 and
occurs in parallel with the monitored
acceleration of relaxation after
β-adrenergic stimulation. Interestingly
the detectable threonine-17
phosphorylation was inhibited in the
presence of the L-type Ca 2+ channel
blocker, verapamil. These data favor a
spatial Ca 2+ signal for activation of
CaM kinase activity near the L-type
Ca 2+ channel. Indeed, L-type Ca 2+
channel activators, like BayK8644 and
BayY5959, induce phosphorylation of
threonin-17 in the absence of
adrenergic stimulation. A dissociation
of β2-adrenoreceptor signaling from
the cAMP regulatory systems has
been evaluated for phosphorylation of
cytoplasmic proteins, like troponin I
and C protein, but not for
phosphorylation of the L-type Ca 2+
channel in the canine heart.
Our data on the short-term regulation
by catecholamines in nonfailing and
failing human hearts clearly shows
that, in the post-adenylyl cyclase
signaling pathway of the failing heart,
the phosphorylation of phospholamban
is impaired, followed by disturbances
in Ca 2+ sequestration, thereby
prolonging the diastolic phase of each
cardiac cycle. Elucidating the
selective activation of β2-adrenergic
receptors in the human heart allowed
us to clearly show that β2-adrenoceptor
activation is mediated by activation of
PKA and phosphorylation of
phospholamban at serine-16 and
threonine-17, as well as of troponin I
and C protein. These data are relevant
to strategies for therapeutic
intervention(s) in patients with endstage
heart failure.
CaMKII in heart function and
cardiac remodeling
P. Karczewski and B. Hoch in
cooperation with J. Bohlender,
Franz Volhard Clinic, Humboldt
University Berlin; A. Remppis,
University of Lübeck; R. Meyer,
German Heart Institute Berlin;
S. Hatem, INSERM Paris
The multigene family of
Ca 2+ /calmodulin-dependent protein
kinases II (CaMKII), as a universal
mediator in Ca 2+ signaling, is involved
in many of the functions of eukaryotic
cells. CaMKII is a multimer
consisting of 8 to 12 subunits encoded
by four different genes (α,β,δ,γ). As a
major regulator of Ca 2+ homeostasis,
CaMKII is essential for heart
function. Despite its importance, little
is known about the isoforms of
CaMKII expressed in the heart and
their specific function in the normal
and diseased myocardium. In
extension of our previous work, we
have characterized four isoforms of
the δ-class, the dominant cardiac
CaMKII, during rat heart
development. Our data have
established that the isoform δ 3 is
characteristic for the adult
myocardium. Furthermore, δ 4 so far
characterized as the skeletal muscle
isoform, is typically expressed in the
embryonic and neonatal rat heart and
becomes down-regulated during
postnatal development. In
hypertensive rat strains, which
develop a compensated cardiac
hypertrophy, δ 4 is re-expressed
whereas the δ 3 transcript levels fall.
These alterations are accompanied by
changes in the δ-CaMKII protein
content in membrane vesicles of the
SR.