Gallon, Clare E.

Marston, Steven B.

Messer, Andrew E.

Elsevier - Publisher Connector 

Tuesday, March 3, 2009 501adifferential PKCdelta targeting to multiple myofibrillar proteins other than TnI.
Further elucidation of alternative PKCdelta signaling to the myofilments is on-
going. Our results suggest Src-dependent phosphorylation of PKCdelta consti-
tutes an alternative mechanism that allows for stimulus-specific PKCdelta ezy-
mology and myofibrillar targeting within the myocardium.
2580-Pos Board B550
Protein Kinase A-based Modulation Of Ca2þ Sensitivity In Skinned
Skeletal Muscle Fibers Reconstituted With Cardiac Troponin
Norio Fukuda1, Douchi Matsuba1, Takako Terui1, Jin O-Uchi1,
Hiroyuki Tanaka2, Takao Ojima2, Iwao Ohtsuki1, Shin’ichi Ishiwata3,
Satoshi Kurihara1.
1The Jikei University School of Medicine, Tokyo, Japan, 2Hokkaido
University, Hakodate, Japan, 3Waseda University, Tokyo, Japan.
It is well known that protein kinase A (PKA) decreases Ca2þ sensitivity in car-
diac muscle via phosphorylation of troponin I (TnI). In the present study, we di-
rectly tested whether PKA-based phosphorylation of cardiac TnI universally
modulates Ca2þ sensitivity regardless of the type ofmuscle, by taking advantage
of our Tn exchange technique (Terui et al., J Gen. Physiol. 131;275-283:2008).
Troponins were extracted from porcine ventricular and rabbit fast skeletal mus-
cles (Ca2þ sensitivity: former < latter). Without Tn exchange, PKA decreased
Ca2þ sensitivity in cardiac (porcine ventricular) muscle, associated with en-
hanced phosphorylation of TnI. Reconstitution of cardiac muscle with the skel-
etal Tn complex (sTn) not only increasedCa2þ sensitivity but abolished the PKA
effect, suggesting that phosphorylation of TnI, but not ofmyosin-binding protein
C, is primarily responsible for the PKA-based reduction in Ca2þ sensitivity. Re-
constitution of rabbit psoasmusclewith the cardiac Tn complex (cTn) decreased
Ca2þ sensitivity, as previously reported by us (Terui et al., J Gen. Physiol.
131;275-283:2008). PKA decreased Ca2þ sensitivity in cTn-reconstituted skel-
etal muscle, and subsequent exchange for sTn restored Ca2þ sensitivity to the
original level. A similar result was obtained when skeletal muscle was reconsti-
tutedwith the hybridTn complex (i.e., cTnI-cTnC-sTnT), suggesting that the tro-
ponin I-C complex, but not TnT, is essential for PKA-based modulation of Ca2þ
sensitivity. Thesefindings support the notion that PKA-based phosphorylation of
TnI universally modulates Ca2þ sensitivity regardless of the type of muscle.
2581-Pos Board B551
Cardiac Troponin I Threonine 144 phosphorylation: impact on myofila-
ment function
Gerrie P. Farman, Kittipong Tachampa, Pieter P. de Tombe.
University of Illinois at Chicago MC901, Chicago, IL, USA.
Threonine 144 (T144) in the inhibitory region of cardiac troponin I (cTnI)
is an important site for PKC mediated phosphorylation in the heart. In ad-
dition, presence of this residue is sufficient to impart length dependent ac-
tivation (LDA) properties onto the cardiac sarcomere (Tachampa Circ. Res.,
2008). Here we studied the functional impact of a charge mutation so as to
mimic phosphorylation of this residue (T144E). Wild-type (WT) or cTnI-
T144E containing recombinant troponin (cTn) complexes were exchanged
for endogenous cTn in skinned rat cardiac trabeculae. Force and ATPase
activity were measured as function of [Ca2þ] at short (2.0 mm) or long
(2.2 mm) sarcomere length. T144E induced decreased maximum force de-
velopment (Fmax) and Calcium sensitivity (EC50), increased cross-bridgeWT cTn T144E cTn
EC50 2.0 (mM) 4.05 0.3 5.9 5 0.4
EC50 2.2 (mM) 3.55 0.2 5.2 5 0.3
Fmax 2.0 (mN/mm2) 25.15 4.7 13.05 1.7
Fmax 2.2 (mN/mm2) 37.25 4.7 24.55 2.7
Tension cost 2.0 7.75 0.5 12.05 1.3
Tension cost 2.2 6.35 0.5 9.4 5 1.2
DEC50 0.65 0.1 0.7 5 0.2cycling rate (tension cost) but, in contrast, did not affect LDA (DEC50).
We conclude that T144E affects cross-bridge cycling and recruitment inde-
pendent of sarcomere length.
2582-Pos Board B552
Effects of Cardiac Troponin C Mutants on TnC-TnI interaction and its
modulation by PKA phosphorylation
Dan Wang, F. Steven Korte, Charles Luo, An-yue Tu, Michael Regnier.
University of Washington, Seattle, WA, USA.We are considering several cardiac TnC mutants as potential therapeutic
strategies for cardiomyopathies. To judge their potential to improve in
situ function it is important to understand how these mutants affect TnC-
TnI interaction and its modulation by PKA-mediated phosphorylation of
TnI Ser 23, Ser 24. In this study, we are characterizing two cardiac TnC
mutations, Leu48Glu (L48Q) and Ile61Glu (I61Q), with increased and de-
creased (respectively) Ca2þ binding affinity. In previous studies we showed
these mutations resulted in increased (L48Q) and decreased (I61Q) Ca2þ
sensitivity of steady state force in skinned rat trabeculae. To determine if
these changes in Ca2þ sensitivity were due to altered TnC-TnI interactions
we generated a structural marker by attaching IANBD to Cys84 in the N-
lobe of cTnC. Half-maximal IANBD fluorescence saturation of Ca2þ bind-
ing occurred at pCa7.42 for L48Q cTnC, 7.38 for wild-type (WT) cTnC
and 7.30 for I61Q cTnC. In both the absence and presence of saturating
Ca2þ (0.6 mM TnC) IANBD fluorescence increased with increasing TnI
and saturated at different [TnI] in the order L48Q, WT, I61Q. Fluorescence
half-maximal saturation occurred at 0.26mM(saturating Ca2þ) and 0.25 mM
(no Ca2þ)TnI for L48Q cTnC, 0.78 mM and 0.49mM for WT cTnC , and
1.45 mM and 0.69 mM I61Q cTnC according to the exponential function
fit of the data. However, preliminary experiments suggest that when PKA
phosphorylated [cTnI] was titrated to cTnC, in both the absence and pres-
ence of saturating Ca2þ, IANBD fluorescence enhancement with L48Q may
be impaired. The data thus far suggest that single amino acid mutations that
alter Ca2þ binding affinity of TnC can influence interaction with TnI and
its modulation by PKA mediated phosphorylation of Ser 23, Ser 24.
Supported by HL65497 to MR.
2583-Pos Board B553
Analysis of Cardiac Myofibrillar Troponin I Phosphorylation in Normal
and Failing Human Hearts Using Phos-Tags
Andrew E. Messer, Clare E. Gallon, Steven B. Marston.
Imperial College - NHLI, London, United Kingdom.
Recently, we have used phosphate-affinity SDS-PAGE gels containing Phos-
tag-acrylamide (a phosphate-chelating molecule), to determine the level of car-
diac troponin I (cTnI) phosphorylation in human myofibrillar extracts. The
Phos-tag moiety binds to, and retards, the mobility of phosphoproteins through
the gel and results in the separation of the phosphoprotein bands according to
their phosphorylation level.
Samples from time-courses of in-vitro PKA catalytic subunit-treated recombi-
nant human cTnI andmyofibrillar extracts from non-failing donor, hypertrophic
obstructive cardiomyopathy (HOCM) and end-stage failing human heart tissue
were analysed by phosphate-affinity SDS-PAGE. Separate gel bands corre-
sponding to 1P, 2P, 3P, 4P and 5P cTnI were observed for the PKA-treated re-
combinant cTnI. Western blotting probed with the anti-cTnI antibody 14G5
and several different site specific phospho-cTnI antibodies demonstrated that
all five of these phospho-species bound to a Ser24P-specific antibody, while
a Thr144P-specific antibody only reacted with the 3P, 4P and 5P phospho-
species of cTnI.
We observed 3 phospho-species of cTnI in the human heart tissue extracts,
which correspond to 0P, 1P and 2P cTnI. Ratios of 0P cTnI were signifi-
cantly higher in failing and HOCM (both 6354%) compared to donor
(852%) while ratios of 2P were significantly lower (failing ¼ 652%,
HOCM ¼ 852%, donor 7356%). Western blots demonstrated that in hu-
man heart cTnI phosphorylation of Ser23/24 was mainly present in the 2P
species (with a very small proportion in the 1P species) and that there was
no phosphorylation at Thr144. Calculated levels of total cTnI phosphoryla-
tion in both HOCM (0.3750.03, n¼50, p<0.0001) and failing heart
(0.3850.03, n¼24, p<0.0001) were significantly reduced from levels in do-
nor heart (1.6550.04, n¼38) and were comparable to previously deter-
mined measurements obtained from Pro-Q Diamond phosphoprotein gel
staining.
2584-Pos Board B554
Effect of Troponin I Ser23/24 Bis-Phosphorylation on Ca2þ-Sensitivity is
Dependent on PKA Phosphorylation of Other Contractile Proteins
Viola Kooij, Jolanda van der Velden, Ger J.M. Stienen.
Laboratory for Physiology, Institute for Cardiovascular Research, VU
University Medical Center, Amsterdam, Netherlands.
Upon b-adrenergic stimulation, protein kinase A (PKA) enhances cardiac Tro-
ponin I (cTnI) phosphorylation at ser23/24. PKA treatment leads to a decrease
in myofilament Ca2þ-sensitivity. However, the specific effect of PKA-mediated
phosphorylation of cTnI in human myocardium is unclear since PKA phosphor-
ylates a broader set of contractile proteins, in particular myosin binding protein
C (cMyBP-C).


Analysis of Cardiac Myofibrillar Troponin I Phosphorylation in Normal and Failing Human Hearts Using Phos-Tags 

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Analysis of Cardiac Myofibrillar Troponin I Phosphorylation in Normal and Failing Human Hearts Using Phos-Tags

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