text:
Effects of chronic atrial fibrillation on active and passive force generation in human atrial myofibrils.
ABSTRACT Chronic atrial fibrillation (cAF) is associated with atrial contractile dysfunction. Sarcomere remodeling may contribute to this contractile disorder.
Here, we use single atrial myofibrils and fast solution switching techniques to directly investigate the impact of cAF on myofilament mechanical function eliminating changes induced by the arrhythmia in atrial myocytes membranes and extracellular components. Remodeling of sarcomere proteins potentially related to the observed mechanical changes is also investigated.
Myofibrils were isolated from atrial samples of 15 patients in sinus rhythm and 16 patients with cAF. Active tension changes following fast increase and decrease in [Ca(2+)] and the sarcomere length-passive tension relation were determined in the 2 groups of myofibrils. Compared to sinus rhythm myofibrils, cAF myofibrils showed (1) a reduction in maximum tension and in the rates of tension activation and relaxation; (2) an increase in myofilament Ca(2+) sensitivity; (3) a reduction in myofibril passive tension. The slow beta-myosin heavy chain isoform and the more compliant titin isoform N2BA were up regulated in cAF myofibrils. Phosphorylation of multiple myofilament proteins was increased in cAF as compared to sinus rhythm atrial myocardium.
Alterations in active and passive tension generation at the sarcomere level, explained by translational and post-translational changes of multiple myofilament proteins, are part of the contractile dysfunction of human cAF and may contribute to the self-perpetuation of the arrhythmia and the development of atrial dilatation.
- [Show abstract] [Hide abstract]
ABSTRACT: During atrial fibrillation (AF) intracellular Ca(2+) signaling in atrial myocytes changes substantially. This 'remodeled' intracellular Ca(2+) homeostasis plays an important role in the development of the contractile dysfunction and the changes in atrial electrophysiology (contractile and electrical remodeling) that are characteristic of AF. Recent studies also show that unstable intracellular Ca(2+) signaling (i.e. increased Ca(2+) sparks and Ca(2+) waves) is present in atrial myocytes from AF patients and that it might contribute to cellular arrhythmogenic mechanisms that help maintain the arrhythmia. It is currently not well understood how and when unstable Ca(2+) signaling develops during the progression of AF, or if, in cases of structural heart disease, it even precedes the onset of AF. Current work therefore in particular aims to elucidate the molecular and sub-cellular mechanisms underlying the arrhythmogenic intracellular Ca(2+) signaling instability in AF. As treatment of AF remains difficult, the identification of novel targets for counteracting or preventing arrhythmogenic Ca(2+) signaling is an important part of AF research. It is therefore important to recognize which phase of AF is addressed in a specific research (and ultimately treatment) approach. Here we review and critique the distinct alterations in intracellular Ca(2+) signaling during the progression of AF from initial intracellular Ca(2+) overload to the remodeling process. We address Ca(2+) signaling after cardioversion of the arrhythmia and its potential role in the recurrence of AF. We propose that altered Ca(2+) signaling during AF progression consists of three phases 1.) Ca(2+) Overload, 2.) Remodeling, 3.) Steady State. Similarly, after AF termination three distinct phases of 'recovery' of intracellular Ca(2+) handling occur. 4.) Calcium Unloading, 5.) Reverse Remodeling and 6.) Full Recovery. While there is evidence that unstable Ca(2+) signaling is part of phases 1, 3 and 4, phase 2 (remodeling) appears to have a more stabilizing function on Ca(2+) signaling ('Ca(2+) silencing'). This has important implications for the timing and type of pharmacological intervention, especially for new compounds aimed at intracellular 'Ca(2+) stabilization'.Journal of Molecular and Cellular Cardiology 01/2013; · 5.15 Impact Factor - Francesca Stillitano, Giuseppe Lonardo, Gabriele Giunti, Martina Del Lungo, Raffaele Coppini, Valentina Spinelli, Laura Sartiani, Corrado Poggesi, Alessandro Mugelli, Elisabetta Cerbai[Show abstract] [Hide abstract]
ABSTRACT: Despite the evidence that the hyperpolarization-activated current (If ) is highly modulated in human cardiomyopathies, no definite data exist in chronic atrial fibrillation (cAF). We investigated the expression, function, and modulation of If in human cAF. Right atrial samples were obtained from sinus rhythm (SR, n = 49) or cAF (duration >1 year, n = 31) patients undergoing corrective cardiac surgery. Among f-channel isoforms expressed in the human atrium (HCN1, 2 and 4), HCN4 mRNA levels measured by RT-PCR were significantly reduced. However, protein expression was preserved in cAF compared to SR (+85% for HCN4); concurrently, miR-1 expression was significantly reduced. In patch-clamped atrial myocytes, current-specific conductance (gf ) was significantly increased in cAF at voltages around the threshold for If activation (-60 to -80 mV); accordingly, a 10-mV rightward shift of the activation curve occurred (P < 0.01). β-Adrenergic and 5-HT4 receptor stimulation exerted similar effects on If in cAF and SR cells, while the ANP-mediated effect was significantly reduced (P < 0.02), suggesting downregulation of natriuretic peptide signaling. In human cAF modifications in transcriptional and posttranscriptional mechanisms of HCN channels occur, associated with a slight yet significant gain-of-function of If , which may contribute to enhanced atrial ectopy.Journal of Cardiovascular Electrophysiology 06/2013; · 3.48 Impact Factor - SourceAvailable from: Margus EimreMart Roosimaa, Taavi Põdramägi, Lumme Kadaja, Arno Ruusalepp, Kalju Paju, Raivo Puhke, Margus Eimre, Ehte Orlova, Andres Piirsoo, Nadežda Peet, Frank N Gellerich, Enn Seppet[Show abstract] [Hide abstract]
ABSTRACT: Cardiac energy metabolism with emphasis on mitochondria was addressed in atrial tissue from patients with overload-induced atrial dilation. Structural remodelling of dilated (D) atria manifested as intracellular accumulation of fibrillar aggregates, lipofuscin, autophagy and signs of myolysis. Despite impaired complex I dependent respiration and increased diffusion restriction for ADP, no changes regarding adenylate and creatine kinase occurred. We observed 7-fold overexpression of HK2 gene in D atria with concomitant 2-fold greater activation of mitochondrial oxygen consumption by glucose, which might represent an adaption to increased energy requirements and impaired mitochondrial function by effectively joining glycolysis and oxidative phosphorylation.Mitochondrion 12/2012; · 3.52 Impact Factor
Page 1
Elisabetta Cerbai, Jolanda van der Velden and Corrado Poggesi
Toniolo, Maurice Drost, Giulia Mearini, Lucie Carrier, Alessandra Rossi, Alessandro Mugelli,
Alexandra Belus, Nicoletta Piroddi, Cecilia Ferrantini, Chiara Tesi, Olivier Cazorla, Luana
Atrial Myofibrils
Effects of Chronic Atrial Fibrillation on Active and Passive Force Generation in Human
Print ISSN: 0009-7330. Online ISSN: 1524-4571
Copyright © 2010 American Heart Association, Inc. All rights reserved.
is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Circulation Research
doi: 10.1161/CIRCRESAHA.110.220699
2010;107:144-152; originally published online May 13, 2010;
Circ Res.
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Page 2
Effects of Chronic Atrial Fibrillation on Active and Passive
Force Generation in Human Atrial Myofibrils
Alexandra Belus, Nicoletta Piroddi, Cecilia Ferrantini, Chiara Tesi, Olivier Cazorla, Luana Toniolo,
Maurice Drost, Giulia Mearini, Lucie Carrier, Alessandra Rossi, Alessandro Mugelli, Elisabetta Cerbai,
Jolanda van der Velden, Corrado Poggesi
Rationale: Chronic atrial fibrillation (cAF) is associated with atrial contractile dysfunction. Sarcomere remodeling
may contribute to this contractile disorder.
Objective: Here, we use single atrial myofibrils and fast solution switching techniques to directly investigate the
impact of cAF on myofilament mechanical function eliminating changes induced by the arrhythmia in atrial
myocytes membranes and extracellular components. Remodeling of sarcomere proteins potentially related to the
observed mechanical changes is also investigated.
Methods and Results: Myofibrils were isolated from atrial samples of 15 patients in sinus rhythm and 16 patients
with cAF. Active tension changes following fast increase and decrease in [Ca2?] and the sarcomere length–passive
tension relation were determined in the 2 groups of myofibrils. Compared to sinus rhythm myofibrils, cAF
myofibrils showed (1) a reduction in maximum tension and in the rates of tension activation and relaxation; (2)
an increase in myofilament Ca2?sensitivity; (3) a reduction in myofibril passive tension. The slow ?-myosin heavy
chain isoform and the more compliant titin isoform N2BA were up regulated in cAF myofibrils. Phosphorylation of
multiple myofilament proteins was increased in cAF as compared to sinus rhythm atrial myocardium.
Conclusions: Alterations in active and passive tension generation at the sarcomere level, explained by translational
and post-translational changes of multiple myofilament proteins, are part of the contractile dysfunction of human
cAF and may contribute to the self-perpetuation of the arrhythmia and the development of atrial dilatation. (Circ
Res. 2010;107:144-152.)
Key Words: myosin ? Titin ? cardiac MyBP-C ? cardiac troponin ? atrial light chain-2
pronounced atrial contractile dysfunction and self-
perpetuation of the arrhythmia (reviewed elsewhere1). The
persistence of atrial contractile disorder after cardioversion to
sinus rhythm (SR) in patients with cAF has long been
reported2and may have dramatic consequences as it favors
thromboembolic events.3In spite of its clinical significance,
the exact mechanism of cAF-induced contractile dysfunction
is poorly understood.
Most investigations into the contractile dysfunction of cAF
remodeled atria have concentrated on structural alterations4,5
or disorders of excitation–contraction coupling.6,7Recent
investigations high-light the role of sarcomere protein mod-
hronic atrial fibrillation (cAF) is characterized by elec-
tric, structural, and contractile remodeling that leads to
ifications in human cAF-associated contractile dysfunc-
tion.8–11However, the functional impact of myofilament
protein changes in cAF is weakly documented because it is
difficult to obtain consistent measurements of functionally
relevant parameters on human cardiac preparations.
Studies on single myofibrils can significantly document
changes in the mechanical performance of human cardiac
sarcomeres because these preparations can be obtained in
large amounts from very small cardiac samples.12,13Single
myofibrils are the smallest units of the contractile apparatus
that retain the organized myofilament lattice and its entire
ensemble of associated proteins. Mechanical measurements
of myofibril force combined with rapid perfusion switching
techniques have been developed recently to investigate fast
Original received December 15, 2008; resubmission received March 22, 2010; revised resubmission received April 16, 2010; accepted May 5, 2010.
In April 2010, the average time from submission to first decision for all original research papers submitted to Circulation Research was 15.2 days.
From the Center of Molecular Medicine (CIMMBA) (A.B., N.P., C.F., C.T., A.M., E.C., C.P.) and Departments of Physiology (A.B., N.P., C.F., C.T.,
C.P.) and Pharmacology (A.M., E.C.), University of Florence, Italy; INSERM U-637 (O.C.), CHU Arnaud de Villeneuve, Montpellier, France; Inserm,
U974, Institut de Myologie (L.C.), and Université Pierre et Marie Curie-Paris 6, UMR-S974, CNRS, UMR7215, Institut de Myologie, IFR14 (L.C.), Paris,
France; Department of Anatomy and Physiology (L.T.), University of Padua, Italy; Institute for Cardiovascular Research, (M.D., J.v.d.V.), VU University
Medical Center, Amsterdam, The Netherlands; Institute of Experimental and Clinical Pharmacology and Toxicology (G.M., L.C.), University Medical
Center Hamburg-Eppendorf, Hamburg, Germany; and CardioSurgery Division (A.R.), Careggi Hospital, Florence, Italy.
This manuscript was sent to Masao Endoh, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Correspondence to Corrado Poggesi, Department of Physiology, University of Florence, Viale Morgagni, 63, 50134 Florence, Italy. E-mail
© 2010 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.110.220699
144 at UNIV DI FIRENZE on November 16, 2012http://circres.ahajournals.org/Downloaded from
Page 3
kinetic events related to cross-bridge action and regulation in
human cardiac myofibrils.12–14
In this study, sarcomere mechanisms underlying passive and
active tension generation and relaxation of atrial myocardium
are dissected and compared in myofibrils from surgical samples
of cAF and control (SR) patients. In the same samples, we also
determined the myosin heavy chain (MHC) and titin isoform
expression and the phosphorylation level of several myofilament
proteins. The results show that myofilament proteins play a
direct role in the altered atrial mechanics associated with cAF
suggesting that sarcomere remodeling also contribute to the
progressive nature of the arrhythmia.
The investigation conforms with the principles outlined in the Declara-
tion of Helsinki and is approved by the local Ethics Committee (no.
2006/0023797). Samples of atrial appendages were obtained following
informed consent from 15 SR patients (67?2 years; 7?;) and 16 cAF
patients (66?2 years; 9?;) undergoing open heart surgery. In the cAF
patients, established atrial fibrillation (AF) was documented for at least
8 months. No significant difference was present in ejection fraction and
left atrial diameter between cAF and SR patients. Details for each
patient group are given in Online Table I, available in the Online Data
Supplement at http://circres.ahajournals.org.
Mechanical Measurements in Myofibrils
Fresh surgical samples were collected in a cold sterile saline solution
for myofibril isolation according to previously described methods.12
Techniques for mechanical measurements in human cardiac myofi-
brils were as previously described.12–14Briefly, myofibrils were
transferred to a temperature controlled chamber filled with relaxing
solution (pCa8, 15°C). The selected myofibril was horizontally
mounted (initial sarcomere length 2.2 to 2.3 ?m) between a
cantilever force probe and a glass needle mounted on the lever arm
of a length control motor. Myofibrils were activated and relaxed by
rapid solution switching between 2 continuous streams of solutions.
Ionic strength of the experimental solutions was 200 mmol/L and pH
7.0. All solutions contained a MgATP-regenerating system and a
cocktail of protease inhibitors.12,13To avoid the effects on myofibril
force and force kinetics of variable levels of contaminant inorganic
phosphate (Pi) in the solutions, [Pi] was reduced to less than
5 ?mol/L using a Piscavenging system.12,13
Sarcomeric Protein Analysis
Myosin heavy chain isoforms
Polyacrylamide gel electrophoresis was used to determine MHC iso-
form composition (MHC-? and MHC-?) after denaturation in sodium
dodecyl sulfate (SDS-PAGE) following a procedure described by
stained with Coomassie Blue for quantitative analysis. Each band
(MHC-? or MHC-?) was expressed as percent of the total MHC.
Titin Isoforms
Analysis of titin content in SR and cAF samples was adapted from
Cazorla et al.16Titin content was analyzed with SDS-PAGE (2.5% to
7% acrylamide gradient gels) and stained with 0.1% Coomassie
Blue. The integrated optical density of MHC and titin peaks (both
N2B and N2BA isoforms) were determined on wet gel images to
measure both the total amount of titin relative to MHC and the ratio
N2BA:N2B. For each sample a range of loadings was electropho-
resed on the same gel. The optical density of titin and MHC peaks
were determined and plotted against their loading volume. The linear
part of this relation was fitted with a line regression and the slope
determined. The slope ratio of titin:MHC was taken as relative
amount of titin in the samples and used to determine the N2BA:N2B
ratio. Western blotting was performed with the antibodies Z1/Z2
(kindly provided by Dr S. Labeit, University of Mannheim, Ger-
many) to label the N-terminal of titin.
ProQ Phosphostaining
Atrial tissue samples from 8 SR and 8 cAF patients were TCA
(trichloroacetic acid)-treated as described previously to fix phosphor-
ylation status of myofilament proteins.17Phosphorylation status of
myofilament proteins separated on a gradient gel was determined
using Pro-Q Diamond phosphostaining.17All protein signals were
within the linear range and were corrected for protein content
determined by SYPRO Ruby staining.
Western Immunoblotting
Gel electrophoresis and Western immunoblotting were performed as
described previously10,18to analyze content and phosphorylation of
Active Tension Generation and Relaxation
Thin bundles of myofibrils obtained from atrial samples of 7 SR
and 8 cAF patients were maximally calcium activated at pCa 4.5
and relaxed at pCa 8.0 by rapid solution change. Representative
force responses of SR and cAF myofibrils to full activation-
relaxation cycles at 15°C are shown in Figure 1A. Average data
for maximal isometric tension (P0) and kinetic parameters of
maximal tension generation (kACT, rate constant of tension
generation following Ca2?activation; and kTR, rate constant of
tension redevelopment following release–restretch applied to the
myofibril under steady-state conditions of force generation) for
both SR and cAF myofibrils are in the Table.
P0in SR myofibrils was close to that previously reported for
control human atrial myofibrils.12P0was significantly depressed
(?30%) in cAF compared to SR myofibrils. Both kTRand kACT
were markedly slower (?50%) in cAF than in SR myofibrils
(Table). The difference is evident in Figure 1B (left) where
tension activation traces of SR and cAF myofibrils are normal-
ized to maximal tension and then superimposed.
Non-standard Abbreviations and Acronyms
cMyBP-C cardiac myosin binding protein-C
cardiac troponin
rate constant of tension generation following rapid Ca2?
rate constant of tension generation following rapid mechani-
cal perturbation
rate constants of the "slow" and "fast" phases of tension
decline following rapid Ca2?removal
myosin heavy chain
Hill coefficient, slope of the pCa–tension relationship
maximal Ca2?-activated tension
pCa at which active tension is 50% of P0
protein kinase
sinus rhythm
slow skeletal troponin I
atrial fibrillation
chronic atrial fibrillation
Belus et alMyofibril Mechanics in Chronic Atrial Fibrillation
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As shown in Figure 1A, for both SR and cAF myofibrils,
active tension fully relaxed on step reduction of [Ca2?] below
the contraction threshold (pCa 8.0). The time course of full
tension relaxation is shown in Figure 1B (right) where the
normalized relaxation transients are superimposed on a faster
time scale. In agreement with previous observations on
skeletal and cardiac myofibrils,19relaxation of both SR and
cAF myofibrils is biphasic starting with a slow linear force
decay (rate constant, slow kREL) followed by a fast exponen-
tial relaxation phase (rate constant, fast kREL). It has been
shown that the slow force decay lasts as long as the
sarcomeres remain isometric, whereas the fast exponential
phase follows the "give" of one or more sarcomeres and is
dominated by intersarcomere dynamics.19Average values for
the rates of both relaxation phases (Table) indicate that
relaxation kinetics were slower or tended to be slower in cAF
than in SR myofibrils. Fast kRELwas reduced (by 35%
P?0.05) and slow kRELtended to decrease (by 25% P?0.1)
in cAF versus SR myofibrils.
MHC Isoform Expression
Slower force kinetics at maximal activation in the cAF myofi-
brils are consistent with previously described8,9,20cAF induced
changes in the motor protein. Of the 2 MHC isoforms expressed
in human cardiac muscle MHC-? is associated with higher
MHC-?. Both SR and cAF myofibrils coexpressed MHC-? and
MHC-? isoforms (Figure 2A). As expected from previous
characterization of human atrial tissue21MHC-? represented the
largest fraction (?50%) of the total amount of MHC. The
Figure 1. Force activation and relax-
ation of SR and cAF myofibrils. A,
Representative examples of maximum
tension activation and full relaxation in
response to sudden pCa changes by
fast solution switching in SR (left) and
cAF (right) myofibrils. Lower traces
show fast release–restretch protocol for
kTRregistration. B, Time courses of ten-
sion activation (left, kACT) and relaxation
(right, slow and fast kREL) of SR and cAF
myofibrils superimposed after normaliza-
tion to maximal tension (same traces as
in A on faster time base).
Generation and Relaxation in Myofibrils from Seven SR and
Eight cAF Patient Samples
Mean Data for Passive Tension and Active Tension
P Student
t Test
Resting tension, mN ? mm?2
Active tension generation
P0, mN ? mm?2
kACT, sec?1
kTR, sec?1
Active tension relaxation
Slow phase duration, ms
Slow kREL, sec?1
Fast kREL, sec?1
Resting sarcomere length, 2.25?0.01. Data are means?SE (no. of myofibrils).
125?7 (52)
3.73?0.18 (54)
3.55?0.10 (46)
90?7 (61)
2.00?0.10 (72)
1.90?0.10 (64)
126?6 (47)
0.52?0.04 (47)
16?1 (47)
139?8 (61)
0.40?0.04 (61)
10?1 (62)
Figure 2. MHC isoform expression in SR and cAF myofibrils.
A, Representative SDS-PAGE of the MHC isoform region of SR
and cAF samples. B, Relative distribution of the MHC isoforms
in SR and cAF atrial myofibrils (means?SE, n?8). C, Total MHC
content expressed relatively to actin.
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MHC-? fraction was larger in SR samples (70%) compared to
cAF (59%) (Figure 2B, n?8 for both groups, P?0.05). Total
MHC, measured as MHC/actin ratio for each atrial sample, was
the same in SR and cAF myofibrils (Figure 2C). The lower
content in MHC-? in cAF myofibrils confirms the previously
reported shift of MHC isoforms from MHC-? to MHC-? for
cAF human atrial myocardium.8,9,20
Ca2?Sensitivity of Active Tension
Ca2?sensitivity of tension was investigated by activating
myofibrils with various pCa solutions then assembling the
pCa–tension relationship. Each preparation was exposed to up to
4 different pCa solutions. pCa 4.5 determined maximum Ca-ac-
tivated tension and pCa 8 fully relaxed the preparation between
each activation. Values of force at any given pCa were normal-
ized to maximal force at pCa 4.5. pCa–tension points were fit to
a Hill equation and the pCa at which tension was half maximum
(pCa50) and the Hill coefficient (nH; slope of the pCa–tension
relationship) were determined for each group.
pCa–tension relationships for SR and cAF myofibrils are
given in Figure 3, with representative experimental traces at
indicated pCa values. The pCa–tension relationship for cAF
was clearly shifted to the left compared to that of SR
myofibrils. Average pCa50was 5.83?0.10 in cAF, which was
significantly different (P?0.05) from pCa50of SR myofibrils
(5.61?0.03). The average nHwas 2.16?0.30 in SR and
1.44?0.43 in cAF myofibrils (P?0.05).
Sarcomeric Protein Phosphorylation
To investigate if the higher Ca2?sensitivity of force in cAF
can be ascribed to differences in myofilament protein phos-
phorylation between SR and cAF sarcomeres, myofibril
proteins were separated on 1D gradient gels and stained with
SYPRO Ruby and Pro-Q Diamond (Figure 4). On average, no
significant difference was found in cTnI phosphorylation
between SR and cAF, whereas phosphorylation of cMyBP-C,
desmin, cTnT, and atrial light chain-2 was significantly
higher in cAF compared to SR samples.
The increase in cMyBP-C phosphorylation found in human
cAF using the Pro-Q Diamond phosphostaining (Figure 4B)
is in sharp contrast with previously reported decrease in
cMyBP-C phosphorylation measured with specific phospho-
cMyBP-C antibody10in human cAF atria. To determine
whether the opposite result was attributable to the different
measurement methods we analyzed the content and phos-
phorylation of cMyBP-C and cTnI of our cAF and SR
samples using Western immunoblotting. The analysis re-
vealed no changes in cMyBP-C (Figure 5A) and cTnI (Figure
5B) protein content in cAF samples compared to SR. In
accordance with ProQ analysis of protein phosphorylation,
the specific antibodies directed against phosphorylated pro-
tein kinase (PK)A sites in cMyBP-C (Ser282) and cTnI
(Ser23/24) showed higher cMyBP-C phosphorylation in cAF
compared to SR (P?0.08, Figure 5A) and confirmed no
difference in cTnI phosphorylation at Ser23/24 (Figure 5B).
The previously reported decrease in cMyBP-C phosphor-
ylation in cAF10may reflect atrial dilatation rather than being
a component of cAF because, at variance with the present
study (see Online Table I), in the previous study10the atrial
size of SR patients was much less than that of cAF patients
(see the table in the article by El-Armouche et al10). Consis-
tent with this explanation, a recent study in goat models of
atrial dilatation and atrial fibrillation22has reported that
reduction in PKA phosphorylation of cMyBP-C is a distinc-
tive feature of atrial dilatation.
Passive Tension
Passive tension at optimum overlap was lower in cAF
compared to SR myofibrils (Table), suggesting that passive
stiffness differs in the 2 myofibril types. To better investigate
passive properties, the force responses of relaxed SR and cAF
myofibrils to various ramp elongations were recorded and the
steady-state sarcomere length–resting tension relationship
determined in the 2 myofibril groups (Figure 6). Sarcomere
length and resting tension were measured 20 seconds after
each length change was completed, ie, when most of the
stress relaxation was over. Details of the length elongation
protocol applied to myofibrils are shown in the inset of Figure
6 together with tension traces for a representative SR myofi-
bril. The average sarcomere length–passive tension relation-
ships of SR and cAF myofibrils (Figure 6) are evidence that
passive stiffness is significantly reduced in cAF myofibrils.
Titin Isoform Expression
Mammalian cardiac muscle coexpresses 2 titin isoforms, a
short N2B and a long, more compliant, N2BA titin.16–23
Considering that titin is the only significant source of resting
Figure 3. pCa–tension relationships of SR and cAF myofi-
brils. Top, Representative traces from cAF and SR myofibrils at
indicated pCa values. Bottom, Mean pCa–tension relationships
for cAF (E) and SR myofibrils (f). Tension values are normalized
to those measured at pCa 4.5. Data points are means?SE of 6 to
12 myofibrils from 4 SR and 4 cAF patients. Data are fit to a modi-
fied Hill equation (continuous lines): P?P0/(1?10[nH{pCa-pCa50}]).
Belus et al Myofibril Mechanics in Chronic Atrial Fibrillation
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tension in single myofibrils, we investigated whether the
lower passive stiffness found in cAF myofibrils correlates
with changes in titin isoform expression.
In Figure 7A, SDS-PAGE reveals that human atrium
coexpresses N2B and N2BA titin isoforms; this is confirmed
by Western blotting. Titin degradation product T2 was hardly
detectable and T3 undetectable in our human atrial samples
indicating a good titin preservation. As shown in Figure 7B,
SR atrial tissue (n?6) expressed more N2BA (61%) than
N2B (39%) and cAF (n?6 patients) expressed even more
N2BA than SR (78%, P?0.01). The titin/MHC ratio was the
same in SR and cAF patients (Figure 7C). Therefore, the total
content of titin was not different in SR and cAF atrial tissue
but the mean expression of N2BA in cAF was increased at the
expense of N2B.
Passive and active mechanics of human atrial myofibrils are
significantly altered in cAF indicating that myofilament
changes contribute to the atrial contractile dysfunction that
persists after cardioversion. The decrease in passive stiffness
in the cAF myofibrils can be entirely explained by the shift in
titin isoform expression. Besides contributing to the altered
mechanics of cAF atria, changes in sarcomere diastolic
properties likely participate in the progressive atrial dilatation
that often accompanies cAF. The contractile alterations found
in cAF myofibrils can be only partly explained by the shift in
the isoform expression of the cardiac motor protein. Altered
phosphorylation of multiple myofilament proteins are likely
related to most contractile alterations. Changes associated
with increased myofilament Ca2?sensitivity may play a role
in the self-perpetuation of cAF.
The observed mechanical changes seem to be a component
of cAF rather than atrial dilatation because the atrial size of
cAF patients, although relatively large, is not significantly
different from that of SR patients (see Online Table I).
Changes in Titin and Diastolic Stiffness
To our knowledge titin isoform expression has never been
studied in human atrial myocardium. We find that the
N2BA:N2B expression ratio in the atrial myocardium of SR
patients is 1.64?0.27, much higher than that reported for
ventricular myocardium of normal human hearts (0.56?
0.06).24Interestingly, in humans as in other big mammals,16
the expression of N2BA dominates in atrial myocardium. It is
an important finding that the N2BA isoform is significantly
upregulated at the expense of the N2B isoform in cAF
(N2BA:N2B 3.80?0.50). As expression shifts toward the
N2BA isoform, because of the different compliance of the 2
titin isoforms, passive myofibril stiffness is expected to
decrease. Consistent with this notion, passive stiffness of cAF
myofibrils is significantly reduced (on average 2.5-fold)
compared to SR myofibrils.
It has been reported that the length dependence of activa-
tion of cardiac muscle (that is the mechanism underlying the
Figure 4. Myofilament protein phos-
phorylation. A, Tissue samples (20
?g/lane) were separated on a 4% to
15% gradient gel stained with SYPRO
and Pro-Q Diamond. ProQ Diamond–
stained signals (cMyBP-C; desmin;
cTnT; cTnI; and atrial light chain 2 [ALC-
2]) were divided by SYPRO-stained pro-
tein signals to correct for differences in
protein loading. PM indicates molecular
weight marker (PeppermintStick phos-
phoprotein marker in which ovalbumin
and ?-casein are phosphorylated). B,
Student's t test analysis revealed signifi-
cantly higher phosphorylation of
cMyBP-C, desmin, cTnT, and atrial light
chain-2 in cAF (n?8) compared to SR
(n?8). Values are given relative to SR
group, which was set to 1. *P?0.05, cAF
vs SR.
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Starling's law of the heart) is titin-based and is reduced in
preparations that express high levels of N2BA titin.25One can
speculate, therefore, that length dependence of activation is
reduced in cAF myofibrils because of the increase in N2BA
titin. A reduction in the length dependence of activation will
contribute to cAF induced atrial contractile dysfunction and
possibly to progressive dilation. Progressive atrial dilation in
cAF26may contribute to self-perpetuation of the arrhyth-
mia.27The mechanism behind cAF related increase in atrial
size is unclear but increase in N2BA expression and reduction
in myofibril resistance to elongation may contribute to it.
Alterations in Contractile Function
The major changes in contractile function of cAF compared
to SR myofibrils are (1) a marked reduction in the maximum
rate of tension generation, (2) a significant decrease in
maximum active tension, and (3) a significant increase in
myofilament Ca2?sensitivity.
In both SR and cAF myofibrils, kACTwas the same as kTR.
This similarity suggests that kACTis not limited by the rate with
which thin filaments are switched on by Ca2?; rather it predom-
inantly reflects the rate with which crossbridges enter their force
generating states. Both kACTand kTRwere markedly reduced
(?50%) in cAF compared to SR myofibrils indicating a slower
cross-bridge turnover rate. Consistent with the present results, a
significant reduction in kTRwas previously reported in perme-
abilized human atrial myocytes from cAF patients.9Relaxation
kinetics, determined by sudden Ca2?removal from myofibrils,
predominantly reflects the apparent rate with which attached
crossbridges leave force-generating states.19Relaxation rates
were slower (25% to 35%) in the cAF myofibrils than in the SR
myofibrils implying that both cross-bridge attachment and de-
tachment rates contribute to the slower overall cross-bridge
turnover in cAF myofibrils.
Increase in the relative amount of the slow MHC-? isoform
expressed in cAF versus SR myofibrils (41% versus 30%),
though smaller than in previous reports (63% versus 25%20;
41% versus 23%9), directly accounts for the reduction in
activation and relaxation kinetics of cAF compared to SR
myofibrils. The negative impact of the MHC isoform change
on the power output and velocity of atrial contraction may
contribute to atrial contractile dysfunction in cAF.
Maximal Ca2?activated tension was reduced by ?30% in
cAF compared to SR myofibrils. Similar effect of cAF on
maximal active tension (33% reduction) was recently reported
for permeabilized human atrial myocytes,9though in that study
the difference between cAF and SR maximal tension was not
significant. A 75% reduction in twitch tension has been reported
in intact atrial trabeculae from cAF patients compared to SR
patients.6In that study, however, following positive inotropic
Figure 5. cMyBP-C and cTnI protein
content and phosphorylation at PKA
sites. Western blot analysis did not
reveal reduced content or degradation of
cMyBP-C (A) and cTnI (B). In accor-
dance with ProQ analysis of protein
phosphorylation, cMyBP-C phosphoryla-
tion at Ser282 was higher in cAF than in
SR (A), whereas phosphorylation at
Ser23/24 in cTnI did not differ between
SR and cAF. NF indicates nonfailing
ventricular donor sample included as
positive control (B). Values are given rel-
ative to SR, which was set to 100%.
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interventions (high extracellular [Ca2?], postrest potentiation)
the difference in twitch tension decreased to ?15% consistent
with the AF-induced reduction in myofibril density per unit
cross sectional area of the trabeculae (-14%). Those results
suggested that most of the atrial contractile dysfunction of cAF
is associated with impaired Ca2?handling whereas the loss of
atrial myofibrils plays a minor role. Here we directly determined
the maximal tension of isolated myofibrils and eliminate con-
tamination by either AF-induced excitation–contraction cou-
pling dysfunction or myofibrillar loss. Thus, in cAF myofibrils
reduction in maximal active tension must be attributable to
defects in the myofilament themselves.
The shift in MHC expression toward the slow ? isoform in
the myofibrils of AF patients is an unlikely explanation for
reduction in maximal tension of cAF myofibrils; maximal
tension does not differ in human atrial and ventricular
myofibrils that express quite different patterns of MHC
isoforms.12Though maximal active tension loss of cAF
myofibrils may come from multiple sources, present results
support a role for altered phosphorylation of myofilament
proteins (see below).
We found evidence that regulation of contraction at the
myofilament level is also altered in cAF. pCa50of active tension
was significantly increased, whereas the Hill coefficient (nH)
significant change in Ca2?sensitivity of tension development
has been previously reported for permeabilized human cAF
preparations,8,9though a significant increase in the Ca2?sensi-
tivity of ATP consumption under isometric conditions has been
found in the same cAF preparations.8This latter observation
seems consistent with the present results, although a discrepancy
between the Ca2?sensitivity of force and isometric ATPase is
difficult to explain.
Several mechanisms, mostly leading to changes in regulatory
proteins associated with thin and thick filaments, can impact the
pCa–tension relationship of atrial myofibrils. cAF is accompa-
nied by re-expression of fetal genes in the atria.3,4The predom-
inant TnI isoform expressed by the fetal human heart is the slow
skeletal (ssTnI) isoform28,29; this imparts higher Ca2?sensitivity
to cardiac sarcomere.30Although re-expression of ssTnI has
been described in the early stages of AF in human atria and in a
goat model of the disease, ssTnI was undetectable in atrial
samples from patients with cAF.31A relation has been found in
human atria between cAF and the activity of calpain-1,32a
protease involved in degradation of contractile proteins,33in-
cluding cTn.34Degradation of cTn by calpain has been reported
to occur in both human cAF and a cell model for tachypacing-
induced remodeling.11The predominant cTnI degradation prod-
uct (cTnI1 to 192) has been shown to significantly increase Ca2?
sensitivity of human cardiac myofibrils.14However, in the
present and previous studies9,31no changes in troponin content
were observed and cTnI degradation products were undetectable
in atrial samples from cAF patients.
The most likely explanation for the increased Ca2?sensitivity
and reduced maximal force resides in the complex changes in
myofilament protein phosphorylation found in cAF versus SR
samples. As previously reported,9,10phosphorylation of cTnI—the
atria. Our analysis revealed, instead, significantly higher phos-
phorylation of other myofilament proteins in cAF. CaMKII
(Ca2?/calmodulin-dependent protein kinase II) is overexpressed
in human cAF35and this may explain why cMyBP-C phosphor-
ylation is increased because Ser282 is both a PKA and CamKII
site. Increased phosphorylation of cMyBP-C in the absence of
Figure 6. Passive tension in SR and cAF myofibrils. Average
sarcomere length–passive tension relationships for SR (f, con-
tinuous line) and cAF (E, dashed line) myofibrils. Data are
means?SE of 9 to 15 myofibrils from 6 SR and 6 cAF patients.
In the inset, representative passive tension responses (lower
traces) to ramp elongations (upper traces) of SR myofibril in
relaxing conditions (pCa 8.0).
Figure 7. Titin isoform expression in SR and cAF atrial myo-
cardium. A, SDS-PAGE of SR and cAF myocardium. Rat car-
diac degraded control (CD) was coelectrophoresed for isoform
reference. Top right, Expanded titin region with evidence that
degradation product T2 is hardly detectable and T3 undetect-
able in the human atrial samples. Bottom right, Western blot
using the antibody Z1/Z2 for isoform band confirmation. B, Titin
isoform percent distribution in SR and cAF (means?SE, n?6,
P?0.01). C, Total titin content, expressed relatively to MHC in
SR and cAF atrial tissue (means?SE, n?6, P?0.9).
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increased PKA phosphorylation of cTnI may increase myofibril
Ca2?sensitivity,36though the specific effect of cMyBP-C
phosphorylation on Ca2?sensitivity of tension is at present
unresolved.37Increased atrial light chain-2 phosphorylation by
myosin light chain kinase or PKC has been associated with
increased myofilament Ca2?sensitivity,38,39whereas increased
cTnT phosphorylation by PKC has been implicated in reduced
maximal force generating capacity.40As conclusion, present
results suggest that the increased Ca2?-responsiveness and the
lower maximal tension of cAF myofibrils cannot be explained
by contractile protein degradation or isoform shifts but result
from the complex interplay between changes in the phosphory-
lation status of multiple myofilament proteins.
The fact that phosphorylation of multiple myofilament
proteins is increased favors a role for altered phosphatase
rather than kinase activity, which would result in more
specific protein phosphorylation changes. A role for altered
phosphatase activity in cAF has been suggested in a previous
study10and warrants further investigation of protein phospha-
tases and their cellular compartmentalization.
In intact cAF myocardium, increased myofilament Ca2?
sensitivity may partly counteract the decrease in active force
generation and impaired excitation–contraction coupling but
may have detrimental effects on relaxation. Moreover, in-
crease in Ca2?sensitivity of cAF myofibrils may contribute
to electric remodeling and self-perpetuation of atrial arrhyth-
mia. Because Ca2?binding to the troponin complex repre-
sents the largest component of dynamic Ca2?buffering
during the cardiac cycle, intracellular Ca2?transients may
significantly change with altered myofilament Ca2?sensitiv-
ity.41,42This, in turn, may lead to action potential remodeling
and altered intracellular Ca2?handling to create both an
arrhythmogenic substrate and a trigger in atrial myocardium.
In isolated mouse heart myofilament Ca2?-sensitization by
drugs or troponin mutations associated with familial hyper-
trophic cardiomyopathy are reported to induce arrhythmias
by (1) shortening the effective refractory period and the
action potential duration, (2) slowing conduction velocity,
and (3) predisposing to early after-depolarizations and trig-
gered activity.43
Clinical Implications
This study provides new insights into the alterations of
myofilament function in cAF that may help us understand
persistent atrial contractile dysfunction, a major contributor to
atrial thrombogenesis. In cAF remodeled sarcomeres, we
identified altered titin isoform expression and increased
myofilament Ca2?sensitivity as contributors to progressive
and self-perpetuating arrhythmia. If mechanoelectric feed-
back between increased myofilament Ca2?sensitivity and
increased propensity for arrhythmias will be definitely estab-
lished, restoration of sarcomere Ca2?sensitivity (likely via
restoration of myofilament protein phosphorylation levels)
may become a novel therapeutic option for AF treatment.
Sources of Funding
This work was supported by the 6th and 7th Framework Programs of
the European Union (STREP Projects "NORMACOR," contract
LSH M/CT/2006/018676 to E.C.; "BIG-HEART," grant agreement
241577 to C.P., L.C., J.v.d.V.; Marie Curie EXT-014051 to L.C.);
the Deutsche Forschungsgemeinschaft (FOR-604/2, CA 618/2 to
L.C.); and Ministero Istruzione Universita ` e Ricerca, Italy
(PRIN2008 to A.M.).
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Novelty and Significance
What Is Known?
● Chronic atrial fibrillation (cAF) is associated with persistent atrial con-
tractile dysfunction, a major contributor to atrial thrombogenesis.
● Mechanisms responsible for impaired contractility are poorly defined
and available therapies do not address this dysfunction.
● MoststudiesfocusonalterationsinatrialmyocyteCa2?handling,butwe
and others emphasize the role of myofilament protein remodeling.
What New Information Does This Article Contribute?
● We demonstrate that diastolic and systolic sarcomere mechanics and
myofilament Ca2?sensitivity are altered in atrial myofibrils from
cAF patients.
● These mechanical changes are explained by shifts in protein isoforms
and by increased phosphorylation of multiple myofilament proteins.
● Myofilament remodeling is part of atrial contractile dysfunction in
human cAF and probably contributes to the progressive and
self-perpetuating nature of the arrhythmia.
It is hypothesized that maladaptive remodeling of the myofila-
ments is responsible, at least in part, for human cAF-associated
atrial contractile dysfunction. To document the functional impact
of myofilament protein changes in human cAF we dissected the
sarcomere diastolic and systolic properties of single atrial
myofibrils from surgical samples of cAF and control patients.
cAF myofibrils show (1) a reduction in diastolic stiffness, (2) a
reduction in maximum active tension and in the rates of
contraction and relaxation, and (3) an increase in myofilament
Ca2?sensitivity. These mechanical changes are associated with
changes in myofilament proteins. In cAF remodeled sarcomeres
we identify altered titin and myosin isoform expression and
increased levels of phosphorylation of multiple proteins. The
latter finding suggests that altered phosphatase activity leads to
increased Ca2?sensitivity that, in turn, may contribute to the
self-perpetuation of the atrial arrhythmia. These results show
that translational and post-translational changes in myofilament
proteins play a direct role in the altered atrial mechanics
associated with cAF and contribute to the progression of the
arrhythmia. Restoration of sarcomere Ca2?sensitivity (likely via
restoration of myofilament protein phosphorylation levels) may
become a novel therapeutic option for AF treatment.
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The investigation conforms with the principles outlined in the Declaration of Helsinki and is
approved by the local Ethics Committee (n. 2006/0023797). Samples of atrial appendages were obtained
following informed consent from 15 SR patients and 16 cAF patients undergoing open heart surgery. In the
cAF patients established AF was documented for at least 8 months. Samples from SR patients were used as
controls. No significant difference was present in ejection fraction and left atrial size between cAF and SR
patients. Details for each patient group are given in Online Table I.
Myofibril preparation
Fresh surgical samples were collected in a cold sterile saline solution for myofibril isolation.
Myofibril suspensions were prepared according to previously described methods1-2.
Mechanical measurements
Techniques for mechanical measurements in myofibrils were as previously described1-3. Briefly,
myofibrils were transferred to a temperature controlled chamber filled with relaxing solution (pCa8, 15°C).
The selected myofibril was horizontally mounted between a cantilever force probe (compliance: 2–6 nm.nN-
1) and a glass needle mounted on the lever arm of a length control motor. Initial sarcomere length (SL) was
adjusted to optimum myofilament overlap (2.2-2.3 µm). Average SL and width of mounted myofibrils were
measured on video images acquired through a CCD camera (2000x; phase contrast optics).
Isometric force was measured photoelectronically by recording the force probe deflection.
Myofibrils were activated and relaxed by rapid solution switching between two continuous streams of
solutions. Release-restretch protocols were applied to myofibrils in relaxing solution to measure passive
tension or in activating solution at the contraction plateau (Fig 1) to measure the rate of tension
redevelopment (kTR)4.
Ionic strength of the experimental solutions was 200mM and pH 7.0. Measurements were made in
the presence of a MgATP regenerating system (10 mM creatine phosphate, 200 units.ml-1 creatine kinase).
To avoid the effects on myofibril maximal force and force kinetic of variable level of contaminant inorganic
phosphate (Pi) in the solutions, [Pi] was reduced to less than 5µM using a Pi scavenging system1-3. All
solutions contained a cocktail of protease inhibitors1-3.
Sarcomeric protein analysis
Myosin heavy chain isoforms
Polyacrylamide gel electrophoresis was used to determine MHC isoform composition (MHCα and MHCβ)
after denaturation in sodium dodecyl sulphate (SDS-PAGE).
After mechanical measurements were completed the remaining myofibril suspensions were centrifuged and
the pellets were re-suspended in 100µl of Laemmli buffer and boiled for 5 min at 80ºC. Appropriate
quantities of the protein suspension were loaded onto gels (almost 1µg of total protein). Cardiac MHC
isoforms were separated on 8% Polyacrylamide slab gels after denaturation in SDS following a procedure
described by Talmadge and Roy4. Slabs gel 18cm wide, 16cm high and 1mm thick were ran at 4°C for 40 h
(2 h at 70V then at 170V for the time remaining). The gel thickness was increased (1mm instead of 0.75mm)
in order to reduce the resistance; the lower voltage at a prolonged running time allowed us to achieve the
resolution required for the separation of the two cardiac MHC isoforms. Gels were silver stained (Bio-Rad
Silver stain plus) for isoform recognition or stained with Coomassie Blue for quantitative analysis.
The Coomassie Blue gels were digitized with a scanner EPSON 1650 at a resolution of 1200 dpi.
Each line was characterised by a Brightness-Area Product (BAP) with a constant threshold after black/white
inversion using Adobe Photoshop.
Each band (MHC-α or MHC-β) was expressed as percent of the total MHC.
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Titin isoforms
Analysis of titin content in SR and cAF samples was adapted from Cazorla et al.5. Human atrial
tissue was pulverized in liquid nitrogen and solubilised for 3 min at 60°C in Laemmli buffer supplemented
with 8M urea. Titin content in SR and cAF samples were analysed with SDS-PAGE (2.5-7% acrylamide
gradient gels) and stained with 0.1% Coomassie Blue.
Wet gel images were acquired with a Kodak Image Station 2000R and analysed using Kodak 1D
image analysis software. The integrated optical density of MHC and titin peaks (both N2B and N2BA
isoforms) were determined to measure both the total amount of titin relative to MHC and the ratio
N2BA:N2B. For each sample a range of loadings was electrophoresed on the same gel. The optical density
of titin and MHC peaks were determined and plotted against their loading volume. The linear part of this
relation was fitted with a line regression and the slope determined. The slope ratio of titin:MHC were taken
as relative amount of titin in the samples and used to determine the N2BA:N2B ratio. Western blotting was
performed with the antibodies Z1/Z2 (kindly provided by Dr Labeit) to label the N-terminal of titin.
ProQ Phosphostaining
Atrial tissue samples from 8 SR and 8 cAF hearts were TCA (tri-chloro acetic acid)-treated as described
previously to fix phosphorylation status of myofilament proteins6. Phosphorylation status of myofilament
proteins was determined using Pro-Q Diamond phosphostaining (Molecular Probes)6. Samples were
separated on a gradient gel (Criterion Tris-HCl 4-15% gel, BioRad) and proteins were stained for one hour
with Pro-Q Diamond Phosphoprotein Stain. Fixation, washing and de-staining were performed according to
the manufacturer's guidelines. Staining was visualized using the LAS-3000 Image Reader (FUJI; 460
nm/605 nm Ex/Em; 2 min illumination) and signals were analyzed with AIDA. All protein signals were
within the linear range. Subsequently gels were stained overnight with SYPRO Ruby stain (Molecular
Probes) and visualized with the LAS-3000 (460 nm/605 nm Ex/Em; 2 s illumination). Since illumination of
Pro-Q Diamond-stained gels for 2 seconds did not reveal any signal, the signals obtained upon SYPRO Ruby
staining are not tainted by Pro-Q signals. All protein phosphorylation signals were corrected for protein
content determined by SYPRO staining.
Western Immunoblotting
Gel electrophoresis and Western immunoblotting was performed as described previously7-8
to analyze content and phosphorylation of myosin binding protein C and troponin I. Shortly, for the
analysis of cMyBP-C 15µg of proteins were loaded on a 10% acrylamide/bisacrylamide (29:1) gel
and electro-transferred onto nitrocellulose membrane. Membranes were stained with polyclonal
antibodies directed against cMyBP-C (MyBP-C motif 1:2500, pSer282-cMyBP-c 1:1000). After
incubation with HRP-secondary antibody (Sigma), the signal was revealed with ECL Plus
(Amersham) and acquired with the ChemiImagar TM 5500 (Alpha Innotech). For troponin analysis
0.5 µg and 5 µg sample was loaded to analyse expression (antibody 8I7, 1:15.000, Spectral
diagnostics) and phosphorylation (pSer23/24, 1:1000, Cell signaling), respectively. Troponin values
were normalized to actin stained on the same blots.
Statistical analysis
Data are presented as means ± SE. Differences between group means were compared by unpaired
Student t test. P<0.05 was considered statistically significant.
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Page 13
ONLINE TABLE I: Patient data
Total number of patients
Gender, n of female
Age, years
Body mass index, kg/m2
Reason for surgery:
• CAD, n
• MVD/AVD, n
• CAD+ MVD/AVD, n
NYHA functional class at time of surgery:
• I, n
• II, n
• III, n
• IV, n
Left atrial diameter (mm)
CAD, coronary artery disease; MVD, Mitral valve disease requiring valve replacement; AVD, Aortic valve
disease requiring valve replacement; LVEF, left ventricular ejection fraction. *P<0.05, nonpaired Student t
test for continuous variables and Х2 test for categorical variables.
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Page 14
1. Piroddi N, Belus A, Scellini B, Tesi C, Giunti G, Cerbai E, Mugelli A, Poggesi C. Tension generation
and relaxation in single myofibrils from human atrial and ventricular myocardium. Pflügers Arch.
2. Belus A, Piroddi N, Scellini B, Tesi C, D'Amati G, Girolami F, Yacoub M, Cecchi F, Olivotto I, Poggesi
C. The FHC-associated myosin mutation R403Q accelerates tension generation and relaxation of human
cardiac myofibrils. J Physiol. 2008;586:3639-3644.
3. Tesi C, Colomo F, Nencini S, Piroddi N, Poggesi C. The effect of inorganic phosphate on force
generation in single myofibrils from rabbit skeletal muscle. Biophys J. 2000;78:3081–3092.
4. Brenner B. Effect of Ca2+ on cross-bridge turn over kinetics in skinned single rabbit psoas fiber:
implication for regulation of muscle contraction. Proc Natl Acad Sci USA. 1988;85:3265-3269.
5. Cazorla O, Freiburg A, Helmes M, Centner T, McNabb M, Wu Y, Trombitás K, Labeit S, Granzier H.
Differential expression of cardiac titin isoforms and modulation of cellular stiffness. Circ Res.
6. Zaremba R, Merkus D, Hamdani N, Lamers JMJ, Paulus WJ, dos Remedios C, Duncker DJ, Stienen
GJM, van der Velden J. Quantitative analysis of myofilament protein phosphorylation in small cardiac
biopsies. Proteomics Clinical Applications. 2007;1;1285-1290.
7. Duncker DJ, Boontje NM, Merkus D, Versteilen A, Krysiak J, Mearini G, El-Armouche A, de Beer VJ,
Lamers JMJ, Carrier L, Walker LA, Linke WA, Stienen GJM, van der Velden J. Prevention of
myofilament dysfunction by beta-blocker therapy in post-infarct remodeling. Circulation – Heart Failure.
8. El-Armouche A, Boknik P, Eschenhagen T, Carrier L, Knaut M, Ravens U, Dobrev D. Molecular
determinants of altered Ca2+ handling in human chronic atrial fibrillation. Circulation. 2006;114:670-680.
at UNIV DI FIRENZE on November 16, 2012 http://circres.ahajournals.org/Downloaded from


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After this weekend, you may be thinking that Justin Timberlake is the next great comedian of our time, but trust me. HE IS SO. MUCH. MORE. In this Rihanna video for her song, "Rehab," it's all about the sexy. Angry sexy. In the video, Rihanna is a lady who lives in a trailer and can't afford pants. Justin is presumably her lover and/or wayward Hell's Angels member, who enjoys taking outdoor showers (because they can't afford indoor plumbing).
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it's finally here! the brand new rihanna video for her hot single rehab which features cutie justin timberlake - watch the hotness below! popbytes over & out for now...xxoo ... Read Post
Last month we got a sneak peek at Rihanna and Justin Timberlake filming a steamy video for her single "Rehab" in the desert, and now here is the final product. Justin doesn't have a huge part in the song, but both of the superstars ... Read Post
Justin Timberlake and Rihanna got to work on a new video for her song "Rehab" in LA yesterday, and from Rihanna's fishnets and leotard, we can expect something pretty steamy from these two. The pop stars are fresh off of spending th... Read Post

subset: commoncrawl


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Greenville, SC -- (ReleaseWire) -- 04/01/2013 -- Designing a new website keeping SEO needs in mind is the need of the hour; however, the process of designing a SEO friendly website is not widely known and very few have the right expertise to get the job done. Keeping these needs in mind, ActionAffiliates, one of the leading SEO firms based in Greenville South Carolina, recently announced its new web design services.
Click here to know more about web design services by ActionAffiliates.
ActionAffiliates caters to the needs of individuals as well as enterprises for web design solutions. The official website of the company lists out all details about the web design packages offered. The Web Design Greenville SC services are available to all the local as well as global customers.
The company offers highly affordable website design packages available on Internet. ActionAffiliates takes orders from all kinds of customers including individual clients as well as businesses and enterprises. Talking about the company's web design services, the market head of ActionAffiliates said, "Through our website design services, we want to serve the customers who are looking for one-stop destination for all the web-based needs. Right from designing a website to optimizing it for consistently driving targeted traffic, our experienced team can help in just about everything that a website owner needs."
ActionAffiliates works towards integrating website design and SEO in every new website it designs. Design of every new website is started keeping the end requirements under consideration. The CEO of ActionAffiliates was quoted as saying - "We keep the focus of every project right at the thick of all our actions. Every project that we work on, is result-driven and it is through our rigorous research and vast experience that we have managed to have a huge list of satisfied clients all across the globe."
All the Web Site Design Greenville SC packages by ActionAffiliates are competitively-priced keeping the needs of new website owners, and small entrepreneurs in mind. Every undertaken project is completed keeping turnaround time in mind and the company has a reputation of always meeting the deadline. Christian Jackson, President of ActionAffiliates said, "Every project we undertake is given equal importance. A team of designer is allocated to every small and big project that we take up. This is the reason we have a wide variety of clients from every niche of business, and we have always delivered the work on time."
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To know more about ActionAffiliates visit http://www.actionaffiliatesgreenvillesc.com/


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POPSUGAR Celebrity Jennifer Aniston Pictures of Jennifer Aniston by Celebrity 9/14/10 0 Shares Print 0 0 0 0 0 Previous Photo 1 of 6 View on One Page ADVERTISEMENT ( more slides) Next Share This Link Copy 1 Pictures of Jennifer Aniston Previous Photo 1 of 6 View on One Page ADVERTISEMENT ( more slides) Next 0 0 0 0 0 Read More Jennifer Aniston Jennifer Aniston Eddie Redmayne Gushes About Hugging Julia Roberts, 'Stalking' Jennifer Aniston at SAG Awards (VIDEO) 1 day ago But Really, What Were Meryl, Jennifer, and Julia Talking About? 2 days ago Poll: How Low Can You Go at the SAG Awards? 3 days ago We Guarantee You've Never Laughed This Hard at Jennifer Aniston Before 5 days ago The Secret to Getting Red-Carpet-Worthy Skin Like Jennifer Aniston's 5 days ago Memorable SAG Awards Snaps to Get You Excited For the Show 5 days ago How Many Stars Can You Spot in the Trailer For She's Funny That Way? 6 days ago Get the scoop with our daily newsletter Enjoy the latest news on the celebs you love. A Listers TV Movies Young Hollywood Sign up with 4 years I'm bored of the PR games and showmances. Link Love Cobie Smulders is out at Sundance three weeks after giving birth — Us Weekly Reese Witherspoon has one cute mini me — HuffPost Celebrity Idris Elba talks James Bond rumors — Lainey Gossip Justin Bieber hides a new haircut — Dlisted Amanda Peet thought Game of Thrones was an awful idea — ET David Beckham talks Bieber's Calvin Klein ads — JustJared Jessica Alba shows off her workout routine — Hollywood Tuna Diana Ross to have mini Vegas residency — Pink Is the New Blog Emile Hirsch gets into trouble at Sundance — The Superficial Taylor Swift shuts down her hackers —Newser Latest Celebrity
Eddie Redmayne Gushes About Hugging Julia Roberts, 'Stalking' Jennifer Aniston at SAG Awards (VIDEO) 1 day ago
Real Housewives of Orange County Season 10 Cast Revealed: All Women Returning, Plus Two More! Us Weekly 11.3K Shares
Amal Clooney back in court today arguing for Armenia in case against Dogu Perincek Lainey Gossip 27 Shares


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Woolly mammoth skeleton sold at UK auction
The skeleton of an Ice Age woolly mammoth fetched £189,000 ($300,000, 239.000 euros) at auction Wednesday as it went under the hammer in Britain with a host of other rare or extinct species.
The skeleton of an Ice Age woolly mammoth fetched £189,000 ($300,000, 239.000 euros) at auction Wednesday as it went under the hammer in Britain with a host of other rare or extinct species.
A portion of a Columbian mammoth skull and tusks have been uncovered in Idaho, and experts say a rare entire skeleton might be buried there.
(Phys.org) —A team of researchers studying narwhal and beluga whales in their native Arctic ecosystem has found what appears to be a correlation between testes mass and tusk length in narwhals—suggesting ...
Two critically-endangered Sumatran elephants have been found dead in Indonesia's Aceh province with their tusks missing, police said Monday.
A man who was having little luck catching salmon decided to look for fossils over the weekend and found a wooly mammoth tusk in the same Alaska location where his mother found one 22 years ago.
Those who want to protect elephants and rhinoceroses in Africa often face dangerous criminal traffickers who are bold, enterprising and well-equipped, leaders said at the US-Africa summit this week.
The game ranger with a rifle slung across his shoulders follows a bush trail through South Africa's famed Kruger national park, alert to the slightest sound or movement.
An auction house in Cannes, France, sold around 50 elephant tusks, or one tonne of ivory, on Saturday, breaking its previous record in terms of volume.
Hong Kong customs have seized HK$7.9 million (US$1 million) worth of illegal ivory hidden in luggage on a flight bound for Cambodia, officials said Tuesday.
Hong Kong on Thursday began destroying nearly 30 tonnes of ivory seized from smugglers in the world's largest such operation, a major step in the fight against the illegal trade in elephant tusks.
Cambodian customs on Friday seized more than three tonnes of ivory—the country's largest-ever haul of elephant tusks—hidden in a container of beans.
Poachers are slaughtering Tanzania's elephants for their ivory at such alarming rates that the population could be completely wiped out in just seven years, conservationists told a conference Friday.
Organised crime gangs in East Africa are generating staggering profits smuggling ivory and rhino horn with impunity, experts say, threatening both an irreplaceable wildlife heritage and key tourism industries.
(Phys.org) —The narwhal is a toothed whale that inspires legend-like notions as the ocean's unicorn and sea warrior, with the male's long tusk that can reach 2.6 m, protruding straight out. While scientists ...
A French auction of legal ivory broke records on Saturday, with prices reaching up to 1,000 euros a kilogramme ($630 a pound), according to Alexander Debussy, associate director of auction house Cannes Encheres.


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In Australia, if the smoking doesn't kill, the new cigarette packets might.
SYDNEY, Australia — Australia's smokers will be finding it even tougher to light up after a raft of tough new government legislation further tightened the country's already stringent restrictions on smoking.
On April 29, the Australian government — virtually overnight — announced a 25 percent hike in cigarette tax.
And in a world first, Australia has set out plans to become the first country to have plain cigarette packaging as of July 1, 2012. Cigarette packs will carry no logos, color or font variation. Instead the pack will bear the brand name and a graphic photo depicting the gruesome consequences of smoking.
"The new branding for cigarettes will be the most hard-line regime in the world and cigarette companies will hate it," Prime Minister Kevin Rudd said in a press conference.
The measure has the backing of the World Health Organization. WHO claims that packets convey a brand image such as toughness, which appeals to teenagers who are still forming their identity.
As Rudd predicted, tobacco companies responded almost immediately.
Imperial Tobacco Australia was concerned about what would happen to its brand recognition: "Introducing plain packaging just takes away the ability of a consumer to identify our brand from another brand and that's of value to us," Cathie Keogh, the brand's spokesperson told ABC News. Keogh said Imperial Tobacco were considering its legal options. "It really affects the value of our business as a commercial enterprise and we will fight to support protecting our international property rights."
British American Tobacco Australia said it would also fight the plain-package ruling. "It commodifies our brand," said spokesperson Louise Warburton. "It will mean that the only differentiation between types of cigarettes is price, and it will drive down prices." Warburton believes the move will also inspire counterfeit products. "Our industry is already losing 12 percent of the market share to knock-off products," said Warburton. "It costs taxpayers AU $600 million ($532 million) annually."
Australian Health Minister Nicola Roxon told ABC News the legislation would be written to withstand a lawsuit by the tobacco industry. "We won't be put off by the fact that tobacco companies won't like this action."
Smoking is the most preventable cause of death and disease in Australia, according to the federal government. Every year, 15,000 Australians die as a result of the habit. In 2007, 16.6 percent of Australians over the age of 14 were smokers and the government wants the rate to drop to 10 percent by 2018. Rudd plans to generate 5 billion Australian dollars ($4.4 billion) in the next four years from cigarette taxes and wants to use this money to fund overhauls of hospitals and the health system.


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Part-time evening students are admitted in the fall of each academic year. The part-time evening division program is a four-year course of study consisting of 14-week fall and spring semesters. A seven-week summer session with a limited course schedule is offered. A part-time evening student is required to take between 8 and 12 credits per semester and may take up to 6 credits in the summer session and 2 credits in the winter session. The following sequence of courses is for students enrolling since fall 2011. For the sequence of courses for students enrolled prior to fall 2011, consult the Student Programs, Policies & Procedures manual. Schedule Evening classes are scheduled Monday through Thursday, with an occasional course offered on a Friday. Classes vary in length from one to two hours and are scheduled between 6:00 and 10:00 p.m. Some weekend classes are also offered, and they may be scheduled for several hours at a time. Classes The prescribed sequence of lockstep courses for part-time evening students is: First semester (fall) courses:
Second semester (spring) courses:
Third semester (fall) courses:
Fourth semester (spring) courses:
Advanced Required Courses In addition to the previously listed lockstep courses, all students must successfully complete the following courses prior to graduation:
Summer Term The law school offers a limited number of courses during one seven-week summer session. Summer classes meet during the day and in the evenings. Students can enroll in up to 8 units per session for full-time students and 6 units per session for part-time students.
View the Texas A&M School of Law 2014-2015 JD Prospectus


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New Living Translation (NLT) | New International Version (NIV) |
14 The Spirit lifted me up and took me away. I went in bitterness and turmoil, but the LORD's hold on me was strong. | 14 The Spirit then lifted me up and took me away, and I went in bitterness and in the anger of my spirit, with the strong hand of the LORD upon me. |
Scripture quotations marked NLT are taken from the Holy Bible, New Living Translation, copyright 1996. Used by permission of Tyndale House Publishers, Inc., Wheaton, Illinois 60189. All rights reserved. (New Living Translation - The Bible Online) | Scripture quoted by permission. Quotations designated (NIV) are from THE HOLY BIBLE: NEW INTERNATIONAL VERSION®. NIV®. Copyright © 1973, 1978, 1984 by Biblica. All rights reserved worldwide. (New International Version Bible Online) |


text: RubRub. gamesBookmark the permalink. Release date: Oct 5th, 2010 Price: Free Version: 1.0 Seller: chungwhae cho Buy Now STP SCORE: Not yet reviewed Description Related Games Fallen Lords Dream Quest Naval Tactics: Captains of the Spanish Main Talisman Hitman Go Lords of Waterdeep Agricola Talisman Prologue HD STRATEGO – Official strategy board game Wake the Cat


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Wed March 19, 2014
Brit Uses Shakespeare To Exact Revenge
By David Greene
Originally published on Wed March 19, 2014 10:28 am
Edd Joseph bought a game console online, but he never received it. So he took revenge by texting 37 full Shakespeare plays to the seller's phone. That's nearly 30,000 messages.