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Deranged myofilament phosphorylation and
function in experimental heart failure with
preserved ejection fraction
Nazha Hamdani1, Kalkidan G. Bishu2, Marion von Frieling-Salewsky1,
Margaret M. Redfield2, and Wolfgang A. Linke1*
1
Department of Cardiovascular Physiology, Ruhr University, MA 3/56, D-44780 Bochum, Germany; and
2
Mayo Clinic and Foundation, Rochester, MN, USA
Received 16 April 2012; revised 14 November 2012; accepted 28 November 2012; online publish-ahead-of-print 4 December 2012
Time for primary review: 28 days
Aims Heart failure (HF) with preserved ejection fraction (HFpEF) is a major cause of morbidity and mortality. Key altera-
tions in HFpEF include increased left ventricular (LV) stiffness and abnormal relaxation. We hypothesized that myo-
filament protein phosphorylation and function are deranged in experimental HFpEF vs.normal myocardium. Such
alterations may involve the giant elastic protein titin, which contributes decisively to LV stiffness.
Methods
and results
LV tissue samples were procured from normal dogs (CTRL) and old dogs with hypertension-induced LV hypertrophy
and diastolic dysfunction (OHT/HFpEF). We quantified the expression and phosphorylation of myofilament proteins,
including all-titin and site-specific titin phosphorylation, and assessed the expression/activity of major protein kinases
(PKs) and phosphatases (PPs), myofilament calcium sensitivity (pCa
50
), and passive tension (F
passive
) of isolated per-
meabilized cardiomyocytes. In OHT vs.CTRL hearts, protein kinase-G (PKG) activity was decreased, whereas PKCa
activity and PP1/PP2a expression were increased. Cardiac MyBPC, TnT, TnI and MLC2 were less phosphorylated and
pCa
50
was increased in OHT vs.CTRL. The titin N2BA (compliant) to N2B (stiff) isoform-expression ratio was
lowered in OHT. Hypophosphorylation in OHT was detected for all-titin and at serines S4010/S4099 within titin-
N2Bus, whereas S11878 within proline, glutamate, valine, and lysine (PEVK)-titin was hyperphosphorylated. Cardio-
myocyte F
passive
was elevated in OHT, but could be normalized by PKG or PKA, but not PKCa, treatment.
Conclusions This patient-mimicking HFpEF model is characterized by titin stiffening through altered isoform composition and
phosphorylation, both contributing to increased LV stiffness. Hypophosphorylation of myofilament proteins and
increased calcium sensitivity suggest that functional impairment at the sarcomere level may be an early event in
HFpEF.
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Keywords Diastolic heart failure †Hypertrophy †Titin †Passive stiffness
1. Introduction
Heart failure (HF) is a major cause of mortality and morbidity and a
frequent reason for hospital admission in the USA and Europe.
1
More than 50% of HF patients have a left ventricular (LV) ejection
fraction (EF) .50% and are referred to as patients with HF with a
preserved EF (HFpEF). Typically, HFpEF patients show impaired LV
filling resulting from abnormal relaxation and increased LV diastolic
stiffness.
2,3
The factors contributing to the increased LV passive stiff-
ness include cardiac hypertrophy, fibrosis,
4
abnormal Ca
2+
-handling,
3
or deranged expression/phosphorylation of the elastic sarcomere
protein titin.
5–7
Cardiac titin is expressed as stiff N2B isoform (3000 kDa) and more
compliant N2BA isoform (3200– 3700 kDa),
8
and the N2BA:N2B ex-
pression ratio partly defines myofibrillar passive stiffness.
9–11
Patho-
logically increased N2BA:N2B ratios have been found in end-stage
human HF with reduced EF (HFrEF).
9,11
In human HFpEF, the propor-
tion of compliant N2BA titin is also increased, but cardiomyocytes
have been found to be stiffer than normal.
7
The increased cardio-
myocyte passive tension (F
passive
) may be due to deranged titin
*Corresponding author. Tel: +49 234 3229100; fax: +49 234 3214040, Email: wolfgang.linke@rub.de
Published on behalf of the European Society of Cardiology. All rights reserved. &The Author 2012. For permissions please email: journals.permissions@oup.com.
Cardiovascular Research (2013) 97, 464–471
doi:10.1093/cvr/cvs353
by guest on January 12, 2016Downloaded from
phosphorylation. A cardiac-specific segment in titin, the N2Bus, is
phosphorylated by protein kinase (PK)A
12,13
and cGMP-activated
protein kinase-G (PKG),
14
an effect augmented by PDE5A-inhibitor
sildenafil, in vivo.
15
This titin modification reduces cardiomyocyte
F
passive
,
6,7,12 –15
whereas a deficit in phosphorylation at the N2Bus-titin
site would increase F
passive
.
7,14
However, if the elastic titin segment is
phosphorylated at a different site, the proline, glutamate, valine, and
lysine-(PEVK) domain, by PKCa, titin-based stiffness increases.
16
PKCais elevated in HF
17
and hyperphosphorylation of the PEVK
domain could increase F
passive
in failing hearts.
18
Thus, while titin phos-
phorylation changes most probably alter F
passive
in HF, we are only be-
ginning to understand which sites on titin become more or less
phosphorylated in failing hearts and how these modifications alter dia-
stolic stiffness, particularly in HFpEF.
Cardiac remodelling in HF also involves phosphorylation changes in
other myofilament proteins, notably the regulatory proteins.
19–21
Little
is known about alterations in regulatory protein phosphorylation in
HFpEF, whereas HFrEF has been well studied for such alterations.
19–21
A prominentexample is cardiac troponin I (cTnI), which is largely respon-
sible for myofilament calcium sensitivity. TnI has been found to be hyper-
phosphorylated in HFrEF in some studies, but hypophosphorylated in
others, and similar differences have been reported for other regulatory
proteins.
22–27
A firm cause for these discrepancies is not known.
We speculated that derangements in phosphorylation and function
of cardiac myofilament proteins may occur in experimental dogs with
advanced age and hypertension-induced LV hypertrophy,
15,28
in com-
parison to normal dog hearts. Whereas the dog model has been
shown to mimic some forms of human HFpEF,
15,28
these dogs have
not been studied yet for biochemical and functional properties at
the level of the sarcomere proteins. We find hypophosphorylation
of regulatory proteins and increased Ca
2+
sensitivity of the contractile
apparatus in the experimental HFpEF model. We also detect elevated
F
passive
in HFpEF dog hearts owing to both titin-isoform switching and
altered titin phosphorylation, including site-specific phosphorylation
revealed by novel phospho-specific antibodies. While these altera-
tions are already apparent in the HFpEF model, in vivo cardiac mech-
anical function is still maintained. Our findings provide a novel
mechanistic insight into the remodelling processes during HFpEF de-
velopment and suggest new possibilities for therapeutic interventions
in this syndrome.
2. Methods
A detailed methods and additional data section is provided in the Supple-
mentary material online.
2.1 Animal model, in vivo mechanical analysis,
and tissue sampling
The study employed mongrel dogs (n¼39; Supplementary material
online, Table S1) that were divided into controls [CTRL; aged one year
(‘young’) or 8– 12 years (‘old’)] and old dogs made hypertensive by bilat-
eral renal wrapping (OHT; aged 8–13 years).
28
All dogs underwent echo-
cardiography in the conscious unsedated state. Short-term haemodynamic
studies were performed in CTRL and OHT dogs 8 weeks after renal
wrapping or sham surgery.
28
Animals were anaesthetized using fentanyl
(0.25 mg kg
21
intravenous bolus followed by 0.18 mg kg
21
h
21
) and mid-
azolam (0.75 mg kg
21
intravenous bolus followed by 0.59 mg kg
21
h
21
).
Adequacy of anaesthesia was monitored from the disappearance of the
corneal reflex and jaw tone. Dogs were intubated, ventilated, and given
maintenance saline infusion (3 mL kg
21
min
21
), and they received
autonomic blockade with atropine (1 mg) and propranolol (2 mg kg
21
).
Thoracotomy and pericardiotomy were performed. Under fluoroscopic
guidance, animals were instrumented with a pulmonary artery catheter,
an LV integrated pressure-conductance catheter (Millar), a left atrial and
central aortic high-fidelity pressure transducer (Millar), a pneumatic oc-
cluding device around the thoracic inferior vena cava, and an atrial lead
for pacing at 10– 20 bpm above the sinus rate. LV tissue samples were
procured from CTRL and OHT (8 weeks after renal wrapping), either
by taking full-thickness LV biopsies from the beating heart (n¼7 per
group) or by excising tissue post-mortem (n¼7– 9 per group).
In the beating-heart biopsy group, serial samples were harvested fromdif-
ferent regions of the anterior or anterior lateral wall from seven CTRL and
seven OHT dogs subjected to an identical experimental protocol without
collection of haemodynamic data, because the biopsy and haemostatic
sutures would alter chamber diastolic properties. Biopsy samples were
frozen in liquid nitrogen within seconds and stored at 2808C until use.
The investigation conforms with the Guide for the Care and Use of Labora-
tory Animals published by the US National Institutes of Health (NIH Pub-
lication, 8th Edition, 2011) and was approved by the Mayo Institutional
Animal Care and Use Committee. Dogs were euthanized by intravenous
KCl under deep anaesthesia, consistent with the Panel on Euthanasia guide-
lines for the American Veterinary Medical Association. Hearts were
removed post-mortem, weighed, and the LV sectioned for samples that
were flash frozen in liquid nitrogen. This procedure occurred as quickly
as possible but could take up to 60 min. Samples were stored at 2808C
until use. All data shown for the CTRL post-mortem group were obtained
from old (aged) animals; however, young CTRL hearts procured post-
mortem revealed a similar myofilament protein expression and phosphor-
ylation and cardiomyocyte mechanical properties compared with old
CTRL post-mortem hearts (data not shown).
2.2 Protein analysis
Titin isoform separation. Homogenized myocardial samples were analysed
by 2% sodium dodecyl sulphate–polyacrylamide gel electrophoresis
(SDS–PAGE).
9
Protein bands were visualized using Coomassie blue or
SYPRO Ruby, scanned, and analysed densitometrically.
Total protein phosphorylation assays. Tissue samples (20 mg dry weight/
lane) were separated on 2% SDS – PAGE gels for titin, or on gradient
gels for other myofilament proteins. Gels were stained with Pro-Q
Diamond for 1 h and subsequently with SYPRO Ruby overnight. Phos-
phorylation signals for myofilament proteins on Pro-Q Diamond-stained
gels were normalized to SYPRO Ruby-stained total protein signals.
Immunoblotting. Expression of cMyBPC, phospho-cMyBPC (S282), cTnI,
phospho-cTnI (S23/S24), cMLC2, phospho-cMLC2 (S19), PKCa,
phospho-PKCa, PP1, and PP2a was measured by 15% SDS– PAGE and
western blot, expression of titin phospho-N2Bus (S4010; S4099) and
phospho-PEVK (S11878) by 2% SDS–PAGE and western blot. The position
of titin phosphosites is according to full-length human titin, UniProtKB identi-
fier, Q8WZ42. Affinity-purified phosphospecific and sequence-specific anti-
titin antibodies were custom-made by Eurogentec (Belgium).
2.3 Myocardial protein kinase G and A activity
tests
PKG activity (pmol/min/mg protein) was measured using radiolabeled
ATP. PKA activity (ng mL
21
) was measured using a nonradioactive PKA
kinase activity-assay kit.
2.4 Force measurements on isolated skinned
cardiomyocytes
Cardiomyocytes were demembranated and isolated cells were attached
between a force transducer and a motor.
29
F
passive
was recorded
between 1.8 and 2.4 mm sarcomere length (SL). Ca
2+
sensitivity of the
contractile apparatus (pCa
50
) was determined at 2.2 mm SL.
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2.5. Statistics
The values are given as mean +SEM in each group. Data were tested for
statistically significant differences using the Bonferroni-adjusted t-test,
apart from Figure 6and Supplementary material online, Figure S10,
where the paired Student’s t-test was used. A P-value of ,0.05 was con-
sidered significant.
3. Results
Clinical, haemodynamic (anaesthesia), and conscious echocardiog-
raphy data were available for all groups of dogs (Supplementary ma-
terial online, Table S1). OHT dogs had chronic hypertension as
described previously.
28,30
Both systolic and diastolic blood pressure
were significantly increased in OHT vs.CTRL, as were the LV end-
systolic pressure, the relaxation constant Tau, and the LV weight/
body weight ratio. Hypertrophy was present in OHT, as indicated
by an increased mean cardiomyocyte diameter in this group com-
pared with CTRL (Supplementary material online, Figure S1). LV EF
was unaltered in OHT vs.CTRL dogs. Thus, the OHT dogs showed
typical signs of early HFpEF.
3.1. Hypophosphorylation of myofilament
regulatory proteins in OHT
Myofilament regulatory proteins cMyBPC, cTnI, cTnT, and cMLC2
showed reduced phosphorylation in biopsies of OHT vs.CTRL
hearts, as detected by Pro-Q Diamond/SYPRO Ruby staining
(Figure 1). In post-mortem OHT hearts, cMyBPC, cTnI, and cTnT
were also hypophosphorylated compared with CTRL, whereas
cMLC2 phosphorylation was unaltered (Supplementary material
online, Figure S2). Using western blots for the detection of myofila-
ment protein expression and phosphorylation, we found cTnI phos-
phorylation at S23/S24 to be significantly reduced by .50% in
OHT vs.CTRL, in beating-heart biopsies (Figure 2B) and post-
mortem tissues (Supplementary material online, Figure S3). Further-
more, cMyBPC phosphorylation at S282 was reduced by 80% in
biopsied OHT vs.CTRL samples and cMLC2 phosphorylation at
S19 was reduced by 70% (Figure 2Aand C). Total cMyBPC, cTnI,
and cMLC2 expression remained unaltered in OHT (Figure 2).
3.2. Alterations in expression/activity of
major protein kinases and phosphatases
in OHT
The activity of PKG was reduced in OHT vs. CTRL (Supplementary
material online, Figure S4A and B). PKCaexpression was similar in
CTRL and OHT (Supplementary material online, Figure S4C and D),
but phosphorylation of PKCa(a measure of kinase activity) was
higher in OHT vs. CTRL (Supplementary material online, Figure S4E
and F). PKA activity was not different between sample groups (Sup-
plementary material online, Figure S5). Expression of PP1 (Supplemen-
tary material online, Figure S6A and B) and PP2a (Supplementary
material online, Figure S6C and D) was increased in OHT vs. CTRL.
3.3. Altered calcium sensitivity of
cardiomyocytes in OHT and effect of PKA
treatment
The force–pCa relationship of skinned single cardiomyocytes from
beating-heart biopsies (n, 10–16 cells per group; from 2 to 3 hearts
per group) revealed significantly higher myofilament calcium
sensitivity (pCa
50
) in OHT (5.77 +0.01) vs.CTRL (5.64 +0.02)
(Figure 3A). Maximum Ca
2+
-activated tension was reduced in OHT
(Figure 3A, inset). In cardiomyocytes from post-mortem hearts,
pCa
50
was significantly lower in OHT (5.52 +0.01) than in CTRL
(5.61 +0.01), while maximum Ca
2+
-activated tension was also
reduced (Supplementary material online, Figure S7). The Hill coeffi-
cient indexing the steepness of the force– Ca
2+
curve was decreased
in OHT (1.9+0.2) vs.CTRL (2.5 +0.2) biopsy samples (Figure 3A),
but increased in OHT (3.9 +0.5) vs.CTRL (3.0 +0.8) post-
mortem samples (Supplementary material online, Figure S7). The
Ca
2+
sensitivity of the contractile apparatus was significantly
reduced (P¼0.012) in OHT cardiomyocytes upon incubation with
a PKA catalytic subunit (pCa
50
shift, 0.16 +0.02 units), whereas in
CTRL myocytes this effect was not significant ( pCa
50
shift, 0.06 +
0.01 units) (Figure 3B).
3.4. Titin isoform shift towards N2B
in OHT
By measuring the titin N2BA/N2B isoform composition (Figure 4A;
Supplementary material online, Figure S8A), we found that the mean
N2B proportion in biopsied samples increased from 63.3 +3.3% in
CTRL to 72.6 +3.3% in OHT (Figure 4B), and in post-mortem
Figure 1 Phosphorylation of regulatory myofilament proteins in
biopsy samples from OHT and CTRL dog hearts, by Pro-Q
Diamond/SYPRO Ruby staining. Top panels show representative
SDS–PAGE gels. M, peppermint-stick marker. Graphs show mean
phospholevels of cMyBPC, cTnI, cTnT, and cMLC2, normalized to
their respective total protein level. Heart samples (n¼7 – 9) were
analysed in duplicate. *P,0.05.
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tissues from 57.5 +6.1% in CTRL to 65.9 +5.1% in OHT (Supple-
mentary material online, Figure S8B). A ‘T2’ titin degradation band
was barely detectable in biopsy samples, but was more frequent
and more intense in post-mortem tissues.
3.5. Phosphorylation deficit of all-titin
in OHT and rescue by PKG
All-titin phosphorylation measured by Pro-Q Diamond/SYPRO Ruby
staining decreased by 30% in OHT vs.CTRL biopsies, and by ~60%
in post-mortem tissue (Figure 4C; Supplementary material online,
Figure S8C). Both N2BA and N2B titin isoforms were hypophosphory-
lated in OHT. Importantly, ex vivo phosphorylation by
cGMP-dependent PKG significantly increased all-titin phosphorylation
in OHT, up to the level measured in CTRL (Figure 4C; Supplementary
material online, Figure S8C). This increase was larger in N2B than in
N2BA titin.
3.6. Site-specific phosphorylation at
titin-N2Bus (S4010/S4099) and titin-PEVK
(S11878)
Alterations in all-titin phosphorylation reflect modifications at poten-
tially hundreds of amino acids within titin. Furthermore, the Pro-Q
Diamond stain reportedly fails to detect phosphosites within titin’s
PEVK domain.
18
Using custom-made phosphospecific titin antibodies,
we measured changes in phosphorylation at two conserved serines
within the N2Bus (S4010 and S4099 of full-length human titin) and
at a conserved serine of the PEVK segment (S11878) by western
blot (Figure 5; Supplementary material online, Figure S9). The mean
proportions of titin N2Bus phosphorylation at S4010 (Figure 5A; Sup-
plementary material online, Figure S9A) and S4099 (Figure 5B; Supple-
mentary material online, Figure S9B) were significantly lower in OHT
vs.CTRL. In contrast, the mean proportion of phospho-PEVK
(S11878) was significantly higher in OHT than in CTRL (Figure 5C;
Supplementary material online, Figure S9C). These phosphorylation
changes occurred in both titin isoforms.
Figure 2 Expression and phosphorylation of cMyBPC, cTnI, and
cMLC2, in CTRL and OHT biopsy samples by western blot. Left
panels show phosphorylated, right panels total protein levels; repre-
sentative immunoblots above graphs, which indicate mean phos-
phorylation/expression. (A), cMyBPC (S282) phosphorylation and
cMyBPC expression; (B), cTnI (S23/S24) phosphorylation and cTnI
expression; (C), cMLC2 (S19) phosphorylation and cMLC2 expres-
sion. Data in graphs are normalized to beta-actin signals. Heart
samples (n¼7–9) were analysed in duplicate. *P,0.05.
Figure 3 Myofilament calcium sensitivity of skinned isolated cardi-
omyocytes from biopsy samples. (A), Relative force vs. pCa relation-
ship at 2.2 mm SL of OHT compared with CTRL cardiomyocytes.
Inset, absolute values for actively developed tension vs.pCa. (B),
Relative force vs. pCa relationship for CTRL and OHT cardiomyo-
cytes at 2.2 mm SL, before (black symbols and curves; data taken
from panel A) and after incubation with a PKA catalytic subunit
(red symbols and curves). n¼10– 16 myocytes per group, from
three different hearts per group. *P,0.05.
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3.7. Cardiomyocyte F
passive
is increased
in OHT but lowered by administration of
PKA or PKG
The passive SL–tension relationship of isolated skinned cardiomyo-
cytes (n, 10–16 per group, from 2 to 3 hearts per group) was gener-
ally steeper in OHT than in CTRL (Figure 6; Supplementary material
online, Figure S10). Administration of PKA significantly reduced F
passive
of OHT cells at a SL of 2.2–2.4 mm, sometimes already at shorter SLs
(Figure 6A; Supplementary material online, Figure S10A). Even in cardi-
omyocytes from beating-heart biopsies, PKA significantly lowered
F
passive
also in CTRL (Figure 6A). Additional administration of
cGMP-dependent PKG had no obvious mechanical effect in all
groups (Figure 6A; Supplementary material online, Figure S10A). If
PKG was administered first, F
passive
dropped as seen with PKA first
and additional administration of PKA caused no further F
passive
change in the myocytes (Figure 6B; Supplementary material online,
Figure S10B). We also tested whether administration of PKCaalters
F
passive
, but found no significant effect in both CTRL and OHT
(Figure 6C). These results demonstrate that F
passive
is increased in
OHT, but can be corrected by PKG- or PKA-mediated protein
phosphorylation.
4. Discussion
The number of patients hospitalized for HFpEF grows steadily, but
neither is the aetiology of the disease understood well, nor are effect-
ive treatment strategies available.
1–3
Mechanistic studies on human
Figure 4 Titin-isoform composition and all-titin phosphorylation
in biopsy samples, by Pro-Q Diamond/SYPRO Ruby staining. (A),
Representative titin gels comparing CTRL and OHT samples, and
OHT samples before and after incubation with cGMP-dependent
PKG. Indicated are the N2BA and N2B titin isoforms and titin pro-
teolytic fragment, T2. (B), Mean titin-isoform composition in OHT
and CTRL hearts, measured on SYPRO Ruby-stained gels. Combined
N2BA +N2B signal intensities are 100%. (C) Mean all-titin phos-
phorylation in CTRL and OHT, and in OHT samples incubated ex
vivo with cGMP-dependent PKG. Combined N2BA +N2B phos-
phorylation of CTRL set to 100%. n¼7–9 per group; heart
samples analysed in triplicate. *P,0.05.
Figure 5 Site-specific phosphorylation of titin in biopsy samples by
western blot. Images on left panels show representative immuno-
blots with OHT and CTRL heart tissue using phosphosite-specific
(top) and corresponding sequence-specific (bottom) anti-titin anti-
bodies. PVDF stains are included for comparison. Graphs on right
show mean phosphorylation levels for N2BA and N2B isoforms in
OHT and CTRL, at position S4010 (N2Bus) (A), S4099 (N2Bus)
(B), and S11878 (PEVK domain) (C). (A) and (B) combined
N2BA +N2B signal intensities of the CTRL set to 100%; (C) com-
bined N2BA +N2B signal intensities of OHT set to 100%. Heart
samples (n¼7–9) were analysed in triplicate. *P,0.05.
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HFpEF are limited by the low availability of tissue samples from dis-
eased and healthy control hearts. Unlike hearts in end-stage failure,
which after transplantation can be used for research purposes,
HFpEF hearts usually do not get explanted. Biopsy samples are some-
times obtained from human HFpEF myocardium, but obviously not
from healthy control hearts. Non-transplanted non-failing donor
hearts occasionally become available for research, but their preserva-
tion is highly variable. These limitations with human cardiac tissue
warrant the study of well-defined animal models of HFpEF, which
can be controlled in terms of age, genetic background, or pharmaco-
logical treatment.
In this study, we used an old dog model of hypertrophy-associated
early HFpEF, which shows signs of diastolic dysfunction resembling
those frequently seen in elderly HFpEF patients: impaired LV relax-
ation, unaltered coefficient of LV diastolic stiffness but reduced dia-
stolic capacitance, along with elevated natriuretic peptides, normal
LV volume but increased LV mass and myocardial fibrosis.
28,30
We
detected profound alterations in cardiac myofilament phosphorylation
and function in dog HFpEF compared with normal dog hearts. Hypo-
phosphorylation of sarcomeric proteins in experimental HFpEF pre-
sumably resulted, at least in part, from the increased PP1 and PP2a
expression and the reduced PKG activity. The deranged
phosphorylation in dog HFpEF altered the calcium sensitivity of the
contractile apparatus and increased cardiomyocyte F
passive
. By focusing
on the elastic protein titin, we found that both isoform switching and
altered phosphorylation increased cardiomyocyte F
passive
in HFpEF.
Since hypophosphorylation of sarcomeric proteins persists in end-
stage human HF,
20
a deficit in phosphorylation of these proteins
could be a general property in the transition to HF. In vivo heart func-
tion was modestly impaired in the dog HFpEF model at rest, but
reserve function, including catecholamine responsiveness (not
studied here), could be more strongly affected. In summary, contrib-
uting factors that drive the HFpEF hearts into diastolic dysfunction
likely include biochemical changes at myofilament proteins leading
to increased calcium responsiveness of force generation and elevated
titin-based passive stiffness.
4.1 Hypophosphorylation of regulatory
myofilament proteins and increased pCa
50
in dog HFpEF
Myofilament Ca
2+
sensitivity is decreased after beta-adrenergic
stimulation of cardiac muscle, an effect largely mediated by increased
PKA-dependent phosphorylation of cTnI.
20,21,24,29,31
Because the
Figure 6 Passive tension of skinned cardiomyocytes isolated from biopsy samples, and effect of incubation with PKA, PKG, or PKCa.(A) and (B),
F
passive
at SL 1.8– 2.4 mm recorded on CTRL (red symbols and curves) or OHT myocytes (green symbols and curves) in non-activating buffer (solid
curves; ‘before’), following incubation with a PKA catalytic subunit (dotted curves; ‘after PKA’), and following incubation with PKG and activator cGMP
(dashed curves; ‘after PKG’). (A) PKA administered first, followed by PKG; (B) PKG administered first, followed by PKA. (C), F
passive
at SL 1.8 – 2.4 mm
recorded with CTRL or OHT myocytes in non-activating buffer (solid curves; ‘before’) and following incubation with PKCa(dotted curves; ‘after
PKCa’). n¼10–16 cardiomyocytes per group, from 2 to 3 hearts per group. Curves are three-order regressions. *P,0.05 CTRL‘before’ vs. OHT‘-
before’;
#
P,0.05 OHT‘before’ vs. OHT‘after PKA’ (in A) and OHT‘before’ vs. OHT‘after PKG’ (in B);
†
P,0.05 CTRL‘before’ vs. CTRL‘after PKA’ in
(A) and CTRL‘before’ vs. CTRL‘after PKG’ in (B).
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beta-adrenoceptor density and adenylate cyclase activity are reduced in
HF,
32
hypophosphorylation of cTnI is a usual consequence, particularly
at the PKA
24
(and PKG
33
)-dependent phosphosites, S23/S24. In dog
HFpEF, we found decreased cTnI phosphorylation (including S23/S24
phosphorylation) compared with normal dog hearts, presumably
explaining the increased Ca
2+
sensitivity of skinned cardiomyocytes
from biopsied HFpEF hearts. In line with this interpretation, the
increased Ca
2+
sensitivity of HFpEF (biopsy) cardiomyocytes could
be normalized by ex-vivo PKA treatment. Thus, the dog HFpEF hearts
show alterations in cTnI phosphorylation and myofilament calcium sen-
sitivity resembling those frequently (but not consistently
23,24
) reported
in human HF vs.donor hearts and in animal models of HF.
20,21,29,31
Along this line, b-blockade in HFpEF patients has been associated
with increased cardiomyocyte pCa
50
compared with HFpEF patients
not treated with b-blockers.
29
Furthermore, myofilament pCa
50
changes are unlikely to be due only to cTnI phosphorylation, but also
due to phosphorylation of cMyBPC and cMLC2. We found both
these myofilament proteins, as well as cTnT, to be hypophosphorylated
in biopsy samples of HFpEF dogs. In summary, hypophosphorylation of
regulatory myofilament proteins and increased calcium sensitivity in this
model suggest that functional impairment at the sarcomere level may be
an early event in the development of HFpEF.
Surprisingly, reduced Ca
2+
sensitivity was found in skinned cardi-
omyocytes from HFpEF hearts procured post-mortem, although
phosphorylation of cTnI, cMyBPC, and cTnT was also lowered in
the post-mortem HFpEF group. PKA activity was similar in biopsy
and post-mortem samples, perhaps because all dogs were under
autonomic blockade. The different direction of Ca
2+
-sensitivity
shift in the post-mortem compared with the beating-heart biopsy
group likely originates in events associated with death, such as a cat-
echolamine surge, enzymatic dysfunction, or activation of proteases.
Unlike in biopsy samples, cMLC2 phosphorylation was unaltered in
post-mortem HFpEF vs.CTRL, which might contribute to the differ-
ences in Ca
2+
-sensitivity shift. Additionally, phosphosites in myofila-
ment proteins not tested by us could be modified differently in the
post-mortem and beating-heart groups. Cardiac TnI contains sites
other than S23/S24 which can be phosphorylated and possibly be
important for the Ca
2+
sensitivity,
26
and some functionally relevant
cTnI phosphosites may still be unknown. In conclusion, since we
consider the beating-heart biopsies as the gold standard, the dog
HFpEF hearts have increased Ca
2+
sensitivity. Our results confirm
that degradation processes or other modifications at the time of
death can impact protein phosphorylation and function, which has
implications for the interpretation of data from the samples obtained
post-mortem.
4.2. Titin-isoform switch in HFpEF
vs. HFrEF
The pattern of titin-isoform expression correlates with systolic and
diastolic functional parameters in patients, including LV end-diastolic
wall stiffness,
6
EF, EDV, and ESV.
10
Titin-isoform shift towards the
more compliant N2BA variants occurs in end-stage failing hearts of
patients with ischaemic cardiomyopathy
9
or non-ischaemic dilated
cardiomyopathy (DCM).
10,11
In contrast, we found a modest decrease
in the proportion of N2BA titin in HFpEF dogs. Similarly, a decreased
N2BA proportion has been reported in rapid pacing canine models of
DCM.
34,35
Possibly, then, dog hearts are unique in their remodelling
response to mechanical stress. However, in aortic stenosis patients,
the N2BA proportion was also lowered compared with donor
hearts
36
—although the opposite result was found elsewhere.
7
In
human hypertrophic cardiomyopathy, the cardiac titin-isoform
pattern did not change compared with donor hearts,
37
whereas spon-
taneously hypertensive rats expressed slightly less N2BA proportions
than normotensive rat hearts.
38
In summary, a decreased N2BA:N2B
titin expression ratio may be a frequent (albeit not general) feature of
hypertrophied hearts, including those developing HFpEF. Titin switch-
ing towards the N2B isoform increases cardiomyocyte F
passive
in
HFpEF (this study), switching towards the N2BA isoform decreases
F
passive
in HFrEF.
9–11
4.3. Titin phosphorylation and F
passive
in HFpEF
Like human end-stage failing hearts,
7,14
dog HFpEF hearts showed a
deficit in phosphorylation of all-titin and at S4010 and S4099 within
titin’s cardiac-only N2Bus. Importantly, ex-vivo administration of
cGMP-dependent PKG corrected the all-titin phosphorylation
deficit in HFpEF heart tissue and administration of PKA or
cGMP-dependent PKG reduced the pathologically increased F
passive
of skinned OHT cardiomyocytes to CTRL levels. Since PKA activity
was unaltered among the dog groups, the deficit in all-titin phosphor-
ylation in HFpEF hearts may, at least partly, be a deficit in
PKG-mediated phosphorylation, causing the higher-than-normal
F
passive
. Lowering F
passive
via increased PKG-mediated phosphorylation
at the titin N2Bus, which improves diastolic function in these dog
hearts,
15
could thus be a useful therapeutic approach in HFpEF.
Against the reduced phosphorylation of all-titin, the PKCa-
dependent phosphosite at S11878 within the PEVK-titin segment
was hyperphosphorylated. Active PKCacan be increased in HF
17
and an elevated PKCaactivity was also apparent in the HFpEF
dog hearts. Because PKCa-dependent phosphorylation at titin-
S11878 (PEVK) increases cardiomyocyte F
passive
,
16
hyperphosphory-
lation at this site presumably added to the higher-than-normal F
passive
in HFpEF.
The increased cardiomyocyte F
passive
in dog HFpEF is consistent
with previous reports of elevated F
passive
in HFpEF patients
39
or
those with diabetes
40
or under b-blockade.
29
Regarding HFrEF,
either reduced
9,11
or elevated
7
F
passive
has been observed compared
with donor hearts. The decreased F
passive
in human HFrEF was
explained by a titin-isoform shift towards the compliant N2BA var-
iants,
9,11
the increased F
passive
by depressed titin phosphorylation,
because administration of PKA lowered F
passive
to control levels.
7
These findings underscore the importance of both titin-isoform tran-
sitions and titin phosphorylation changes for cardiomyocyte F
passive
in
chronic HF. In dog HFpEF, the changes in titin phosphorylation may be
more important for altering LV passive stiffness than the relatively
small transitions in titin isoforms. In any case, we conclude that titin-
based F
passive
is increased in dog experimental HFpEF and contributes
to elevated LV passive stiffness, the hallmark of HFpEF.
4.4. Conclusions
This clinically relevant large-animal model of HFpEF is characterized
by cardiac titin-isoform switch towards the stiffer N2B variant, a
deficit in phosphorylation of all-titin and at specific serines within
the N2Bus-titin domain, but hyperphosphorylation at titin’s PEVK
domain. These alterations act synergistically to elevate cardiomyocyte
F
passive
. A stiffer titin may be a key determinant of diastolic dysfunction
N. Hamdani et al.470
by guest on January 12, 2016Downloaded from
resulting from increased LV passive stiffness in HFpEF. Regulatory
myofilament proteins are hypophosphorylated in dog HFpEF,
causing increased Ca
2+
sensitivity. A phosphorylation deficit for myo-
filament proteins could be an early and general event in the transition
to HF, thus unbalancing cardiac mechanical function. Reversing the
phosphorylation deficit by pharmacological manipulation of PK or
phosphatase (PP) signalling pathways may be a useful therapeutic
strategy in HFpEF.
Supplementary material
Supplementary material is available at Cardiovascular Research online.
Acknowledgement
We thank Dr Martina Kru¨ger and Dr Tobias Voelkel for their help
with titin antibody design.
Conflict of interest: none declared.
Funding
This work was supported by a European Union FP7 grant (MEDIA) as well
as a German Research Foundation grant (SFB 1002, TPB3) to W.A.L. and
aEuropean Society of Cardiology grant to N.H.
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