Single histidine-substituted cardiac troponin I
confers protection from age-related systolic
and diastolic dysfunction
Nathan J. Palpant1, Sharlene M. Day1,2, Todd J. Herron1, Kimber L. Converso3,
and Joseph M. Metzger1,2*
1Department of Molecular and Integrative Physiology, University of Michigan Medical School, 1301 E. Catherine Street, 7727
Medical Science II, Ann Arbor, MI 48109-0622, USA;2Department of Internal Medicine, University of Michigan Medical School,
Ann Arbor, MI 48109, USA; and3Department of Pediatrics, University of Michigan Medical School, Ann Arbor, MI 48109, USA
Received 27 November 2007; revised 30 June 2008; accepted 14 July 2008; online publish-ahead-of-print 16 July 2008
Time for primary review: 19 days
Aims Contractile dysfunction associated with myocardial ischaemia is a significant cause of morbidity
and mortality in the elderly. Strategies to protect the aged heart from ischaemia-mediated pump
failure are needed. We hypothesized that troponin I-mediated augmentation of myofilament calcium
sensitivity would protect cardiac function in aged mice.
Methods and results To address this, we investigated transgenic (Tg) mice expressing a histidine-
substituted form of adult cardiac troponin I (cTnI A164H), which increases myofilament calcium sensi-
tivity in a pH-dependent manner. Serial echocardiography revealed that Tg hearts showed significantly
improved systolic function at 4 months, which was sustained for 2 years based on ejection fraction and
velocity of circumferential fibre shortening. Age-related diastolic dysfunction was also attenuated in
Tg mice as assessed by Doppler measurements of the mitral valve inflow and lateral annulus Doppler
tissue imaging. During acute hypoxia, cardiac contractility significantly improved in aged Tg mice
made evident by increased stroke volume, end systolic pressure, and þdP/dt compared with non-
Conclusion This study shows that increasing myofilament function by means of a pH-responsive
histidine button engineered into cTnI results in enhanced baseline heart function in Tg mice over
their lifetime, and during acute hypoxia improves survival in aged mice by maintaining cardiac
Cardiovascular disease (CVD) is the leading cause of morbi-
dity and mortality worldwide, accounting for 16.7 million
deaths globally each year.1–3Advancing age is a major risk
factor for acquiring CVD.4–6Over one-third of all patients
with CVD are 65 years of age or older. Despite significant
advances in medical therapy and technology, ischaemic
heart disease in the elderly is associated with high mor-
tality.1,7Myocardial ischaemia results from inadequate
regional blood flow and leads to reduced contractility,
often leading to heart failure.
Myocardial ischaemia causes a drop in myocardial intra-
cellular pH which desensitizes the myofilament to cytosolic
fluxes, compromising myocyte contractility.8
pH-mediated changes in thin filament proteins, particularly
the interaction between troponin C and troponin I, are
primarily responsible for this reduction in myofilament
calcium responsiveness and resulting decrement in contrac-
tile function.9Troponin I (TnI), the inhibitory subunit of the
ternary troponin complex, is the Ca2þ-sensitive molecular
switch of the myofilament.10During diastole, the C-terminus
of TnI interacts with actin which allows tropomyosin to
sterically inhibit actomyosin
systole, a conformation change in the troponin complex
occurs wherein the C-terminal switch domain of TnI binds
to the hydrophobic patch of the Ca2þsaturated N-terminal
lobe of TnC.11This releases the inhibition on actomyosin
ATPase resulting in cross-bridge cycling and subsequent
Carboxy-terminal charge differences between the neo-
natal and adult isoforms of TnI, slow skeletal TnI and
cardiac TnI, respectively, are responsible for alterations in
pH sensitivity of the myofilament.12,13Recent studies indi-
cate that a critical histidine residue underlies TnI regulation
ATPase activity. During
*Corresponding author. Tel: þ1 734 763 0560; fax: þ1 734 647 6461.
E-mail address: email@example.com
Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2008.
For permissions please email: firstname.lastname@example.org.
Cardiovascular Research (2008) 80, 209–218
of myofilament pH sensitivity (Figure 1A).12–15When cardiac
TnI is modified to contain a histidine at codon 164, it
behaves as a titratable molecular switch regulating myofila-
ment tension development in response to biochemical
changes in the myocyte in vitro and in the whole heart
in vivo.15Importantly, this histidine substitution increases
myofilament sensitivity to activating calcium while retaining
important PKA phosphorylation sites on the N-terminus of
cardiac troponin I (cTnI). PKA-mediated phosphorylation of
TnI is a critical mediator of the b-adrenergic response in
the adult heart.16
Numerous studies have shown that increasing myofilament
calcium sensitivity through mutations in the troponin
complex results in varying forms of cardiomyopathy in the
context of normal physiological growth and function (e.g.
cTnI R193H, cTnT R92Q).17,18In contrast, we have shown
that cTnI A164H has no baseline pathology and significantly
protects cardiac function in response to a variety of patho-
physiological challenges including acute and chronic ischa-
emia.15Adding to the evidence supporting the therapeutic
role of histidine-modified troponin I, this paper is the first
to show that improvement in inotropy through modification
of a myofilament protein attenuates cardiovascular dysfunc-
tion in the aged heart in vivo.
2.1 Mouse model
Generation and analysis of transgenic (Tg) mice expressing a
histidine-modified cTnI A164H with FLAG epitope was previously
described (Figure 1A).15The University of Michigan is accredited
by the American Association of Accreditation of Laboratory Animal
Health Care, and the animal care use programme conforms to
the standards of the National Institutes of Health Guide for the
Care and Use of Laboratory Animals (NIH Pub. No. 85-23). (see
Supplemental Methods online).
2.2 Conductance micromanometry
Measurements of in vivo cardiovascular haemodynamics were
obtained using conductance micromanometry as previously per-
formed by this laboratory15(see Supplemental Methods online).
Two-dimensional, M-mode, Doppler and tissue Doppler echocardio-
graphic images were recorded using the Visual Sonics Vevo 770 high-
resolution in vivo micro-imaging system (see Supplemental Methods
2.4 Adult mouse myocyte isolation and analysis
Adult mouse cardiac myocytes were isolated from 3- to 6-month-old
mice and analysed for SR load as described previously19(see
Supplemental Methods online).
2.5 Immunoblot detection
The stoichiometric ratio of cTnI A164HFLAG to native cTnI was
accomplished by detection with the pan-TnI antibody MAB 1691
(Chemicon; 1:1000). Calcium-handling proteins were detected
Upstate), SERCA2a (MA3-919, ABR), NCX (11-13, Swant), and calse-
questrin (PA1-913, ABR). Phosphorylation status was performed by
Western blot using antibodies directed against the serine 16 phos-
phorylation site on phospholamban (07-052, Upstate) and the
serine 23/24 tandem phosphorylation sites on cardiac troponin I
(4004, Cell Signaling). Indirect immunodetection was carried out
using a fluorescently labelled secondary antibody (Rockland, IRDye
680 conjugated affinity purified; 1:5000). Western blot analysis
was accomplished using the infrared imaging system, Odyssey
(Li-Cor, Inc.) and images analysed using Odyssey software v. 1.2.
For protein detection, samples from young and aged non-transgenic
(Ntg) and Tg mice were run on the same gel. After protein transfer,
the gel was stained with Coomassie for use in quantification to
account for any loading differences.
All results are expressed as mean+SEM. All two-group comparisons
were assessed by two-tailed t-test. All multi-group comparisons
were assessed using two-way analysis of variance (ANOVA) with
Tukey post hoc test. Survival after the in vivo acute hypoxic chal-
lenge was assessed by the Fisher’s exact test.
3.1 cTnIA164H transgene expression increases in
aged murine hearts
Consistent with recent findings,156-month-old mice (line
892) showed high levels of stoichiometric replacement of
endogenous cTnI with cTnI A164H (78.6+3.5%) (Figure 1B).
Ageing of generation F2–F3 Tg mice to 2 years revealed
A164H transgenic mice. (A) Sequence alignment showing location of the
single histidine substitution originally studied in the slow skeletal isoform
of troponin I and subsequently introduced into cardiac troponin I (cTnI
A164HFLAG). Expression cassette of cDNA-encoding cardiac troponin I
A164H with an SV40 polyadenylation (pA) signal was driven by the 5.5 kb
Myh6 promoter. A C-terminal flag epitope was used to aid detection of
cardiac troponin I A164H. (B) Immunoblot and bar graph showing expression
of native cardiac troponin I (filled square) in non-transgenic mice as well as
stoichiometric incorporation of cardiac troponin I A164HFLAG (open square)
relative to endogenous cardiac troponin I (filled square) in transgenic mice
at 6 months (n ¼ 12) and 2 years (n ¼ 6). Values are expressed as mean+
SEM. *P , 0.05 for 6 month vs. 2 year replacement.
Age-dependent stoichiometric replacement in cardiac troponin I
N.J. Palpant et al.
a significant increase in cTnI A164H replacement relative to
native cTnI (94.6+3.1%; P , 0.05, Tg 6 vs. 24 month
expression) as determined by Western blotting (Figure 1B).
3.2 cTnI A164H improves global cardiac function
Systolic and diastolic cardiac function at 4 months, 1, and 2
years of age was assessed by serial echocardiography. Com-
parison of Ntg and Tg mice revealed significant differences
(Figure 2A and B). Compared with Ntg mice, Tg mice had sig-
nificantly higher contractility as measured by the velocity of
circumferential fibre shortening (Vcfc) (Figure 2B). Ejection
fraction correlated with the Vcfcduring this time course
further validating the increased inotropy of Tg mice
(Table 1). Tg mice also had significantly smaller LV diastolic
volume (Vol d) compared with Ntg mice at all ages
(Figure 2B). Although the ageing process had similar
effects on both cohorts resulting in an age-dependent
deterioration of cardiac function and geometry, cTnI
A164H Tg mice had sustained improvements in contractility
and reduced LV cavitary dilation compared with Ntg mice.
These conclusions were supported by ANOVA main effects
for genotype and age with measurements of contractility
(Vcfc), and cardiac geometry (Vol d) (P , 0.05).
Diastolic function was also assessed by conventional
Doppler of the mitral valve early (Ea) and late (Aa) waves
as well as Doppler tissue imaging (DTI) of the early (Ela)
and late (Ala) tissue velocities of the lateral annulus.
Although no differences in diastolic function were observed
at 4 months, age-related decrements in diastolic function
were observed in Ntg mice that were significantly attenu-
ated in Tg mice at 2 years. The lateral annular E wave
(Ela), as measured by DTI, was not statistically different
between groups (Table 1). The ratio of the mitral valve E
waveto thelateral annular
significantly lower in Tg mice compared with Ntg mice
at 2 years of age (Ntg vs. Tg: 51.0+3.8 vs. 37.8+4.1;
P , 0.05) (Figure 2B). Taken together, the progression of
diastolic dysfunction that accompanies the ageing process,
as observed in Ntg mice, was reduced by cTnI A164H result-
ing in an attenuation of diastolic dysfunction in aged Tg
mice. These conclusions were supported by ANOVA main
effects for genotype and age (P , 0.05) as well as an inter-
action effect (P , 0.05) for E/Ela.
implantation of a Millar catheter into the left ventricle for
measurements. Continuous monitoring of cardiac function
revealed significant differences in age-dependent baseline
haemodynamics between Ntg and Tg mice. Measurements
E wave (E/Ela)was
images along the parasternal short axis view showing changes in contractility of non-transgenic and transgenic mice between 4 months and 2 years (scale bar ¼
0.1 s). White bars delineate systole and diastole. (B) Summarized mean data showing age-related differences in cardiac function as assessed by echocardiography
including velocity of circumferential fibre shortening corrected for heart rate (Vcfc), volume at diastole (Vol d), and the ratio of the Mitral E wave to lateral
annular E wave (E/Ela) showing changes in cardiac function in non-transgenic (filled square) and transgenic (open square) mice at 4 months (non-transgenic,
n ¼ 26; transgenic, n ¼ 32), 1 year (non-transgenic, n ¼ 12; transgenic, n ¼ 13), and 2 years (non-transgenic, n ¼ 13; transgenic, n ¼ 15). Values are expressed
as mean+SEM. Two-way ANOVA main effects: age (double dagger) and genotype (single dagger): Vcfc, Vol d, E/Ela, P , 0.05. ANOVA interaction effects between
genotype and age (section symbol): E/Ela, *P , 0.05 for non-transgenic vs. transgenic at 2 years of age. (C) Summarized conductance micromanometry mean
data showing age-related differences in haemodynamic function including the ejection fraction (EF), time constant for relaxation (tau g), and heart rate of non-
transgenic (filled square) and transgenic (open square) mice at 4 months (n ¼ 12–14) and 2 years (n ¼ 3–4) of age. Values are expressed as mean+SEM. *P , 0.05
for non-transgenic vs. transgenic at 24 months. ANOVA main effects: genotype (single dagger): tau g, HR, P , 0.05; age (double dagger): EF, P , 0.05. Ntg, non-
transgenic; Tg, transgenic.
Age-dependent changes in baseline cardiac function by echocardiography and Millar catheterization. (A) Representative M-mode echocardiographic
cTnI A164H prevents age-related cardiac dysfunction
of systolic function, such as the positive derivative of
pressure development (data not shown) and ejection frac-
tion, were comparable between groups (Figure 2C). In
terms of diastolic function, conductance micromanometry
measurements were inconclusive based on the rate constant
for isovolumic relaxation (Tau) which showed either an
increase [Tau Glanz (Figure 2C)] or no change [Tau Weiss
(Table 1)] during the ageing process. A significant decrease
in heart rate was also observed in 2-year-old Tg mice com-
pared with their Ntg littermates (Figure 2C).
Analysis of heart-weight-to-body-weight ratio revealed
significant age-dependent changes (Table 2). At 4 months
of age Tg mice had significantly lower body weights and
heart weights compared with Ntg mice (4 month HW/BW
ratio: 4.6+0.1 vs. 4.0+0.1; Ntg vs. Tg; P , 0.05).
However, at 2 years the HW/BW ratio was similar between
Tg and Ntg mice (2 year HW/BW ratio: 5.6+0.2 vs.
5.9 + 0.4; Ntg vs. Tg).
3.3 cTnI A164H aged mice retain cardiac
haemodynamics during hypoxia
Previous studies have shown that young cTnI A164H Tg mice
sustain cardiac function during an acute hypoxic challenge
and consequently survive significantly longer than Ntg
mice.15To assess the physiological and therapeutic role of
cTnI A164H in the aged murine heart, mice were exposed
to conditions of controlled hypoxia (12% O2) during Millar
catheterization and assessed for global cardiac function
and survival (Figures 3 and 4). After 4 min of hypoxia, raw
traces of LV pressure (LVP), the derivatives of LV pressure
development (dP/dt) and volume, together with their corre-
sponding pressure–volume loops, revealed that aged Ntg
mice have significantly compromised cardiac haemody-
namics compared with aged Tg mice (Figure 3A and B).
Mean haemodynamic data taken at 4 min into the acute
hypoxic challenge and the delta change from baseline
(Table 1) to hypoxia showed improved systolic and diastolic
performance ofTg mice
(Figure 3C). Specifically, compared with their Ntg litter-
mates, LV performance was maintained during hypoxia in
Tg mice as represented by higher stroke work (SW), end sys-
tolic pressure (ESP), and the positive derivative of pressure
development (dP/dt max) (Figure 3C). Compared with base-
line values, aged Tg mice showed improved contractility
during hypoxia compared with a significant decrease in con-
tractile function in aged Ntg mice based on measurements of
stroke work (D SW), and the positive derivative of pressure
development (D dP/dt max) (Figure 3C). The negative
derivative of pressure development (dP/dt min) was also
maintained during an acute hypoxic challenge in Tg mice
(data not shown). Finally, compared to Ntg littermates, Tg
mice were able to sustain global cardiac function during
hypoxia as shown by a higher stroke volume (SV) and
cardiac output (D CO), (Figure 3C). These data show
improved cardiac function in response to hypoxia in Tg
mice compared with their Ntg littermates (Figure 4A).
Consistent with improved haemodynamic function, aged Tg
mice survived significantly longer during an acute hypoxic
challenge than Ntg mice (11.6+1.3 vs. 3.7+1.9 min,
P , 0.05) (Figure 4B).
compared with Ntg mice
3.4 Calcium cycling is different between Ntg and
Functional analysis of calcium handling in acutely isolated
cardiac myocytes showed that under conditions of 1 Hz
pacing, young Tg mice had a reduced calcium transient
amplitude compared with young Ntg myocytes (Figure 5A
and C), consistent with previous reports.15Furthermore,
experiments were performed to measure SR calcium load
by means of acute addition of 20 mM caffeine (Figure 5B
and C). Similar to baseline calcium transients, these data
show that SR calcium load was lower in Tg vs. Ntg mice
(Figure 5A–C). This supports the hypothesis that increased
myofilament calcium sensitivity reduces calcium require-
ments in the cell and may contribute to the protection
of Tg hearts during ageing as well as cardiomyopathic
challenges such as ischaemia/reperfusion injury.15
Heart and body weight analysis
Heart weight (mg)
Body weight (g)
Heart weight (mg)
Body weight (g)
All values are expressed as mean+SEM.
*P , 0.05.
Baseline cardiac function of aged mice
Stroke volume (mL)
(mmHg ? mL)
End systolic pressure
dP/dt min (mmHg/s)
Tau w (ms)
11 440.7+1542.2 12 471.4+2984.3
MV E (m/s)
MV A (cm/s)
For conductance micromanometry measurements n ¼ 3–4/group. For
echocardiography measurements n ¼ 13–15/group. All values are from
animals aged to 2 years. Echocardiography parameters: Doppler tissue
imaging of the early (Ela) and late (Ala) tissue velocities of the lateral
annulus; conventional Doppler imaging of the mitral valve early (MV E)
and late (MV A) filling velocities; ejection fraction (EF). All values are
expressed as mean+SEM.
Ntg, non-transgenic; Tg, transgenic.
*P , 0.05 for Ntg vs. Tg.
N.J. Palpant et al.
3.5 Calcium-handling protein expression and
Western blot analysis was performed to assess changes in
calcium-handling proteins between young (2–4 months)
and aged (24 months) Ntg and Tg mice (Figure 6A and B).
Alterations in the expression of proteins specifically involved
in the regulation of calcium homoeostasis have been
established as one of the factors that contribute to the
These data show that calsequestrin (CSQ), the sodium
calcium exchanger (NCX), and phospholamban (PLN) were
not altered during ageing by two-way ANOVA. In contrast,
SERCA2a was reduced in both Ntg and Tg aged mice based
on a significant ANOVA main effect for age (P , 0.05), a
finding that is consistent with previous studies of the
ageing heart.26,27Furthermore, these data show that the
SERCA2a/PLN ratio was not different between Ntg and Tg
mice during the ageing process (Figure 6B). Although the
SERCA2a levels are elevated in Tg hearts by Western blot
(based on an ANOVA main effect for genotype), in vitro
calcium-handling measurements indicate a reduced level
of SR calcium load in Tg hearts (Figure 5).
for changes in protein phosphorylation (Figure 6A and B).
These data showing serine 16 phosphorylation of phospholam-
ban indicate that aged mice have a higher baseline level of
nificant ANOVA main effect for age. cTnI serine 23/24 phos-
sweeps derived by conductance micromanometry of left ventricular pressure (LVP), left ventricular pressure derivatives (dP/dt), and left ventricular volume
of 2-year-old non-transgenic and transgenic mice during an acute hypoxic challenge (12% O2). (B) Representative pressure–volume loops in vivo of non-transgenic
(grey) and transgenic (black) mice during hypoxia. (C) Mean data showing haemodynamic function as well as the delta change (D, right side axes) from baseline
values for cardiac output (CO), stroke volume (SV), stroke work (SW), end systolic pressure (ESp), and positive pressure derivatives (dP/dt max) derived by Millar
catheterization of 2-year-old non-transgenic (filled square; n ¼ 3) and transgenic (open square; n ¼ 3) mice. *P , 0.05. Values are expressed as mean+SEM. Ntg,
nontransgenic; Tg, transgenic.
In vivo haemodynamic differences between aged non-transgenic and transgenic mice during an acute hypoxic challenge. (A) Two second raw data
cTnI A164H prevents age-related cardiac dysfunction
This study focused on the physiological impact of histidine-
modified cTnI A164H in aged mice. These data provide
new evidence that molecular manipulation of myofilament
calcium sensitivity can preserve heart function during
ageing. Specifically, this study showed that age-related
decrements in baseline cardiac systolic function were signifi-
cantly attenuated in Tg mice compared with Ntg litter-
mates. Echocardiography studies also demonstrated that
age-dependent diastolic dysfunction (elevated E/Ela) was
including end systolic pressure (ESp), stroke volume (SV), cardiac output (CO), stroke work (SW), and the positive derivative of pressure development (dP/dt
max) during the time course of an acute hypoxic challenge for non-transgenic (filled circle; n ¼ 3) and transgenic (open circle; n ¼ 3) aged mice. (B) Summarized
mean survival data and survival curve showing time to systolic heart failure for 2-year-old non-transgenic (n ¼ 3) and transgenic (n ¼ 3) mice. Values are
expressed as mean+SEM. *P , 0.05.
In vivo haemodynamic function and survival of aged mice during an acute hypoxic challenge. (A) Averaged left ventricular haemodynamic function
(left and middle) as well as raw calcium transient traces normalized to the peak amplitude (right). (B) Raw calcium transients after acute addition of 20 mM
caffeine to release sarcoplasmic reticulum calcium (horizontal black bars). (C) Summarized data from calcium-handling experiments (n ¼ 20–24 myocytes
per group). Values are expressed as mean+SEM. *P , 0.05 for non-transgenic vs. transgenic. Ntg, nontransgenic; Tg, transgenic.
Analysis of calcium homoeostasis in young mice. (A) Raw traces of calcium transients from FURA 2AM loaded isolated myocytes during pacing with 1 Hz
N.J. Palpant et al.
attenuated in Tg animals. Importantly, when exposed to an
acute hypoxic challenge in vivo, aged Tg mice maintained
cardiac performance that significantly extended their
survival compared with Ntg mice. These data support the
hypothesis that myofilament-based enhanced function by
single histidine-modified cTnI provides a mechanism for
attenuating age-related decrements in cardiac function.
Increasing calcium sensitivity of the myofilament conse-
quent to alterations in the biochemistry of cTnI has been
well studied.10,12,13,15,16,28A recent report of the crystal
structure of cTnI places Ala164 at the critical switch
domain that is key in regulating myofilament calcium acti-
vation.11Codon 164 is situated at the interface between
the amphiphilic switch region, H3, and the C-terminal actin-
binding domain, H4. These regions are defined as the regulat-
ory segment. In the calcium-saturated state, the entire
regulatory segment of cTnI (residues 137–210) undergoes a
conformational change concomitant with binding of the H3
domain to the conserved N-terminal hydrophobic patch of
TnC. In the slow skeletal isoform of TnI (ssTnI), which is the
foetal/neonatal isoform in mammals, a unique biochemical
characteristic of the switch region was found to confer
pH-dependent increases in calcium sensitivity to the myofila-
ment.29,30In initial studies, mutagenesis experiments ident-
ified a critical histidine at position 132 as responsible for
this pH-dependent functional outcome.12,14,15The physiologi-
cal effects of this mutation in the heart directly reflect the
imidazole moiety which, unique among all the amino acids,
ionizes within the physiological range (pKR¼ 6.0). Numerous
studies have shown that under conditions of acidosis, ssTnI
protects contractility of myocytes in vitro and the whole
heart in vivo.12,14,29–32In light of these findings, we sought
to introduce this pH sensitivity into the switch domain of
cTnI. An engineered histidine modification in codon 164 of
cTnI (A164H) takes advantage of this critical functional
property of ssTnI transposed into the context of cTnI. This
modification takes place without altering important physio-
logical functions of cTnI in regulating global cardiac inotropic
and lusitropic responses to b-adrenergic signalling.15Cardiac
TnI A164H provides a titratable molecular switch mechanism
regulating myofilament tension development in response to
biochemical changes in the adult myocyte. One of the
central hypotheses of this study was that increasing myofila-
ment calcium sensitivity by means of cTnI A164H is a powerful
molecular therapy for attenuating morbidity and mortality
failure in aged mice.
Declining cardiac function contributes to waning health in
geriatric populations.5In many cases these age-related
changes are secondary to coronary artery disease.6Of par-
ticular salience to this study is the propensity for chronic
ischaemia associated with vascular stenosis to contribute to
poor cardiovascular health of the elderly.4A portion of this
study was based on an experimental protocol to impose a
controlled state of hypoxia in aged mice through low
inhaled oxygen. In the clinical setting, hypoxia is usually sec-
ondary to cardio-pulmonary pathologies frequently seen in
geriatric populations. Physiologically these conditions are
usually due to hypoventilation, vascular shunting, venti-
defects. Clinical diseases of the lungs such as COPD, pneumo-
nia, interstitial lung disease, or pulmonary embolic disease
transgenic and transgenic mice. (A) Western blots of calcium-handling
proteins including the sarco-endoplasmic reticulum ATPase (SERCA2a), phos-
pholamban (PLN), the sodium calcium exchanger (NCX), serine 16 phosphoryl-
ation of phospholamban (pPLN), and tandem serine 23/24 phosphorylation of
troponin I (pTnI) for young and aged non-transgenic and transgenic mice. Coo-
massie was used to normalize for protein loading (W, Western; C, Coomassie).
(B) Mean summary data for differences in the expression of calcium-handling
proteins including CSQ, NCX, PLN, SERCA2a, the SERCA2a/PLN ratio, pPLN,
and pTnI. Two-way ANOVA main effects (P , 0.05): age (double dagger),
SERCA2a, and pPLN; and genotype (single dagger), PLN, SERCA2a. Ntg, non-
transgenic; Tg, transgenic.
Changes in protein expression and phosphorylation in non-
cTnI A164H prevents age-related cardiac dysfunction
are common conditions associated with hypoxia which may
lead to secondary ischaemic cardiac injury. The aged heart
is particularly at risk in these settings because of intrinsic
muscle disease and underlying coronary artery disease with
areas of marginally perfused myocardium. These clinically
relevant aetiologies, known risk factors for CVD, provide con-
textual relevance for the key findings of this study. These
important results include the finding that aged cTnI A164H
Tg mice have enhanced cardiac function and extended survi-
val capacity during an acute hypoxic challenge compared
with Ntg mice.
Irrespective of the aetiology, systolic and diastolic dys-
function are strong predictors of heart failure in geriatric
populations.33In this study we show that cTnI A164H Tg
mice have improved contractility during the ageing process
compared with Ntg mice. The equalization of the HW/BW
ratio at 2 years of age neutralizes any heart size-dependent
between groups at this time point. This lends credence to
the conclusion that Tg mice, which retained the same rela-
tive increase in contractility over Ntg mice during the ageing
process, actually underwent an age-dependent and heart
size-independent increase in absolute contractility. This
could be explained in part by the increase in stoichiometric
incorporation of cTnI A164H in old vs. young Tg mice and
could provide mechanistic basis for the observed increase
in contractility at 2 years of age. Additionally, compared
with Tg mice, increased LV cavitary dilation in aged Ntg
mice may also, at least in part, contribute to the divergence
of contractile efficiency at the 2-year time point. A shift
towards higher sarcomere lengths, which occurs with
dilated cardiomyopathies and may be a component of
age-related dysfunction, could reduce the efficiency of con-
tractility based on the Frank–Starling principle.
In addition to systolic dysfunction, decrements in diastolic
function are also characteristic of the ageing process. LV
dilation together with stiffening of the aged myocardium
prevents sufficient relaxation of the heart during the filling
phase of the cardiac cycle. Thus, another key finding of
this study is that cTnI A164H protects diastolic function
during the ageing process. These data indicate that Ntg
mice show evidence of diastolic dysfunction based on a
decrease in the velocity of the lateral annulus during the
early filling phase (Ela) of the LV as well as an increase in
the mitral valve E wave flow velocity to lateral annular E
wave ratio (E/Ela). This latter parameter (E/Ela) shows
the ratio of the inflow velocity to the tissue velocity provid-
ing insight into the elastic properties of the ventricle. In
essence, the E wave velocity controls for cardiac output,
heart rate, and filling so that the ratio (E/Ela) correlates
with left atrial pressure. Here, the E/Ela shows evidence
of the transgene altering the typical progression of diastolic
dysfunction based on a significant interaction effect
(P , 0.05). These data indicate that cTnI A164H Tg mice
are able to retain improved diastolic function during the
ageing process compared with Ntg mice which experience
predictable age-related diastolic dysfunction.
The relationship between cTnI A164H and diastolic per-
formance, however, is complex as indicated by differences
in echocardiographic and micromanometry data at baseline.
Conflicting data based on the measures of isovolumic relax-
ation (Tau) provide inconclusive evidence regarding baseline
diastolic function in Tg mice, which is consistent with pre-
vious findings.15However, the mechanical component of
relaxation during the late (filling) phase of diastole, as
measured non-invasively by DTI indicates that Ntg mice
have compromised viscoelastic properties of the ventricle
resulting in a decline in diastolic function during the
ageing process which is significantly attenuated in Tg mice.
Taking this whole organ functional analysis into consider-
ation, it has been established that, at the sub-cellular
level, changes in the expression of calcium-handling pro-
teins contribute to the progression of pump dysfunction
during ageing.34Our study supports these findings specifi-
cally with regard to the observation that SERCA2a levels
are diminished at 2 years of age in Ntg and Tg mice.
Reduced expression of SERCA2a has been implicated directly
in the progression of age-induced cardiomyopathy26and pro-
vides a sub-cellular basis, at least in part, for the whole
organ diastolic dysfunction observed in this study during
ageing. The lack of any genotypic differences in CSQ, NCX
and the SERCA2a to PLN stoichiometry during ageing
suggests an alternative mechanism for improvement of
cardiac function in the Tg mice during the ageing process.
We propose that this mechanism is predominantly the
result of increasing inotropy by molecular manipulation of
myofilament performance by means of cTnI A164H.
The results of this study show that Tg hearts have reduced
SR calcium loading. Previous reports have found that aged
hearts are more susceptible to calcium overload than
young hearts.3,35,36This suggests that a reduced SR Ca2þ
load may benefit cTnI A164H hearts in ageing, similar to
our recent report in the context of myocardial injury such
as ischaemia/reperfusion.15We hypothesize that the lower
SR Ca2þload and Ca2þtransient are made possible by
myofilament activation enhancement by cTnIA164H. We
propose that there is an interplay between the myofilament
enhancement and SR functionality that could account for
higher SERCA2a levels in Tg hearts.
The cardiac functional readout of these findings at the
sub-cellular level is seen in the whole organ serial echo-
cardiographic analysis of Ntg and Tg mice during the
2-year ageing process. The decline in cardiac contractility
(e.g. EF) increase in LV chamber geometry and development
of diastolic dysfunction (e.g. increased E/Ela) particularly
evident at the 2-year time point are likely the consequence,
at least in part, of the changes observed in calcium-handling
In conclusion, ischaemia-related cardiac dysfunction in
aged populations remains a significant cause of morbidity
and mortality. Therapies for ischaemic heart disease and
heart failure could be directed specifically towards improv-
ing myofilament function. The consequent improvement in
contractility that results from increased myofilament per-
formance may appear to counter the logic of commonly
prescribed therapeutics, which call for the use of beta
blockers that decrease contractility acutely, allowing for
reduced oxygen consumption and energy expenditure.
Although there is proven value in the diverse effects of
beta blockers, we have shown that targeted alteration of
the myofilaments to increase contractile performance is
an effective mechanism for treatment of ischaemic heart
disease and heart failure in small mammals.15This is in
concurrencewith Mann andBristow’sview that
N.J. Palpant et al.
augmentation of myofilament responsiveness to calcium
would improve the force-generating capacity of the sarco-
mere and thus redress global cardiac dysfunction.37The
present study, together with previous work,15strengthens
the hypothesis that altering the functionality of troponin I
is an effective means of specifically augmenting myofila-
ment functionand consequently
contractility. Adding to the growing evidence for the
therapeutic role of histidine-modified TnI in the heart, this
study provides new evidence that specific replacement of
native cTnI with cTnI A164H in the adult heart protects
cardiac function during ageing. We propose that the
progression of pathologic and age-related diminutions in
cardiac function may be improved by myofilament- based
molecular therapeutics for increasing cardiac performance.
Supplementary material is available at Cardiovascular Research
The authors acknowledge support from National Institutes of Health
(RO1 HL 059301 to J.M.) and American Heart Association (0715632Z
We thank Jaime Predmore for technical expertise in mouse
myocyte isolation with which SR calcium load experiments were
performed. We appreciate Dr Mark Russell for his assistance with
interpretation of echocardiography data and Dr Margaret Westfall
for helpful comments. We also thank Dr Samuel Palpant for his
medical advice regarding cardio-pulmonary diseases in geriatric
Conflict of interest: none declared.
1. Michaud CM, Murray CJL, Bloom BR. Burden of disease–implications for
future research. JAMA 2001;285:535–539.
2. Thom T, Haase N, Rosamond W, Howard VJ, Rumsfeld J, Manolio T et al.
Heart disease and stroke statistics–2006 update: a report from the
American Heart Association Statistics Committee and Stroke Statistics
Subcommittee. Circulation 2006;113:e85–e151.
3. Jahangir A, Sagar S, Terzic A. Aging and cardioprotection. J Appl Physiol
4. Lakatta EG. Arterial and cardiac aging: major shareholders in cardio-
vascular disease enterprises. Part III: cellular and molecular clues to
heart and arterial aging. Circulation 2003;107:490–497.
5. Lakatta EG, Levy D. Arterial and cardiac aging: major shareholders in
cardiovascular disease enterprises: Part II: the aging heart in health:
links to heart disease. Circulation 2003;107:346–354.
6. Lakatta EG, Levy D. Arterial and cardiac aging: major shareholders in
cardiovascular disease enterprises. Part I: aging arteries: a ‘set up’ for
vascular disease. Circulation 2003;107:139–146.
7. Deedwania PC. The key to unraveling the mystery of mortality
in heart failure – an integrated approach. Circulation 2003;107:
8. Parsons B, Szczesna D, Zhao J, Van Slooten G, Kerrick WG, Putkey JA
et al. The effect of pH on the Ca2þaffinity of the Ca2þregulatory sites
of skeletal and cardiac troponin C in skinned muscle fibres. J Muscle
Res Cell Motil 1997;18:599–609.
9. Ball KL, Johnson MD, Solaro RJ. Isoform specific interactions of troponin I
and troponin C determine pH sensitivity of myofibrillar Ca2þactivation.
10. Farah CS, Reinach FC. The troponin complex and regulation of muscle
contraction. FASEB J 1995;9:755–767.
11. Takeda S, Yamashita A, Maeda K, Maeda Y. Structure of the core domain
of human cardiac troponin in the Ca2þ-saturated form. Nature 2003;424:
12. Westfall MV, Borton AR, Albayya FP, Metzger JM. Specific charge
differencesin troponinI isoforms
sensitivity of tension in adult cardiac myocytes. Biophys J 2001;80:
13. Dargis R, Pearlstone JR, Barrette-Ng I, Edwards H, Smillie LB. Single
mutation (A162H) in human cardiac troponin I corrects acid pH sensitivity
of Ca2þ-regulated actomyosin S1 ATPase. J Biol Chem 2002;277:
14. Westfall MV, Metzger JM. Single amino acid substitutions define isoform-
specific effects of troponin I on myofilament Ca2þand pH sensitivity.
J Mol Cell Cardiol 2007;43:107–118.
15. Day SM, Westfall MV, Fomicheva EV, Hoyer K, Yasuda S, La Cross NC et al.
Histidine button engineered into cardiac troponin I protects the ischemic
and failing heart. Nat Med 2006;12:181–189.
16. Metzger JM, Westfall MV. Covalent and noncovalent modification of
thin filament action: the essential role of troponin in cardiac muscle
regulation. Circ Res 2004;94:146–158.
17. Davis J, Wen H, Edwards T, Metzger JM. Thin filament disinhibition by
restrictive cardiomyopathy mutant R193H troponin I induces Ca2þ-
independent mechanical tone and acute myocyte remodeling. Circ Res
18. Tardiff JC, Hewett TE, Palmer BM, Olsson C, Factor SM, Moore RL et al.
Cardiac troponin T mutations result in allele-specific phenotypes in a
mouse model for hypertrophic cardiomyopathy. J Clin Invest 1999;104:
19. Coutu P, Bennett CN, Favre EG, Day SM, Metzger JM. Parvalbumin
corrects slowed relaxation in adult cardiac myocytes expressing hyper-
trophic cardiomyopathy-linked alpha-tropomyosin mutations. Circ Res
20. Hasenfuss G, Pieske B. Calcium cycling in congestive heart failure. J Mol
Cell Cardiol 2002;34:951–969.
21. Koretsune Y, Marban E. Cell calcium in the patho-physiology of
ventricular-fibrillation and in the pathogenesis of postarrhythmic
contractile dysfunction. Circulation 1989;80:369–379.
22. Lakatta EG, Sollott SJ. Perspectives on mammalian cardiovascular aging:
humans to molecules. Comp Biochem Physiol A Mol Integr Physiol 2002;
23. Lee JA, Allen DG. Mechanisms of acute ischemic contractile failure of the
heart. Role of intracellular calcium. J Clin Invest 1991;88:361–367.
24. Wehrens XHT, Lehnart SE, Marks AR. Intracellular calcium release and
cardiac disease. Annu Rev Physiol 2005;67:69–98.
25. Kranias EG, Bers DM. Calcium and cardiomyopathies. Subcell Biochem
26. Hasenfuss G, Meyer M, Schillinger W, Preuss M, Pieske B, Just H. Calcium
handling proteins in the failing human heart. Basic Res Cardiol 1997;
27. del Monte F, Hajjar RJ, Harding SE. Overwhelming evidence of the
beneficial effects of SERCA gene transfer in heart failure. Circ Res
28. Solaro RJ, el Saleh SC, Kentish JC. Ca2þ, pH and the regulation of cardiac
myofilament force and ATPase activity. Mol Cell Biochem 1989;89:
29. Westfall MV, Metzger JM. Troponin I isoforms and chimeras: tuning the
molecular switch of cardiac contraction. News Physiol Sci 2001;16:
30. Westfall MV, Albayya FP, Turner II, Metzger JM. Chimera analysis of
troponin I domains that influence Ca2þ-activated myofilament tension
in adult cardiac myocytes. Circ Res 2000;86:470–477.
31. Westfall MV, Rust EM, Metzger JM. Slow skeletal troponin I gene transfer,
expression, and myofilament incorporation enhances adult cardiac
myocyte contractile function. Proc Natl Acad Sci USA 1997;94:
32. Urboniene D, Dias FAL, Pena JR, Walker LA, Solaro RJ, Wolska BM.
Expression of slow skeletal troponin I in adult mouse heart helps to main-
tain the left ventricular systolic function during respiratory hypercapnia.
Circ Res 2005;97:70–77.
33. Redfield MM, Jacobsen SJ, Burnett JC Jr, Mahoney DW, Bailey KR,
Rodeheffer RJ. Burden of systolic and diastolic ventricular dysfunction
in the community: appreciating the scope of the heart failure epidemic.
influence myofilament Ca2þ
cTnI A164H prevents age-related cardiac dysfunction
34. Hunter WC. Role of myofilaments and calcium handling in left ventricular
relaxation. Cardiol Clin 2000;18:443–457.
35. Ataka K, Chen D, Levitsky S, Jimenez E, Feinberg H. Effect of aging on
intracellular Ca2þ, pHi, and contractility during ischemia and reperfu-
sion. Circulation 1992;86(Suppl. 5):II371–II376.
36. Frolkis VV, Frolkis RA, Mkhitarian LS, Shevchuk VG, Fraifeld VE,
Vakulenko LG et al. Contractile function and Ca2þtransport system of
myocardium in ageing. Gerontology 1988;34:64–74.
37. Mann DL, Bristow MR. Mechanisms and models in heart failure: the
biomechanical model and beyond. Circulation 2005;111:2837–2849.
N.J. Palpant et al.