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Abnormal heart rate regulation in murine hearts with familial hypertrophic

cardiomyopathy-related cardiac troponin T mutations

Article  in  AJP Heart and Circulatory Physiology · February 2011

DOI: 10.1152/ajpheart.00247.2010 · Source: PubMed

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Abnormal heart rate regulation in murine hearts with familial hypertrophic
cardiomyopathy-related cardiac troponin T mutations
Jesus Jimenez1 and Jil C. Tardiff1,2
1
Department of Physiology and Biophysics and 2Department of Internal Medicine, Division of Adult Cardiology, Albert
Einstein College of Medicine, Bronx, New York
Submitted 10 March 2010; accepted in final form 30 November 2010
Jimenez J, Tardiff JC. Abnormal heart rate regulation in murine hearts
with familial hypertrophic cardiomyopathy-related cardiac troponin T muta-
tions. Am J Physiol Heart Circ Physiol 300: H627–H635, 2011. First
published December 3, 2010; doi:10.1152/ajpheart.00247.2010.—Mutations
in cardiac troponin T (cTnT), ⌬160E and R92Q, have been linked to
familial hypertrophic cardiomyopathy (FHC), and some studies have
indicated that these mutations can lead to a high incidence of sudden
cardiac death in the relative absence of significant ventricular hyper-
trophy. Alterations in autonomic function have been documented in
patients with hypertrophic cardiomyopathy. We hypothesize that al-
terations in autonomic function may contribute to mutation-specific
clinical phenotypes in cTnT-related FHC. Heart rate (HR) variability
(HRV) has been used to assess autonomic function from an electro-
cardiograph. Nontransgenic, ⌬160E, or R92Q mice were implanted
with radiofrequency transmitters to obtain continuous electrocardio-
graph recordings during 24-h baseline and 30-min recordings after
␤-adrenergic receptor drug injections. Although ⌬160E mice did not
differ from nontransgenic mice for any 24-h HRV measurements,
R92Q mice had impaired HR regulation, as measured by a decrease in
the SD of the R-R interval, a decrease in the low frequency-to-high
frequency ratio, a decrease in normalized low frequency, and an
increase in normalized high frequency. ␤-Adrenergic receptor density
measurements and HRV analysis after drug injections did not reveal
any significant differences for ⌬160E or R92Q mice versus nontrans-
genic mice. Arrhythmia analysis revealed both an increased incidence
of heart block in R92Q mice at baseline and frequency of premature
ventricular contractions after isoproterenol injections in ⌬160E and
R92Q mice. In addition, ⌬160E and R92Q mice exhibited a prolonged
P duration after drug injections. Therefore, between two independent
and clinically severe cTnT mutations within the same functional
domain, only R92Q mice exhibited altered autonomic function,
whereas both mutations demonstrated abnormalities in conduction
and ventricular ectopy.
sudden cardiac death; telemetry; ␤-adrenergic receptors
NATURALLY OCCURRING MUTATIONS in cardiac troponin T (cTnT)
are associated with familial hypertrophic cardiomyopathy
(FHC), an autosomal dominant primary myocardial disorder
(20). FHC patients with the cTnT missense mutation Arg92Gln
(R92Q) or an in-frame deletion of a single Glu residue (⌬160E)
in the TNT1 tail domain have been reported to exhibit signif-
icant degrees of sudden cardiac death, often in the absence of
massive left ventricular hypertrophy or precedent symptoms
(37). Transgenic (TG) murine models with the ⌬160E and
R92Q cTnT mutations that have been generated in our labo-
ratory recapitulate many aspects of human FHC and have
allowed us to longitudinally study these specific FHC muta-
Address for reprint requests and other correspondence: J. C. Tardiff, Depts.
of Physiology and Biophysics and Internal Medicine (Adult Cardiology),
Albert Einstein College of Medicine, 1300 Morris Park Ave., Ullmann Bldg.
316, Bronx, NY 10803 (e-mail: j.tardiff@einstein.yu.edu).
http://www.ajpheart.org 0363-6135/11 Copyright © 2011 the American Physiological Society
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Am J Physiol Heart Circ Physiol 300: H627–H635, 2011.
First published December 3, 2010; doi:10.1152/ajpheart.00247.2010.
tions in depth (3, 4, 10, 14, 21, 31). Although some phenotypic
similarities exist between ⌬160E and R92Q, including dia-
stolic dysfunction, a lack of overt cardiac hypertrophy, and
cardiomyocyte disarray, the mutations are at opposite ends of
the TNT1 tail domain and have been postulated to cause
disease via distinct molecular mechanisms (24).
It is unclear how mutations in cardiac sarcomeric proteins
and the resulting pathogenic process directly contribute to
sudden cardiac death; however, alterations in autonomic func-
tion have been proposed as a potential trigger (26). Autonomic
function can be assessed using noninvasive Holter monitoring
to determine heart rate (HR) variability (HRV) from an ECG.
Impaired autonomic function assessed via a decrease in HRV
has been demonstrated to be an independent risk factor in both
patients with heart failure and myocardial infarction (23). One
clinical finding observed in FHC patients that may suggest
autonomic dysfunction is an abnormal blood pressure response
after exercise and is represented by a failure to appropriately
increase blood pressure or a paradoxical drop in blood pressure
(28). Heradien et al. (11) recently found that patients with
R92W cTnT mutations were predisposed to an abnormal blood
pressure response to exercise that may be due to vagal en-
hancement. Moreover, changes in autonomic function have
been previously reported in patients with hypertrophic cardio-
myopathy (HCM); however, the authors of these studies found
limited value with HRV analysis of this patient population (6,
8, 22, 34, 40). Addressing the obstacles that were faced in
HCM studies may help elucidate the potential use of HRV
analysis to facilitate risk stratification in patients with FHC.
Our previous characterization of our two TG murine models
with the independent cTnT mutations R92Q, and ⌬160E,
suggested a possible mutation-specific alteration in HR regu-
lation by the autonomic nervous system. The present study was
conducted to determine if either of these mutations in cTnT
alters autonomic function, which can be assessed from HRV
analysis. In addition, we measured ␤-adrenergic receptor (␤-
AR) density, measured the response to ␤-AR drugs in these
mice, assessed arrhythmogenesis, and measured ECG param-
eters to determine possible mechanisms for any changes in HR
regulation. Assessment of autonomic function in ⌬160E and
R92Q mice may help establish a link between genotype and
phenotype using noninvasive HR monitoring and may provide
a potential prognostic screen in patients with FHC.
METHODS
Study animals. ⌬160E and R92Q mice were generated as previ-
ously described and compared against non-TG littermates (4, 31).
⌬160E and R92Q were bred on a congenic C57BL/6 background. The
⌬160E TG line expressed 70% of its total cTnT as the mutant form,
whereas the R92Q TG line had 67% cTnT replacement. To eliminate
H627
H628 HEART RATE REGULATION IN cTnT MUTANT HEARTS
the potential effect of sex, only male mice were studied for each
genotype. Twenty-nine 5 ⫾ 0.5-mo-old mice (11 non-TG, 10 ⌬160E,
and 8 R92Q mice) weighing 27 ⫾ 2 g at the time of surgery were
housed in individual cages at 24°C in 12:12-h light-dark cycles in full
compliance with the Public Health Service animal welfare policy and
the American Association for the Accreditation of Laboratory Animal
Care. The animal research protocol was approved by the Albert
Einstein College of Medicine Institute for Animal Studies.
Animal preparation and surgery. Long-term ECG analysis in
conscious, ambulatory mice was obtained with a wireless radiofre-
quency transmitter (model ETA-F20, Data Sciences, St. Paul, MN)
that was implanted in the peritoneal cavity using sterile techniques.
Mice were anesthetized with 1.5% inhaled isoflurane (Baxter Health-
care, Deerfield, IL) for the duration of the surgery. A midline skin
incision was made on the ventral abdomen, and a smaller incision was
made on the right pectoral region. A subcutaneous tunnel from the
abdominal incision to the pectoral incision was generated upon
removing a trochar from its sleeve to feed through the anodal lead. To
generate an ECG lead II configuration, the anodal lead was sutured on
the right pectoral muscle, and the cathodal lead was sutured near the
apex of the heart to the left of the xyphoid process. The 3.9-g
transmitter was inserted into the abdominal cavity and sutured to
the abdominal muscles to anchor it in place. The skin incisions were
sutured, and a warming lamp was used to maintain body temperature
for recovery.
Study protocol. ECG signal recordings began 8 ⫾ 2 days postsur-
gery. Mice had unrestricted access to food and water throughout the
recordings. Twenty-four-hour baseline recordings of undisturbed mice
transpired from 7 PM to 7 PM. To study the effects of various drugs,
intraperitoneal injections of 0.9% saline (IVX Animal Health, St.
Joseph, MO), 1.0 mg/kg body wt isoproterenol (Sigma-Aldrich, St.
Louis, MO) for ␤-AR stimulation, and 1.0 mg/kg body wt propranolol
(Sigma-Aldrich) for ␤-AR challenge were administered on consecu-
tive but separate days. Drug dosages were similar to those in previous
studies (9, 15, 30). A 5-min equilibration period after the injection was
given, and the immediate 30-min segment of the ECG signal was used
for drug analysis.
Data acquisition and analysis. ECG signals were recorded using a
telemetry receiver (Data Sciences) and digitized with 12-bit precision
without a signal filter at a sampling rate of 2 kHz. The Data Sciences
data files were then exported and converted to Chart data files and
analyzed using HRV module version 1.2 of Chart Pro software
version 6.1.2 (AD Instruments, Colorado Springs, CO). Ectopic beats
were defined as those having an R-R interval shorter than 70 ms or
longer than 150 ms and were excluded from analysis. Excluded beats
were not replaced by artificial or interpolated beats. HRV measure-
ment and analysis were carried out based on recommendations from
previously published guidelines (9, 32). Measurements of the ECG
parameters, P-R interval, P duration, and QRS interval, along with
arrhythmia analysis were carried out using ECG module version 2.2 of
Chart Pro software. One-second averages of a 10-s segment taken at
the beginning of the 30-min postinjection recordings were used for the
ECG parameter measurements. A 1-h segment from the 24-h baseline
recording beginning at 10 PM and the 30-min postinjection recordings
were analyzed for arrhythmias.
Time-domain analysis. In the time domain, mean HR, mean R-R
interval (RRmean), SD of all normal R-R intervals (SDNN), and SD of
averages of normal R-R intervals in all 2-min segments of the entire
recording (SDANN) were calculated.
Frequency-domain analysis. In the frequency domain, power spec-
tral density was computed by applying fast Fourier transformation
(FFT). FFT was calculated using 512 points and half-overlap with a
Hanning window. Frequency band cutoffs were set at 1.5–5 Hz for
high frequency (HF), 0.15–1.5 Hz for low frequency (LF), and
0 – 0.15 for very LF (VLF) as well as 0 –5.0 Hz for total power (TP),
as recommended by Thireau et al. (32). Normalized HF (nHF) and LF
(nLF) were calculated by dividing either LF or HF by the difference
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between TP and VLF and multiplying by 100 to account for differ-
ences in TP (9, 31a). The ratio of LF to HF is represented as LF/HF.
Membrane preparation. Protocols for myocardial sarcolemmal
membrane preparation and ␤-AR quantification were adapted from
protocols by Wolf et al. (38). To generate membrane preparations,
hearts were flash frozen after they were extracted and the atria
trimmed away. Hearts were then homogenized in ice-cold homoge-
nization buffer [25 mM Tris·HCl (pH 7.4), 5 mM EDTA, 2 mg/ml
leupeptin, and 2 mg/ml aprotinin, Sigma-Aldrich] and incubated on
ice for 20 min. To remove large tissue fragments and cellular or-
ganelles, the homogenate was centrifuged at 750 g for 5 min at 4°C.
The supernatant was recentrifuged at 36,600 g for 30 min at 4°C, and
the pellet was resuspended in membrane resuspension buffer [75 mM
Tris·HCl (pH 7.4), 4 mM EDTA, and 12.5 mM MgCl2, Sigma-
Aldrich] to a concentration of 1 mg/ml.
␤-AR density binding assay. Myocardial membranes (20 ␮g) from
12 mice (5 non-TG, 3 ⌬160E, and 4 R92Q mice) were incubated for
60 min at 37°C with 125I-labeled cyanopindolol (ICYP; Perkin-Elmer,
Waltham, MA, 80 pM) in a final volume of 400 ␮l binding buffer [10
mM Tris·HCl (pH 7.4) and 5 mM EDTA, Sigma-Aldrich]. Nonspe-
cific binding of ICYP was measured by adding 200 ␮M propranolol.
The entire assay volume was poured over glass fiber filters (Fisher
Scientific, Waltham, MA), and rapid vacuum filtration was used to
separate ICYP bound to membrane protein from free, unbound ICYP.
Radioactivity in the membranes trapped by the filters was measured in
an automatic ␥-counter (Wizard 1470, Perkin-Elmer).
Statistics. Comparisons for all 24-h analysis were done by an
unpaired Student’s two-tailed t-test except for 1-h baseline heart block
and premature ventricular contraction analyses, which were done by a
Mann-Whitney test. Comparisons for drug experiments were all done
by two-way ANOVA with Bonferroni post hoc analysis (Graphpad
Prism version 5.01, La Jolla, CA), and additional post hoc analysis
was performed using the QuickCalcs web calculator (www.graphpad.
com). Data are expressed as means ⫾ SE. Statistical significance was
accepted at the level of P ⬍ 0.05.
RESULTS
Twenty-four-hour HR regulation. To determine the baseline
physiological effects of ⌬160E and R92Q cTnT mutations on
HR parameters, mice were observed for an entire 24-h period
in an undisturbed environment. ⌬160E mice showed a trend
toward decreased HR (566 ⫾ 10 beats/min) and increased
RRmean (107 ⫾ 2 ms), whereas R92Q mice showed the oppo-
site trend with increased HR (591 ⫾ 12 beats/min) and de-
creased RRmean (102 ⫾ 2 ms) compared with non-TG mice
(HR: 579 ⫾ 12 beats/min and RRmean: 105 ⫾ 2 ms). To
observe macroscopic HRV among the mice studied, histogram
analyses of 2-min averages of RRmean over the same 24-h
period showed an increased SD for ⌬160E mice but showed a
decreased SD for R92Q mice compared with non-TG mice
(Fig. 1).
To quantitatively assess beat-to-beat cardiovascular control,
time- and frequency-domain measures of HRV were calculated
from the R-R interval. Of the time-domain measures of HRV
we looked at, SDNN represents the gold standard measurement
of HRV, whereas SDANN is used to measure short-term HRV.
SDNN and SDANN did not differ between ⌬160E (9.4 ⫾ 0.7
and 4.3 ⫾ 0.3 ms, respectively) and non-TG (8.8 ⫾ 0.5 and
4.3 ⫾ 0.3 ms, respectively) mice, whereas R92Q mice had
significantly decreased SDNN (7.3 ⫾ 0.4 ms, P ⬍ 0.05) and
SDANN (3.2 ⫾ 0.2 ms, P ⬍ 0.05; Fig. 2). When evaluating
frequency-domain measures of HRV, LF is thought to have
contributions from both vagal and sympathetic inputs, whereas
 HEART RATE REGULATION IN cTnT MUTANT HEARTS H629
⌬160E mice did not show any significant changes, but R92Q
mice showed significant increases in SDANN and TP (Figs. 4
and 5). Inactivation of ␤-ARs was achieved via propranolol
injections, as shown by the reduced HR in the representative
tracings (Fig. 4, inset). After propranolol injections, non-TG
mice showed a significant increase in RRmean with significant
decreases in HR, nLF, and LF/HF, ⌬160E mice showed a
significant increase in RRmean with a significant decrease in
HR, and R92Q mice showed significant increases in RRmean
and nHF with a significant decrease in HR compared with
saline injections (Figs. 4 and 5). Compared with non-TG mice,
⌬160E mice did not show any significant changes, but R92Q
mice showed significant increases in RRmean and SDNN (Figs.
4 and 5).
Arrhythmia detection and ECG parameters. Arrhythmias
have been linked to sudden cardiac death in FHC patients.
During 1 h of arrhythmia detection in the 24-h baseline
Fig. 1. Histograms of mean R-R intervals (RRmean) demonstrating differences
in macroscopic heart rate (HR) variabilitiy (HRV) of 24-h baseline recordings
recordings, there was no incidence of 2° atrioventricular heart
calculated in 2-min average segments. n ⫽ 6 nontransgenic (non-TG), ⌬160E, block for non-TG or ⌬160E mice; however, R92Q mice
and R92Q mice. showed a significant incidence of heart block compared with
non-TG mice (1.5 ⫾ 0.5 vs. 0 heart block/h, P ⬍ 0.01).
Premature ventricular contractions (PVCs) were sporadically
HF is thought to represent vagal control to the heart; thus, observed at similar rates between non-TG, ⌬160E, and R92Q
LF/HF is thought to assess the sympathovagal balance. LF and mice (0.3 ⫾ 0.2, 0.1 ⫾ 0.1, and 0.3 ⫾ 0.3 PVCs/h, respec-
HF were normalized to account for effect that changes in TP
have on LF and HF (31a). TP, VLF, LF/HF, nLF, and nHF did
not differ between ⌬160E (17 ⫾ 2 ms2, 10.5 ⫾ 1 ms2, 2.6 ⫾
0.4, 62 ⫾ 3, and 37 ⫾ 3, respectively) and non-TG (17 ⫾ 2
ms2, 11 ⫾ 1 ms2, 3.3 ⫾ 0.3, 68 ⫾ 2, and 31 ⫾ 2, respectively)
mice. However, R92Q mice had lower TP (10 ⫾ 2 ms2, P ⬍
0.05), VLF (5.5 ⫾ 0.5 ms2, P ⬍ 0.05), LF/HF (1.6 ⫾ 0.3, P ⬍
0.01), and nLF (50 ⫾ 3, P ⬍ 0.001) and higher nHF (48 ⫾ 3,
P ⬍ 0.001) compared with non-TG mice (Fig. 3).
␤-AR density. Alterations in the autonomic nervous system
can lead to alterations in the ␤-adrenergic signaling cascade,
frequently affecting the quantity and distribution of ␤-ARs on
the sarcolemma. To determine if the alterations in the auto-
nomic nervous system that we observed in 24-h baseline
recordings resulted in alterations to the ␤-adrenergic signaling
cascade, ␤-AR density was characterized in myocardial mem-
branes of non-TG, ⌬160E, and R92Q mice. Neither ⌬160E
(53 ⫾ 6 fmol/mg) nor R92Q (63 ⫾ 3 fmol/mg) mice demon-
strated a significant change in ␤-AR density compared with
non-TG mice (50 ⫾ 5 fmol/mg).
␤-AR drug effects. The inset in Fig. 4 shows representative
HR tracings for non-TG, ⌬160E, and R92Q mice after saline,
isoproterenol, and propranolol injections. Each line represents
the average HR value for the entire 30-min recording of all
animals within a group, illustrating the response to each drug
by non-TG, ⌬160E, and R92Q mice. Given that the ␤-adren-
ergic system is involved in altering HR, transient catechol-
amine release caused by handling of the animal results in a
momentary activation of ␤-ARs and was assessed via saline
sham injections. Continuous activation of ␤-ARs was achieved
via isoproterenol injections, as shown in the HR tracings (Fig.
4, inset). After isoproterenol injections, non-TG mice showed
a significant increase in HR with significant decreases in
SDNN and nLF, ⌬160E mice did not show any significant
Fig. 2. Time-domain measures of HRV of 24-h baseline recordings. A: SD of
changes, and R92Q mice showed a significant increase in HR all normal R-R intervals (SDNN). B: SD of averages of normal R-R intervals
with a significant decrease in RRmean compared with saline in all 2-min segments of the entire recording (SDANN). Values are means ⫾
injections (Figs. 4 and 5). Compared with non-TG mice, SE. N.S., not significant. *P ⬍ 0.05 vs. non-TG mice.

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H630 HEART RATE REGULATION IN cTnT MUTANT HEARTS

Fig. 3. Frequency-domain measures of HRV of 24-h baseline recordings. A: total power (TP; 0.0 –5 Hz). B: very low frequency (VLF; 0.0 – 0.15 Hz).
C: normalized low frequency (nLF). D: normalized high frequency (nHF). E: ratio of low frequency (0.15–1.5 Hz) to high frequency (1.5–5 Hz) (LF/HF). Values
are means ⫾ SE. *P ⬍ 0.05, **P ⬍ 0.01, and ***P ⬍ 0.001 vs. non-TG mice.

tively; ⌬160E and R92Q mice were not significant vs. non-TG
mice). Representative tracings of heart block and a PVC are
shown in Fig. 6, A and B. In the 30-min ECG recordings after
saline and propranolol injections, there were sporadic PVCs
and/or occasions of heart block between non-TG, ⌬160E, and
R92Q mice that occurred at similar rates for all three groups
(Fig. 6, C and D). However, after isoproterenol injections, all
three groups exhibited an increased frequency of PVCs and
heart block. Specifically, both ⌬160E and R92Q mice showed
a significant incidence of PVC events compared with non-TG
mice and compared with saline injection alone (Fig. 6C).
non-TG, ⌬160E, and R92Q mice also showed a significant
incidence of heart block events compared with saline injection
alone (Fig. 6D). The P-R interval (atrioventricular conduction),
P duration (atrial contraction), and QRS interval (ventricular
contraction) were measured after injections of saline, isopro-
terenol, and propranolol. There was a significant increase in the
P-R interval after the propranolol injection for non-TG, ⌬160E,
and R92Q mice compared with the saline injection (Table 1).
There was a significant increase in P duration for ⌬160E mice
compared with non-TG mice after the saline and propranolol
injections (Table 1). There was also a significant increase in the
P duration for R92Q mice compared with non-TG mice after
the isoproterenol and propranolol injections. The only differ-
ence observed for the QRS interval was a significant increase
in duration after the isoproterenol injection compared with the
saline injection for R92Q mice (Table 1).
DISCUSSION
Alterations in autonomic function are thought to be a trigger
for sudden cardiac death, but the underlying mechanism as to

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 HEART RATE REGULATION IN cTnT MUTANT HEARTS H631

Fig. 4. HR and time measures of HRV after saline, isoproterenol, and propranolol injections (n ⫽ 8 non-TG, 6 ⌬160E, and 5 R92Q mice). A: HR. B: RRmean.
C: SDNN. D: SDANN. The inset shows averaged representative HR recordings of non-TG, ⌬160E, or R92Q mice over the 30-min segment after each saline,
isoproterenol, or propranolol injection. Values are means ⫾ SE. *P ⬍ 0.05 and ***P ⬍ 0.001 vs. the saline counterpart; #P ⬍ 0.05 and ##P ⬍ 0.01 vs. non-TG
mice within drug groups.

how autonomic dysfunction leads to sudden cardiac death
remains elusive (26). Clinically, autonomic dysfunction has
already been shown to have prognostic significance in patients
with heart failure and myocardial infarction (23). If assessment
of autonomic function could be used as a noninvasive clinical
test to identify patients within FHC cohorts who are most at
risk for sudden cardiac death, it would have significant benefits
in risk stratification.
In this study, we demonstrated that two independent cTnT
mutations, ⌬160E and R92Q, which both exhibit clinically
severe phenotypes in patients, result in mutation-specific alter-
ations in HR regulation. ⌬160E and R92Q mice have been
extensively studied and been found to have similar phenotypes
as assessed via a broad array of methodologies. In skinned
fibers studies, they both demonstrated increased Ca2⫹ sensi-
tivity at short and long sarcomere length and increased tension-
dependent ATP consumption at short sarcomere length (3, 4,
31). Measures of myocellular mechanics and Ca2⫹ kinetic
studies revealed a decreased rate of contraction, decreased
percent shortening, decreased peak rate of relaxation, de-
creased sarcoplasmic reticulum Ca2⫹ load, decreased baseline
Ca2⫹ levels, decreased peak rate of Ca2⫹ rise and decline,
decreased Ca2⫹ peak amplitude, and decreased sarco(endo)-
plasmic reticulum Ca2⫹-ATPase-to-phospholamban ratios for
both mutations (10). ⌬160E mice differed from R92Q mice in
that ultrastructural analysis revealed extensive Z-band misreg-
istration, myofibrillar lysis, and a disorganized thin filament
structure, whereas R92Q mice generally had preserved sarco-
meric structure but increased lipid deposition and an increased
number of mitochondria. We have determined that of these two
independent mutations on opposite ends of the TNT1 tail
domain, only R92Q results in alterations in HRV. Specifically,

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H632 HEART RATE REGULATION IN cTnT MUTANT HEARTS

Fig. 5. Frequency-domain measures of HRV after saline, isoproterenol, and propranolol injections. A: TP. B: VLF. C: nLF. D: nHF. E: LF/HF. Values are
means ⫾ SE. *P ⬍ 0.05, **P ⬍ 0.01, and ***P ⬍ 0.001 vs. the saline counterpart; ##P ⬍ 0.01 vs. non-TG mice within drug groups.

R92Q mice show a decrease in HRV, as indicated by a
decrease in SDNN and SDANN, whereas ⌬160E mice do not
show any changes in these same parameters. A significantly
smaller SDNN indicates that the R-R interval for R92Q mice
stays within a limited range during a 24-h period compared
with non-TG mice. This can be interpreted as a perturbation to
the natural physiological variability of autonomic control in the
hearts of R92Q mice.
To determine possible contributing factors for a decrease in
HRV, we used FFT to look at frequency-domain measures of
HRV that can assess sympathetic and parasympathetic nervous
system inputs. From previous studies in humans (25) and mice
(9, 30), the LF component is believed to be influenced by both
the sympathetic and parasympathetic nervous systems, whereas
the HF component is mainly influenced by the parasympathetic
nervous system. LF/HF is thought to assess sympathovagal
balance. In two separate studies, one looking at HCM patients
and the other looking at heart failure patients, alterations to the
autonomic nervous system were observed in conjunction with
alterations to LF and HF of the same magnitude as in our R92Q
mice (8, 35). In the study with HCM patients, the authors
concluded that there was a decrease in sympathetic drive,

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 HEART RATE REGULATION IN cTnT MUTANT HEARTS H633

Fig. 6. A and B: representative 1-s ECG traces of a premature ventricular contraction (PVC; A) and heart block (B). C and D: PVC and heart block incidence
rates after saline, isoproterenol, and propranolol injections. C: PVC/30 min; D: heart block/30 min. Values are means ⫾ SE. *P ⬍ 0.05, **P ⬍ 0.01, and
***P ⬍ 0.001 vs. the saline counterpart; #P ⬍ 0.05 and ##P ⬍ 0.01 vs. non-TG mice within drug groups.

whereas the authors of the study with heart failure patients duced LF/HF. It is not entirely understood how or why the LF
actually measured an increase in sympathetic drive in support component decreases in environments where high sympathetic
of their findings. Interestingly, in canine studies measuring activity is expected and/or observed (i.e., severe heart failure
HRV during exercise, where sympathetic drive is known to be and heavy physical exercise). Van de Borne et al. (35) suggests
increased, a decrease in the LF component was observed in that while the “traditional paradigm may hold true for a
conjunction with a decrease in HF (12). In our experiments, we physiological range of autonomic drives,” it may no longer
found that R92Q mice had a significantly reduced nLF com- hold true in cases of extreme stress. Ultimately, the increased
ponent, a significantly elevated nHF, and a significantly re- HR, decreased SDNN, and frequency measures of HRV in
R92Q mice are in agreement with previous studies but require
further assessment to determine which branch of the autonomic
Table 1. Effect of drug injections on ECG parameters
nervous system is involved.
P-R Interval P Duration QRS Interval In previous studies using heart failure models, a high sym-
non-TG mice pathetic drive was correlated with downregulation of ␤-ARs or
Saline 32.5 ⫾ 0.4 8.7 ⫾ 0.2 10.8 ⫾ 0.2 increased circulating catecholamines (13, 17). However, when
Isoproterenol 32.0 ⫾ 0.5 (NS) 9.0 ⫾ 0.2 (NS) 11.3 ⫾ 0.2 (NS) we measured ␤-AR density in our TG mice, we observed no
Propranolol 35.1 ⫾ 0.5† 9.1 ⫾ 0.4 (NS) 10.7 ⫾ 0.3 (NS) changes in ⌬160E or R92Q mice. These results are in agree-
⌬160E mice
Saline 32.2 ⫾ 0.4 (NS) 10.3 ⫾ 0.4‡ 10.1 ⫾ 0.2 (NS)
ment with a study using an HCM rat model but are in contrast
Isoproterenol 31.8 ⫾ 1.3 (NS) 10.3 ⫾ 0.7 (NS) 10.7 ⫾ 0.1 (NS) to studies looking at patients with HCM (5, 16, 29, 39). To
Propranolol 34.3 ⫾ 0.7* 10.7 ⫾ 0.5‡ 10.1 ⫾ 0.2 (NS) further extend our original findings, we performed pharmaco-
R92Q mice logical tests that followed previously published guidelines (9,
Saline 33.9 ⫾ 0.6 (NS) 10.3 ⫾ 0.3 (NS) 10.3 ⫾ 0.2 (NS)
Isoproterenol 34.4 ⫾ 1.1 (NS) 11.2 ⫾ 0.7§ 11.1 ⫾ 0.3*
30, 32). Compared with non-TG mice, R92Q and ⌬160E mice
Propranolol 36.6 ⫾ 0.9* 10.8 ⫾ 0.4‡ 10.1 ⫾ 0.3 (NS) had similar responses to time- and frequency-domain measures
of HRV after a transient catecholamine release (saline injec-
All values are means ⫾ SE (in ms); n ⫽ 8 nontransgenic (non-TG), 6
⌬160E, and 5 R92Q mice. NS, not significant. *P ⬍ 0.05 and †P ⬍ 0.01 vs.
tion), a ␤-AR agonist (isoproterenol), and a ␤-AR antagonist
the saline counterpart; ‡P ⬍ 0.05 and §P ⬍ 0.01 vs. non-TG mice within drug (propranolol). If there was ␤-AR downregulation, we would
groups. have expected to see a blunted response to ␤-AR stimulation or

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H634 HEART RATE REGULATION IN cTnT MUTANT HEARTS
inhibition instead of observing values similar to non-TG mice.
These findings would also suggest that if R92Q mice did have
increased sympathetic drive, the uncoupling occurs further
down the ␤-adrenergic signaling cascade. Overall, these find-
ings suggest that HR dysregulation in R92Q mice is initiated at
the myocellular level.
Alterations in HR regulation have been observed in patients
with HCM, suggesting autonomic nervous system dysfunction.
There is agreement in many studies that overall HRV is
decreased in HCM patients (6, 8, 22, 34, 40). Another common
finding among these studies was depressed parasympathetic
activity, suggesting unopposed sympathetic activity in HCM
patients. Consequently, there has been no clear consensus
among these studies as to the predictive value that autonomic
function has on risk assessment of HCM patients. However, it
is important to note that the clinical profiles and genetic
backgrounds for HCM patients in these studies vary consider-
ably, potentially obscuring the benefits of autonomic function
assessment. We were able to address these potential obstacles
in our highly backcrossed TG mouse models of FHC by
comparing two independent cTnT mutations that have been
extensively studied in previous studies (3, 4, 10, 14, 18, 19, 21,
31, 33). In doing so, we can now determine that the R92Q
cTnT mutation leads to altered HR regulation, and, thus, we
can identify a genotype-specific phenotype using a noninvasive
technique in mice.
Finally, we also sought to determine the arrhythmogenic
potential in our cTnT mutant mice. While ventricular tachyar-
rhythmias have been posited to be a cause of death in FHC
patients, neither nonsustained nor sustained ventricular tachy-
arrhythmias were observed in R92Q or ⌬160E mice in the
present study either at baseline or with isoproterenol injections.
We did, however, observe significant 2° atrioventricular heart
block at a significant rate during a 1-h ECG analysis of the 24-h
baseline recording in R92Q mice that was absent in both
non-TG and ⌬160E mice. In addition, R92Q mice along with
⌬160E mice exhibited a significant incidence of PVCs com-
pared with non-TG mice after isoproterenol injections. In-
creased ventricular ectopy has been linked to mice with myo-
filament Ca2⫹ sensitivity, a characteristic also found in both
R92Q and ⌬160E mice (1). There were no significant changes
in P-R and QRS intervals between ⌬160E and R92Q mice
versus non-TG mice after the drug injections. However, there
were significant increases in the P duration in both ⌬160E and
R92Q mice versus non-TG mice after the drug injections.
Coupled with our previous findings of significant atrial en-
largement in our R92Q mice (31), these results potentially
highlight conduction abnormalities in ⌬160E and R92Q mice.
Interestingly, all the ⌬160E and R92Q mice survived isopro-
terenol injections, unlike mice in a study by Knollmann et al.
(15), where mice carrying the I79N cTnT mutation demon-
strated significant mortality as a result of ECG changes and
heart block (15). There are also case reports linking bradycar-
dias such as heart block with HCM and with being secondary
to underlying conditions such as myocardial ischemia or auto-
nomic dysfunction (7, 36). Another study (27) claimed a
pathological interruption of the heart’s electrical conduction
pathway. Together, these findings highlight the potential proar-
rhythmogenic nature and conduction abnormalities of our
cTnT mutant mice.
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Although limitations exist on extrapolating electrophysio-
logical information from murine models due to such differ-
ences as having more rapid HRs and shorter action potentials
than humans, this is the first study to analyze the role of
autonomic regulation in mice with cTnT mutations. Our find-
ings provide the first example of mutations in cTnT leading to
autonomic dysfunction. Of the two independent cTnT muta-
tions that were studied, only R92Q mutant mice had signifi-
cantly altered HRV in 24-h baseline recordings. In addition,
R92Q and ⌬160E mice did not show a decrease in ␤-AR
density, nor did they have significantly different responses to
␤-AR drugs compared with non-TG mice. We have found that
gene specific, noninvasive physiological measurements of
HRV can provide information on autonomic function in mice
with cTnT mutations associated with FHC.
GRANTS
This work was supported in part by National Heart, Lung, and Blood
Institute Grants 5F31-HL-085917-04 (to J. Jimenez) and R01-HL-075619-05
(to J. C. Tardiff).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
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