Download Titin isoform switching is a major cardiac adaptive response in

Am J Physiol Heart Circ Physiol 295: H366 –H371, 2008.
First published May 23, 2008; doi:10.1152/ajpheart.00234.2008.
Titin isoform switching is a major cardiac adaptive response in hibernating
grizzly bears
O. Lynne Nelson,1 Charles T. Robbins,2 Yiming Wu,3 and Henk Granzier3,4
1
Veterinary Clinical Sciences, College of Veterinary Medicine, 2School of Biological Sciences and Department of Natural
Resource Sciences, and 3Veterinary Comparative Physiology and Pharmacology, Washington State University, Pullman,
Washington; and 4Molecular Cardiovascular Research Program, Department of Molecular and Cellular Biology, University
of Arizona, Tucson, Arizona
Submitted 4 March 2008; accepted in final form 21 May 2008
collagen; bradycardia; diastolic function; echocardiography
state of energy conservation
in response to harsh climatic conditions. Grizzly bears (Ursus
arctos horribilis) do not eat, drink, or urinate and demonstrate
minimal activity during their annual 4 – 6 mo hibernation
period. Cardiovascular adaptations must occur for the myocardium to remain healthy and efficient during a period of extremely low heart rates and cardiac output (6, 7, 8, 12, 26).
Nonhibernators that suffer from bradycardic conditions will
develop cardiac chamber dilation due to volume overload
during the excessively long diastolic pauses (23, 35, 42). Over
MANY MAMMALS UNDERGO A UNIQUE
Address for reprint requests and other correspondence: O. Lynne Nelson,
Diplomate ACVIM (Internal Medicine and Cardiology), Veterinary Clinical
Sciences, Washington State Univ., 100 Grimes Way, Rm. 17, Pullman, WA
99164 (e-mail: [email protected]).
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time, chamber dilation and elevated end-diastolic pressures
lead to congestive heart failure if no intervention is sought (23,
35). However, hibernating grizzly bears tolerate extremely low
heart rates without ventricular chamber dilation (30). The
mechanisms that circumvent chamber dilation are not well
understood, but they might involve increased ventricular stiffness (30).
Molecular changes in sarcomeric and extracellular matrix
proteins relate to myocardial stiffness in many forms of cardiac
disease. In particular, titin, a giant sarcomeric protein (9, 17,
18, 29, 43, 45), and fibrillar collagen types I and III (22, 32, 37,
40) most commonly exhibit changes that affect ventricular
diastolic chamber stiffness. Recent studies in animal models of
dilated cardiomyopathy and pressure overload have revealed
changes in titin isoform expression that increase myocardial
stiffness (44, 45). Total titin content is often unchanged, but the
increased expression of the stiffer N2B (vs. N2BA) isoform
results in higher passive myocardial stiffness (44, 45). Likewise, many chronic cardiac diseases result in an increased
accumulation of interstitial collagen I and III via increased
gene expression and decreased degradation (22, 33). Collagen
metabolism may be stimulated by hemodynamic as well hormonal factors (38, 40). Collagen or titin isoform changes can
adversely affect or compensate for increased chamber stiffness,
and these proteins could be altered in hibernating bears.
We undertook this investigation to evaluate diastolic function and collagen and titin isoform expression in the myocardium of grizzly bears during hibernation compared with bears
during the active period. To avoid the confounding effect of
anesthesia on cardiac function, we evaluated unanesthetized
subjects by echocardiography. We hypothesized that changes
in ventricular compliance occur to avoid remodeling the ventricular chamber during hibernation. Such an adaptation could
prevent the negative consequences of chronic bradycardiainduced dilation and potential congestive heart failure.
MATERIAL AND METHODS
Animals. Four three-year-old subadult female grizzly bears were
used for this study. The mean weights were 91.7 ⫾ 15 kg in early
summer, 120.1 ⫾ 13 kg in late fall, and 95.6 ⫾ 7 kg in late winter,
before emergence from hibernation. The animals were housed at the
Washington State University Bear Research, Education and Conservation Facility. The animals were maintained according to the Bear
Care and Colony Health Standard Operating Procedures, and all
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0363-6135/08 $8.00 Copyright © 2008 the American Physiological Society
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Nelson OL, Robbins CT, Wu Y, Granzier H. Titin isoform
switching is a major cardiac adaptive response in hibernating grizzly
bears. Am J Physiol Heart Circ Physiol 295: H366 –H371, 2008. First
published May 23, 2008; doi:10.1152/ajpheart.00234.2008.—The hibernation phenomenon captures biological as well as clinical interests
to understand how organs adapt. Here we studied how hibernating
grizzly bears (Ursus arctos horribilis) tolerate extremely low heart
rates without developing cardiac chamber dilation. We evaluated
cardiac filling function in unanesthetized grizzly bears by echocardiography during the active and hibernating period. Because both
collagen and titin are involved in altering diastolic function, we
investigated both in the myocardium of active and hibernating grizzly
bears. Heart rates were reduced from 84 beats/min in active bears to
19 beats/min in hibernating bears. Diastolic volume, stroke volume,
and left ventricular ejection fraction were not different. However, left
ventricular muscle mass was significantly lower (300 ⫾ 12 compared
with 402 ⫾ 14 g; P ⫽ 0.003) in the hibernating bears, and as a result
the diastolic volume-to-left ventricular muscle mass ratio was significantly greater. Early ventricular filling deceleration times (106.4 ⫾
14 compared with 143.2 ⫾ 20 ms; P ⫽ 0.002) were shorter during
hibernation, suggesting increased ventricular stiffness. Restrictive
pulmonary venous flow patterns supported this conclusion. Collagen
type I and III comparisons did not reveal differences between the two
groups of bears. In contrast, the expression of titin was altered by a
significant upregulation of the stiffer N2B isoform at the expense of
the more compliant N2BA isoform. The mean ratio of N2BA to N2B
titin was 0.73 ⫾ 0.07 in the active bears and decreased to 0.42 ⫾ 0.03
(P ⫽ 0.006) in the hibernating bears. The upregulation of stiff N2B
cardiac titin is a likely explanation for the increased ventricular
stiffness that was revealed by echocardiography, and we propose that
it plays a role in preventing chamber dilation in hibernating grizzly
bears. Thus our work identified changes in the alternative splicing of
cardiac titin as a major adaptive response in hibernating grizzly bears.
CARDIAC TITIN ISOFORM SWITCHING IN HIBERNATING BEARS
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13 parameters were assessed for statistical differences. Table 1 lists
the echo-derived parameters and the definitions of each.
Tissues. Tissues were harvested from 12 animals euthanized for
reasons unrelated to this project. All animals were healthy. Six
samples were collected for each group (active and hibernating). Each
group had three males and three females. The mean age for the
hibernating group was nine years. The mean age for the active group
was 10.5 years. The age range was 3–21 years in each group. Note that
no significant sex difference was found in titin and collagen expression and that no age dependence was detected either (within the
available age range). Tissues sampled from active bears were collected during June, July, or August. Tissues samples from hibernating
bears were collected in January or February. Sample collecting procedures adhered to guidelines provided by the IACUC, WSU College
of Veterinary Medicine. Tissues were collected immediately after
euthanization. Samples were obtained from left ventricular free wall
(separated into endo-, ecto-, and mesocardium), snap frozen in liquid
nitrogen, and stored at ⫺80°C until use. Left ventricular mesocardium
was used for this study because it represents the majority of the wall
and thus is likely to be the main determinant of left ventricular
function. We also analyzed titin isoform expression in the epicardium
and endocardium of six bears (4 hibernating and 2 active bears) and
found no significant difference between the different layers, unlike the
epi-endo gradient that was reported in previous work (5). Thus data
from the mesocardium (reported in Titin gel electrophoresis) are
likely to well represent the titin expression level in the left ventricular
wall.
Collagen I and III immunohistochemistry. We used an immunofluorescence method adapted from Neagoe et al. (29). Briefly, left
ventricular mesocardium tissues were cut into 6-␮m sections using a
freezing microtome (active, n ⫽ 6; and hibernating, n ⫽ 6). Each
sample was processed for both collagen type I and III according to
manufacturer’s instructions (US Biological, Swampscott, MA). Specificity for the antibodies was previously validated for numerous
species (US Biological). Briefly, the collagen type I antibody (C751017T; US Biological) was diluted 1:200 in PBS. The antibody was
added to each section and incubated for 60 min. The samples were
washed in PBS for 10 min. FITC-conjugated anti-mouse antibody
(11904-79L; US Biological) was added to the sample and incubated
for 60 min. The samples were rewashed in PBS for 10 min and then
mounted in glycerol containing 1 mM 1,4 phenylenediamine. The
same procedure was used for collagen type III antibody (C7510 –39P;
US Biological). A negative control was also run for each sample using
FITC-conjugated secondary antibody without collagen primary antibody. The samples were examined using fluorescent microscopy
(FITC filter, Zeiss Axioskop 2; Peabody, MA) under ⫻63 oil immersion, and images were captured with a digital camera (Zeiss AxioCam; Peabody, MA) as tagged image file format (TIFF) files at 1,016
ms exposure. Images were acquired of each muscle sample of collagen type I, collagen type III, and negative control. Fluorescence was
quantified using an NIH-inspired public domain program (ImageJ)
(11). The fluorescence quantification was performed by converting the
red, blue, and green channels into separate eight-bit grayscale images.
The green images were evaluated using the histogram function assessing the color intensity on a scale of 0 to 256. The intensity values
were averaged to yield one value for each collagen type and a negative
control for each sample.
Titin gel electrophoresis. SDS-agarose electrophoresis studies were
performed as previously described (44A). Wet gels were scanned and
analyzed with one-dimensional scan software (Scanalytics). The integrated optical density of N2BA titin, N2B titin, total titin, and
myosin heavy chain (MHC) was determined as a function of the
volume of the solubilized protein sample that was loaded (a range of
volumes was loaded on each gel). The slope of the linear range of the
relation between integrated optical density and loaded volume was
obtained for each protein. The N2BA-to-N2B ratio was calculated as
the slope of N2BA titin divided by the slope of N2B titin.
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procedures were approved by the Washington State University (WSU)
Institutional Animal Care and Use Committee (IACUC) based on the
National Institutes of Health (NIH) guidelines. Hibernation began the
last week of October when feeding ceased and ended the second week
of March when feeding resumed. In early October, food was gradually
reduced until completely withdrawn in late October. Water was
available ad libitum, and straw was provided for bedding. Bears
hibernated in pairs in unheated pens (3 m ⫻ 3 m ⫻ 2.5 m) with
continuous access through a small door to an outdoor area (3 m ⫻
5 m ⫻ 5 m) that was covered on all sides to minimize external noise
and stimulation. Because the pens were open to the outside, the bears
experienced daily light and temperature fluctuations.
The four bears were hand raised, human socialized, and trained for
echocardiography. The bears have a comfortable routine of handling
and were fully conscious for this study. These unanesthetized bears
were used to avoid the potential confounding effects of anesthesia on
in vivo assessment of cardiac function parameters by echocardiography. Diastolic function was measured in all the bears of the study at
three different periods during the active phase of the year (June, July,
and August) and the hibernation period (December, January, and
February).
Echocardiography. All bears underwent a complete transthoracic
echocardiographic examination that included two-dimensional, Mmode, spectral, and color-flow Doppler evaluations. Echocardiography was performed by the same person (O. Lynne Nelson) using
commercially available equipment (Siemens Acuson Ultrasound;
Cypress, Bothell, WA). The bears were imaged in sternal recumbency
with forelegs positioned cranially to optimize the parasternal thoracic
windows. Standard imaging planes (canine) and function calculations
have been previously described (37) and were performed in accordance with the American Society of Echocardiography (4, 15, 34).
Video images were captured using a commercially available digital
echocardiography software program, and data was collected offline
using a workstation (Siemens Acuson Ultrasound; Cypress). The
following echocardiographic parameters were collected: end-diastolic
volume index (milliliters), the maximum diastolic volume by modified
Simpson’s rule divided by the body mass in kilograms; stroke volume
index (milliliters), volumetric calculated stroke volume divided by
body mass in kilograms; percent left ventricular ejection fraction,
volumetric calculation of left ventricular ejection by modified Simpson’s rule; cardiac index (milliseconds per minute per kilogram),
cardiac output divided by body mass in kilograms; left ventricular
muscle mass (grams), calculated by area length method; left ventricular end-diastolic volume-to-left ventricular mass ratio, end-diastolic
volume divided by left ventricular muscle mass; left ventricular wall
relaxation time (milliseconds), the time recorded for mechanical
relaxation as identified by the left ventricular free wall excursion on
the M-mode exam (point of maximal excursion in systole to first point
of minimal excursion in diastole); left ventricular wall relaxation rate
(meters per second), the rate of mechanical relaxation as identified by
the slope created by connecting the two points above; left ventricular
isovolumic relation time (milliseconds), the time from aortic valve
closure to mitral valve opening; left ventricular early filling-to-atrial
contraction ratio, the ratio of the velocity of left ventricular early
filling to the velocity of atrial contraction; deceleration time of early
left ventricular filling (milliseconds), time required for maximal left
ventricular filling velocity to decelerate to baseline; pulmonary venous systolic-to-diastolic flow velocity ratio, maximal pulmonary
venous systolic velocity divided by maximal diastolic velocity; and
pulmonary venous diastolic flow deceleration rate (meters per second), time required for maximal pulmonary venous diastolic flow
velocity to decelerate to baseline. The mean of four consecutive
measurements was considered average for each parameter during each
data collection period. To account for differences in body weight,
volume parameters were normalized to body weight (American
Society of Echocardiography Index of Published Guidelines, http://
asecho.org/Guidelines.php). Of the echocardiography data collected,
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CARDIAC TITIN ISOFORM SWITCHING IN HIBERNATING BEARS
Statistical analysis. Analysis was performed using commercial
statistical software (version 15, Minitab statistical software). Repeatedmeasures ANOVA was used to compare echocardiography parameters and collagen quantification among the two groups, active and
hibernating bears. Assumptions of ANOVA were satisfied; a P value
of ⬍0.05 was considered significant. Because of the large number of
echocardiography parameters, and possible multiplicity issues therein,
a Bonferroni adjustment was made. The stricter P value threshold of
ⱕ0.003 was considered significant for the echocardiography parameters. Paired t-tests were conducted to evaluate differences in total
titin and N2BA-to-N2B ratios between hibernating and active bears. A
P value of ⬍0.05 was considered significant. All data are presented as
means ⫾ SD.
RESULTS
DISCUSSION
Our goal was to study cardiovascular adaptations that allow
the myocardium to remain healthy and efficient during a period
of extremely low heart rates and cardiac output. Our study is
the first that uses unanesthetized active and hibernating grizzly
bears to avoid the confounding effects of drugs on cardiac
function. For example, a previous study on anesthetized bears
revealed opposing results in ejection fraction (decreased in
hibernation), and heart rate, compared with those of the unanesthetized bears. Systolic and diastolic function parameters
Table 1. Echocardiographic parameters recorded in conscious grizzly bears during summer active and hibernation period
Parameters
Active
Hibernation
P Value
Heart rate, beats/min
End diastole
Interventricular septal wall thickness, cm
Left ventricular free wall thickness, cm
Left ventricular internal dimension, cm
End-diastolic volume, ml
End-diastolic volume index, ml/kg
Stroke volume, ml
Stroke volume index, ml/kg
Left ventricular ejection fraction, %
Cardiac index, ml 䡠 min⫺1 䡠 kg⫺1
Left ventricular wall relaxation rate, m/s
Left ventricular wall relaxation time, ms
Left ventricular isovolumic relaxation time, ms
Left ventricular inflow early filling-to-atrial contraction ratio
Deceleration time of early ventricular filling, ms
Pulmonary venous systolic-to-diastolic flow velocity ratio
Pulmonary venous diastolic flow deceleration rate, m/s
83.8⫾12
18.7⫾8.4
⬍0.0001
1.5⫾0.04
1.3⫾0.08
6.0⫾0.09
219.3⫾15.6
2.4⫾0.14
127.5⫾4.5
1.4⫾0.04
66.4⫾5.2
117.7⫾14.2
0.09⫾0.02
123.3⫾24
63.4⫾10.7
1.4⫾0.2
143.2⫾20.3
1.1⫾0.3
3⫾0.7
1.3⫾0.07
1.1⫾0.1
5.6⫾0.3
188.9⫾6.2
1.9⫾0.13
110.3⫾6.4
1.2⫾0.02
65.8⫾3.9
33.6⫾6.1
0.04⫾0.01
268⫾38.8
97.4⫾15.7
4.9⫾1.1
106.5⫾14.6
0.4⫾0.06
1.4⫾0.3
0.008
0.05
0.196
0.06
0.710
0.03
0.968
0.736
⬍0.0001
⬍0.0001
⬍0.0001
⬍0.0001
0.003
0.002
⬍0.0001
0.003
Values are means ⫾ SD; n ⫽ 4 grizzly bears during the summer active and 4 grizzly bears during the hibernation periods. Note that to account for differences
in the size of the bears, various indexes are shown normalized to body mass. A P value of ⱕ0.003 was considered significant. End-diastolic volume index is
the diastolic volume ⫼ body mass in kilograms; stroke volume index is the stroke volume ⫼ body mass in kilograms; and cardiac index is the cardiac output ⫼
body mass in kilograms.
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Echocardiography. Hibernating bears had significantly
lower heart rates (19 vs. 84 beats/min) and cardiac output
indexes (33.6 vs. 117.7 ml䡠min⫺1 䡠kg⫺1) than the active bears
(Table 1). (Note that the heart rates given for mean rates were
an average of 6 measures during the course of the echocardiogram. The heart rate that was used to calculate cardiac output
was the instantaneous rate at the time of the Simpson’s calculation. Because of the fact that the bears have sinus arrhythmia
during hibernation, the mean heart rate and the rate used for
cardiac output were not exactly identical.) We found that the
end-diastolic volume index, stroke volume index, and left
ventricular ejection fraction were not different between hibernating and active bears (Table 1). The diastolic volume-to-left
ventricular muscle mass ratio was significantly greater in
hibernating bears (Fig. 1A). Thus total volume was not different between the two groups, but left ventricular muscle mass
was significantly lower (Fig. 1B). The left ventricular wall
relaxation rate was decreased and the left ventricular and
isovolumic relaxation times were longer, but the deceleration
time of early ventricular filling was shorter in hibernating bears
(Fig. 2). The ratio of early ventricular filling to late ventricular
filling of the atrial contraction was significantly increased in
hibernating bears. The ratio of pulmonary venous systolic to
diastolic flow was significantly lower and the pulmonary venous diastolic rate was significantly faster in hibernating bears
(Fig. 3). Thus heart rate and relaxation rates were slower, but
early ventricular filling ended sooner in hibernating bears,
indicating increased ventricular stiffness.
Collagen I and III. The mean collagen type I values were
36.7 ⫾ 6.0 in hibernating bears and 33.9 ⫾ 4.3 in active bears
(P ⫽ 0.4). The mean collagen type III values were 48.7 ⫾ 11.6
in hibernating bears and 45.7 ⫾ 7.2 in active bears (P ⫽ 0.6).
Collagen type I and III comparisons were not statistically
significant between the two groups of bears. Likewise, the ratio
of collagen type I to collagen type III was not significantly
different between groups. The mean ratio in hibernating bears
was 0.8 ⫾ 0.2 compared with 0.7 ⫾ 0.2 (P ⫽ 0.79) in active
bears.
Titin protein expression. High-resolution SDS-agarose gels
revealed prominent N2B and N2BA bands in both active and
hibernating bears. The densitometric gel analysis revealed no
difference in the total titin-to-MHC ratio between the two
groups (active, 0.19 ⫾ 0.06; and hibernating, 0.18 ⫾ 0.02; P ⫽
0.7). The mean ratio of N2BA to N2B titin was 0.73 ⫾ 0.07 in
the active bears and decreased significantly to 0.42 ⫾ 0.03
(P ⫽ 0.006) in the hibernating bears (Fig. 4). Thus total titin
was not different between active and hibernating bears, but the
mean expression of the stiffer N2B isoform was increased at
the expense of the more compliant N2BA isoform.
CARDIAC TITIN ISOFORM SWITCHING IN HIBERNATING BEARS
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Fig. 1. Left ventricular (LV) end-diastolic
volume-to-muscle mass ratio (milliliters/
grams; A) and muscle mass (grams; B) in
active and hibernating conscious bears (n ⫽
4). Total volume was not different between the
2 periods, but LV muscle mass was significantly lower. All data are presented as
means ⫾ SD. P ⱕ 0.003, significant.
Fig. 2. LV early filling deceleration time in an active (A) and hibernating (B)
bear. a, ventricular filling waveform due to atrial contraction; DTE, deceleration time of early ventricular filling waveform; E, early ventricular filling
waveform. Note: in the hibernating bear with profound bradycardia, the atrial
contraction waveform seen is from the previous diastolic cycle.
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disease (27). Higher ratios are typically associated with elevated myocardial wall stress and are known to trigger compensatory ventricular muscle hypertrophy (31). Instead, reduced cardiac mass is present in hibernating bears, which
might be due to the absence of food intake during hibernation
or decreased activity.
Diastolic function parameters revealed prolonged relaxation
times during hibernation and reduced early left ventricular
filling waveform deceleration time (DTE). Increased relaxation
times have been associated with bradycardia (32), but the
reduced DTE that we found is unique (Fig. 2). DTE is known to
be an accurate indicator of ventricular stiffness in numerous
cardiac conditions and is a clinically accepted standard measure of left ventricular operating stiffness (4, 15). The restrictive filling pattern of pulmonary venous inflow in hibernating
bears was also consistent with increased ventricular stiffness
(Fig. 3), which mirrored the early ventricular filling waveform
and DTE. Chamber stiffness relates to how much pressure is
required to fill the ventricle for a given volume of fluid. The
stiffer the chamber, the more rapid the pressure rises inside the
chamber and the earlier the filling phase is completed (2, 3,
15). Left ventricular stiffness is governed by a number of
factors that include ventricular geometry (particularly hypertrophy) and myocardial stiffness (largely related to tissue
Fig. 3. The ratio of pulmonary venous systolic to diastolic flow (PVs/d) was
significantly lower in hibernating bears (B). A: active bear. The reduced PVs/d
is consistent with a hemodynamic restrictive ventricular filling pattern. S, atrial
filling from the pulmonary venous system during cardiac systole; D, atrial and
ventricular filling from the pulmonary venous system during cardiac diastole
(early filling).
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are known to be affected by anesthetics (19, 36). Even though
the use of unanesthetized bears is challenging, it is clear that
the echocardiography of conscious, well-adjusted subjects
more accurately represents true cardiac physiology.
As expected, the hibernating bears had significantly lower
heart rates and cardiac output than active bears (Table 1).
Ventricular volumes were not different from the active period
to hibernation, but left ventricular muscle mass was significantly lower during hibernation (Fig. 1). Because ventricular
mass returns to baseline during the next active period (unpublished data), it is possible that cardiac myocytes atrophy during
hibernation and hypertrophy during the active period. However, myocyte death and regeneration might participate to
some extent since these cellular processes have been documented in certain pathological states (1). The finding of reduced cardiac mass (⬃26% change) during hibernation is in
contrast to that of skeletal muscle, which demonstrates limited
atrophy (⬍10% change) with disuse (24, 39). These findings
suggest interesting differences between adaptations in cardiac
versus skeletal muscle that warrant future follow-up studies
highlighting an independent measure of cardiomyocyte size.
Systemic arterial pressures were not obtained in these conscious bears, but blood pressure did not differ between active
and hibernating anesthetized bears in an earlier study (30). The
ventricular volume-to-mass ratio is an important clinical determinant of ventricular wall stress in humans with cardiac
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CARDIAC TITIN ISOFORM SWITCHING IN HIBERNATING BEARS
collagen content and titin expression) (4, 9, 17, 18, 33). With
the consideration that myocardial hypertrophy is not detected
in hibernating bears, it is likely that the increased ventricular
stiffness involves changes in either collagen or titin. Our
findings indicate that collagen expression was not different
between active and hibernating bears, suggesting that collagen
played no major role in altering ventricular stiffness characteristics. We found no difference in the total titin-to-MHC ratio
between the two groups, but the mean expression of the stiffer
N2B isoform was increased at the expense of the more compliant N2BA isoform in hibernating bears (Fig. 4), indicating
that changes in titin expression might explain the increased
ventricular stiffness. Preliminary data suggest that ⬃1 wk after
feeding starts, at the beginning of the active period, echo
parameters have mostly recovered to those of active bears.
However, additional data are needed to confirm and extend this
result, including exploring the role titin and collagen in the
recovery process.
Hibernating rodents have demonstrated opposing results
in titin expression (6, 43). Some of the differences between
our study and that of hibernating rodents may be due to
conditions of study (microgravitation for rodents), difference between cardiac and skeletal muscle adaptations, or
possibly due to species differences. For example, in contrast
to hibernating ground squirrels, hibernating bears do not
show degradation of fast muscle fibers (20). Hibernating
bears are also unique in that they are less hypothermic
(33°C) than hibernating rodents (5°–7°C) (21). Thus calciumhandling dynamics may be affected to a lesser degree in bears
than in rodents.
Titin is a giant elastic protein that spans form Z-disk to
M-line. The I-band region of titin is extensible and functions as
a molecular spring that develops passive force in sarcomeres
that are stretched beyond their slack length. The difference in
the molecular mass of N2BA and N2B isoforms (⬃3.3 vs.
⬃3.0 MDa) is largely due to the differential splicing of the
tandem Ig and the PEVK segments of titin that comprise the
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ACKNOWLEDGMENTS
We thank Caroline Benoist, Abbey Burgess, Jennifer Fortin, Gary Radamaker, Justin Teisburg, and Pam Thompson for excellent technical assistance.
Present address of Y. Wu: Dept. of Internal Medicine, Univ. of Iowa, Iowa
City, IA.
GRANTS
This work was supported by the Autzen Foundation, the Washington State
University Bear Research Center, and National Heart, Lung, and Blood
Institute Grant HL-062881.
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Fig. 4. SDS-agarose gel electrophoresis of titin expression in active (n ⫽ 6)
and hibernating (n ⫽ 6) grizzly-bear hearts. N2BA-to-N2B expression ratio is
significantly lower in hibernating bears. A, active period; H, hibernating;
MHC, myosin heavy chain.
molecular spring. As a result, the molecular spring is shorter in
N2B titin, and a given change in sarcomere length gives rise to
a titin-based force that is higher for N2B titin (its shorter
extensible region results in higher fractional extensional) than
for the longer N2BA isoform (9). Thus the upregulation of
N2B titin can explain the increased wall stiffness in hibernating
grizzly bears.
Correlations between titin expression and ventricular systolic and diastolic function have been observed in humans (14,
28). In dilated cardiomyopathy, increased N2BA expression
resulted in a decrease in ventricular stiffness, which may be
important in reducing ventricular filling pressures for a given
volume (reducing symptoms of congestive heart failure) (3).
However, although diastolic function improved, increased
N2BA expression has been correlated with a negative effect on
systolic function (14, 16). This can be explained by the finding
that titin-based passive force enhances calcium sensitivity.
Thus the calcium sensitivity effect is reduced when N2BA-toN2B ratios are high (14, 25). In contrast to human dilated
cardiomyopathy, an increased calcium sensitivity is predicted
in hibernating grizzly bears due to the upregulation of N2B
titin.
Changes in titin isoform expression might be an adaptive
response that occurs during hibernation to allow the myocardium to remain healthy and efficient during a period of extremely low heart rates and cardiac output. A possible explanation for the functional significance of increased ventricular
stiffness may be that it is compensatory for the reduced muscle
mass relative to volume and the profound bradycardia. Increased ventricular wall stress and prolonged diastolic filling
are typically associated with dilation/remodeling of the chamber. A chamber that is stiffer is less likely to distend or dilate.
Increases in ventricular stiffness would have to be well balanced with rising ventricular and atrial pressures in diastole. If
diastolic filling pressures increased greatly, congestion would
occur. There is no evidence of increased left atrial pressures
in hibernating bears since the left ventricular isovolumic
relaxation period is prolonged (this period is classically
shortened with high atrial pressures). Diastolic dysfunction
and congestive failure become particularly relevant with
exercise and activity (10). Thus inactivity in hibernating
bears might promote tolerance of increased ventricular stiffness. If the lower N2BA-to-N2B ratios are associated with
enhanced calcium sensitivity and systolic function as in
humans, it then follows that any decline in diastolic function
might be offset by improved systolic function during hibernation. Thus we propose that changes in alternative splicing
of cardiac titin constitute an adaptive response in hibernating grizzly bears.
CARDIAC TITIN ISOFORM SWITCHING IN HIBERNATING BEARS
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