Am J Physiol Heart Circ Physiol 298: H890–H897, 2010.
First published December 18, 2009; doi:10.1152/ajpheart.00805.2009.
Mechanisms underlying hypothermia-induced cardiac contractile dysfunction
Young-Soo Han,1 Torkjel Tveita,3,4 Y. S. Prakash,1,2 and Gary C. Sieck1,2
Departments of 1Physiology and Biomedical Engineering and 2Anesthesiology, Mayo Clinic College of Medicine, Rochester,
Minnesota; and 3Department of Anesthesiology, Institute of Clinical Medicine, and 4Department of Medical Physiology,
Institute of Medical Biology, University of Tromso, Tromso, Norway
Submitted 25 August 2009; accepted in final form 14 December 2009
calcium sensitivity; troponin I phosphorylation; rat
from accidental hypothermia is often complicated by cardiovascular instability ranging from minor cardiac output depression to fatal circulatory collapse, the latter
termed “rewarming shock.” However, the pathophysiological
mechanisms of posthypothermic cardiovascular instability remain elusive, leaving mortality after accidental hypothermia
between 50% and 80% (21, 23).
Experimental hypothermia research has disclosed that hypothermia may induce cardiac dysfunction (4), and, depending on
level of severity, cardiac contractile dysfunction will be an
essential determinant in cardiovascular instability during rewarming. Thus, the determination of the pathophysiological
mechanisms underlying hypothermia-induced cardiac dysfunction will be critical to comprehensively treating hypothermiainduced myocardial failure in the clinical setting.
The present study focused on possible hypothermia-induced
changes in regulatory processes at the level of cardiac sarcomeric proteins. It is well known that the functional dynamics of
cardiac troponin C (cTnC) is not only dominated by the
reception of Ca2⫹ but also the transduction of this Ca2⫹binding signal by cardiac troponin I (cTnI) and cardiac tropo-
REWARMING PATIENTS
Address for reprint requests and other correspondence: G. C. Sieck, Dept. of
Physiology and Biomedical Engineering, Mayo Clinic and Foundation, 200
First St. SW, Rochester, MN 55905 (e-mail: [email protected]).
H890
nin T (cTnT) (28, 29). Phosphorylation of these sarcomeric
proteins tunes the intensity and dynamics of cardiac contraction and relaxation independent of membrane Ca2⫹ fluxes to
meet physiological demands (28, 29). The phosphorylation of
cTnI at Ser23/24 represents a well-established physiological
mechanism for reduced myofilament Ca2⫹ sensitivity after
␤-adrenergic stimulation and the activation of PKA. Previous
studies (7, 10, 11, 34, 41, 43– 45) have reported that changes in
the cTnI phosphorylation status of myofibrillar proteins underlie myocardial contractile dysfunction under various pathophysiological conditions, such as ischemia-reperfusion injury,
hypertrophy, sepsis, and heart failure. A prevailing notion is
that physiological states reflect a homeostatic distribution of
phosphorylation of sarcomeric proteins, whereas pathophysiological states reflect a disturbance in this homeostasis with
maladaptive distribution of these phosphorylations (27). Accordingly, we hypothesized that hypothermia and rewarming
lead at least to changes in cardiac myofilament Ca2⫹ sensitivity
and/or in the phosphorylation level of cTnI, thus contributing
to reduced contractility.
To test this hypothesis, intact left ventricular (LV) rat
papillary muscles were loaded with fura-2 AM to follow
changes in the intracellular Ca2⫹ concentration ([Ca2⫹]i), and
twitch force was evaluated in papillary muscle preparations
maintained at 15°C for 1.5 h before being rewarmed to baseline
temperature (30°C). Hypothermia at 15°C was chosen to
mimic the core body temperature (15°C) of rats used in our
previous in vivo rewarming shock experiments (16, 17, 37). In
skinned papillary muscle fibers, we assessed maximal Ca2⫹activated force (Fmax) and Ca2⫹ sensitivity of force. PKAmediated phosphorylation of cTnI at Ser23/24 and the total
phosphorylation of cTnI were determined using Western blot
analysis and two-dimensional (2-D) gel electrophoresis, respectively.
MATERIALS AND METHODS
The Institutional Animal Care and Use Committee of Mayo Clinic
approved the experimental protocols and care of the animals. Adult
male Sprague-Dawley rats (250 –350 g) were injected intramuscularly
with ketamine (60 mg/kg) and xylazine (2.5 mg/kg) for anesthesia.
Isolation of LV papillary muscle. Hearts from anesthetized rats
were quickly removed and placed in a custom-made dish continuously
circulated with aerated Krebs-Henseleit (KH) solution containing (in
mM) 118.4 NaCl, 4.7 KCl, 2.0 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25
NaHCO3, and 10 glucose. Papillary muscles from the LV were gently
excised and immediately mounted between a force transducer and a
length change manipulator in a quartz cuvette continuously perfused
with aerated KH solution (Guth Muscle Research System) (14). The
average muscle length was 3 mm and diameter was ⬃1.2 mm,
allowing for proper gas exchange with the perfusate (26). Force
normalized to cross-sectional area (CSA) was expressed as milliNewtons per millimeter squared.
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Han YS, Tveita T, Prakash YS, Sieck GC. Mechanisms underlying
hypothermia-induced cardiac contractile dysfunction. Am J Physiol Heart
Circ Physiol 298: H890–H897, 2010. First published December 18, 2009;
doi:10.1152/ajpheart.00805.2009.—Rewarming patients after profound
hypothermia may result in acute heart failure and high mortality
(50 – 80%). However, the underlying pathophysiological mechanisms
are largely unknown. We characterized cardiac contractile function in
the temperature range of 15–30°C by measuring the intracellular Ca2⫹
concentration ([Ca2⫹]i) and twitch force in intact left ventricular rat
papillary muscles. Muscle preparations were loaded with fura-2 AM
and electrically stimulated during cooling at 15°C for 1.5 h before
being rewarmed to the baseline temperature of 30°C. After hypothermia/rewarming, peak twitch force decreased by 30 – 40%, but [Ca2⫹]i
was not significantly altered. In addition, we assessed the maximal
Ca2⫹-activated force (Fmax) and Ca2⫹ sensitivity of force in skinned
papillary muscle fibers. Fmax was decreased by ⬃30%, whereas the
pCa required for 50% of Fmax was reduced by ⬃0.14. In rewarmed
papillary muscle, both total cardiac troponin I (cTnI) phosphorylation
and PKA-mediated cTnI phosphorylation at Ser23/24 were significantly increased compared with controls. We conclude that after
hypothermia/rewarming, myocardial contractility is significantly reduced, as evidenced by reduced twitch force and Fmax. The reduced
myocardial contractility is attributed to decreased Ca2⫹ sensitivity of
force rather than [Ca2⫹]i itself, resulting from increased cTnI phosphorylation.
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ALTERATIONS IN Ca2⫹ SENSITIVITY AFTER HYPOTHERMIA/REWARMING
Fig. 1. Experimental protocol showing the four experimental steps. CTL,
control.
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relaxing solution. The sarcomere length of the fibers was set to ⬃2.2
␮m using a calibrated monocular microscope (⫻20 long focal length
lens for positioning the whole fiber strip inside the quartz cuvette and
⫻40 lens for adjusting the sarcomere length, respectively) at 30°C.
Varied pCa solutions (6.2, 6.0, 5.8, 5.6, 5.4, 5.2, 5.0, 4.5, and 4.0)
were made by mixing the activating solution with the relaxing solution on the basis of a computer program-determined ratio (9). [Ca2⫹]
in the cuvette perfusing a permeabilized muscle strip was sequentially
varied from pCa 8.0 to 4.0 to obtain force-pCa relationships. ForcepCa relationships were fitted with the following Hill equation:
F ⫽ Fmax
1
关1 ⫹ 10nH共pCa⫺pCa50兲兴
where F is the steady-state force at various pCa, pCa50 is the pCa
required for 50% of Fmax, and nH is the Hill coefficient. pCa50 and nH
were used as an index of myofibrillar Ca2⫹ sensitivity on the force
development and an index of cooperativity of myofibrillar proteins,
respectively. Fibers were subjected to a test activation/relaxation cycle
(at pCa 4.0 and 8.0, respectively) before the measurement of forcepCa relationships.
Western blot analysis. To analyze the phosphorylation of cTnI, a
separate set of experiments was performed using LV papillary muscle
samples (5– 8 mg) from time-matched control and rewarmed groups.
All steps were carried out at 4°C to avoid protein degradation. Tissue
samples were homogenized in buffer solution containing the following: 20 mM Tris (pH 8.0), 0.15 mM NaCl; 1% Nonidet P-40, 1%
Triton X-100, 1% sodium deoxycholate, 10 mM EDTA, and 10 mM
sodium azide. Homogenized samples were solubilized in a sample
rotator, placed in a cold room for 20 min, and spun in a refrigerated
centrifuge at 10,000 g for 10 min. Protein content was assayed by a
Lowry assay (Bio-Rad DC protein Assay). Protein (50 ␮g) was
denatured in 1⫻ sample buffer at 100°C for 3 min, fractionated by
SDS-PAGE, and transferred to a polyvinylidene difluoride (PVDF)
membrane (Bio-Rad). Transferred proteins on separate PVDF membranes were detected with specific antibodies for either total cTnI
[polyclonal antibodies produced with the following epitope: NH2terminal (102–110) and COOH-terminal (170 –180), Cell Signaling,
Santa Cruz, CA] or phospho-cTnI at Ser23/24 (Cell Signaling) and
visualized by a highly sensitive enhanced chemiluminescent substrate
for detecting horseradish peroxidase on immunoblots (Pierce Biotechnology, Rockford, IL). The bands were quantitatively analyzed with
molecular imaging software (version 4.0.1, Kodak).
2-D gel electrophoresis. To complement the Western blot analysis
results, which only showed cTnI phosphorylation at specific Ser23/24
residues, total cTnI phosphorylation in time-matched control and
rewarming groups was determined using 2-D gel electrophoresis
(isoelectric point for cTnI: 9.6). The spots were quantitatively analyzed with ImageQuant software (version 5.2, Amersham Bioscience,
Piscataway, NJ). The detailed protocols for 2-D gel electrophoresis
were previously described (25). In brief, LV papillary muscle samples
(5– 8 mg) from control and rewarmed groups were extracted with 7 M
urea, 2 M thiourea, 4% CHAPS, 0.5% immobilized pH gradient (IPG)
buffer (pH 7–11), and 40 mM DTT. Extracts were treated with a 2-D
clean-up kit (Amersham Biosciences), loaded with rehydration buffer
solution containing 7 M urea, 2 M thiourea, 2% CHAPS, 0.5% IPG
buffer, and 0.002% bromophenol blue, and then kept in a freezer
(⫺70°C). pH 7–11NL IPG dry gel strips (Amersham Biosciences)
laid on rehydration buffer were rehydrated overnight. After rehydration, proteins were isoelectrically focused in a Protein IEF Cell system
(Bio-Rad) equipped with a cup loading tray. After isoelectric point
separation or isoelectric focusing (IEF), gel strips were equilibrated in
6 M urea, 75 mM Tris · HCl (pH 8.8), 30% glycerol, 2% (wt/vol) SDS,
and 0.002% bromophenol blue with 130 mM DTT and 135 mM
iodoacetamide before undergoing SDS-PAGE for protein separations
by molecular weight. After SDS-PAGE using 12.5% Tris · HCl criterion precast gels (Bio-Rad), the gels were silver stained for the
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Simultaneous measurement of intracellular Ca2⫹ transients and
twitch force. The horizontally mounted papillary muscle was loaded
with 10 ␮M fura-2 AM for 1.5 h in oxygenated KH solution containing Pluronic F-127 [0.01% (wt/vol)], which facilitated the loading of
fura-2 AM into the muscle fibers at room temperature (20 –22°C) (26,
42). After being loaded, the muscle was washed out for 10 min and
continuously perfused with KH solution aerated with 95% O2-5%
CO2 throughout the experiment. Afterward, the muscle was electrically stimulated at 0.5 Hz at the baseline temperature of 30°C, during
which muscle length was adjusted for maximal twitch force. During
hypothermia at 15°C, the papillary muscle was stimulated at 0.25 Hz
to imitate the hypothermia-induced change in heart rate of the intact
heart at this temperature. Twitch force and the corresponding ratio
signals from fura-2 fluorescences at 340- and 380-nm excitation were
recorded using a real-time data-acquisition program (LabVIEW, National Instruments, Austin, TX). To minimize photobleaching, the
excitation shutter was only opened during data recording. The emission fluorescence signal from the fura-2-loaded muscle was filtered
through a 510-nm filter and collected by a photomultiplier tube.
Background fluorescence from the unloaded muscle was subtracted
from the corresponding fura-2 fluorescence signals at 340 and 380 nm.
[Ca2⫹]i was calculated from the ratio of fluorescence signals (ratio ⫽
340/380 nm) using the equation described by Grynkiewcz et al. (13).
Experimental protocol. All experiments started at baseline temperature (30°C), which kept the progressive fura-2 loss small enough to
carry out the 2.5-h experimental protocol (26). Twitch force and
[Ca2⫹]i transients were recorded at the following specific steps:
baseline temperature before the tissue was cooled (30°C, step 1),
initial hypothermia (15°C, 0 h, step 2), hypothermia maintained for
1.5 h (15°C, 1.5 h, step 3), and after the tissue was rewarmed to the
baseline temperature (30°C, step 4). The duration of cooling and
rewarming the muscle tissue was 30 min. Time-matched controls were
maintained at 30°C for 2.5 h. The cooling/rewarming protocol is
shown in Fig. 1. Hypothermia at 15°C was chosen to mimic the core
body temperature (15°C) of rats used in our previous in vivo rat model
of rewarming shock (16, 17, 37).
Permeabilized papillary muscle fibers. In a separate set of experiments, LV papillary muscles from the control and rewarming groups
were teased into thin strips (⬃1 mm in length and ⬍200 ␮m in
diameter) in relaxing solution containing the following: 85 mM K⫹, 5
mM MgATP, 1 mM Mg2⫹, 7 mM EGTA, and 70 mM imidazole, with
a total ionic strength of 150 mM and pH 7.0 with propionic acid
(HPr). The activating solution contained the following: 0.1 mM
CaPr2, 85 mM K⫹, 5 mM MgATP, 1 mM Mg2⫹, 7 mM EGTA, and
70 mM imidazole, with a total ionic strength of 150 mM and pH 7.0
with HPr. A computer program was used to calculate the free Ca2⫹
concentration for the activating and relaxing solutions. Dissected thin
fibers were permeabilized in relaxing solution containing an additional 1% (vol/vol) Triton X-100 for 1 h at room temperature (20 –
22°C). Subsequently, the permeabilized fibers were mounted in the
Guth Muscle Research System (14) and rinsed for 10 min with
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ALTERATIONS IN Ca2⫹ SENSITIVITY AFTER HYPOTHERMIA/REWARMING
analysis of cTnI phosphorylation or transferred to a PVDF membrane
for Western blot analysis to identify TnI phosphorylation spots.
Statistical analysis. All results are presented as means ⫾ SE. To
statistically analyze the changes in twitch force and [Ca2⫹]i in the
time-matched control group and in the hypothermia/rewarming
groups within experimental steps, one-way ANOVA was used. When
significant differences were observed, Student’s t-test with the Bonferroni correction was used as a post hoc analysis. For the comparison
of the time-matched control group versus hypothermia/rewarming
groups, unpaired two-tailed Student’s t-test was used. Differences
were considered significant at P ⬍ 0.05.
RESULTS
Table 1. Amplitude, kinetic parameters, and time-matched control values for peak [Ca2⫹]i transients and twitch force at
each experimental step
Amplitude
[Ca2⫹
]i, nM
Force, mN/mm2
Time to Peak, ms
Time to 50% Decay, ms
Step 1: baseline (0 h)
[Ca2⫹]i transient
Control
Twitch force
Control
812.41 ⫾ 133.82
925.20 ⫾ 51.65
[Ca2⫹]i transient
Control
Twitch force
Control
1,025.85 ⫾ 192.91*
910.31 ⫾ 135.25
[Ca2⫹]i transient
Control
Twitch force
Control
1,190.66 ⫾ 205.17*
917.24 ⫾ 42.89
7.71 ⫾ 1.29
8.28 ⫾ 1.30
57.03 ⫾ 3.06
52.01 ⫾ 2.83
118.13 ⫾ 7.82
116.02 ⫾ 6.99
177.19 ⫾ 5.32
204.50 ⫾ 9.71
86.04 ⫾ 6.53
101.80 ⫾ 4.45
162.09 ⫾ 8.55*
45.50 ⫾ 1.72
407.97 ⫾ 13.36*
103.40 ⫾ 5.84
681.89 ⫾ 30.23*
178.50 ⫾ 29.50
324.51 ⫾ 22.62*
81.80 ⫾ 7.22†
166.99 ⫾ 8.48*
50.02 ⫾ 2.84
382.51 ⫾ 11.38*
92.20 ⫾ 4.37†
625.98 ⫾ 19.30*
192.05 ⫾ 15.34
279.00 ⫾ 18.41*
70.80 ⫾ 3.34†
Step 2: initial hypothermia (0.5 h)
13.23 ⫾ 2.22*
7.57 ⫾ 0.98
Step 3: maintained hypothermia (2.0 h)
13.43 ⫾ 2.30*
7.63 ⫾ 0.75
Step 4: rewarming to baseline temperature (2.5 h)
2⫹
[Ca ]i transient
Control
Twitch force
Control
973.99 ⫾ 154.89
815.86 ⫾ 78.49
4.73 ⫾ 0.68*
7.31 ⫾ 0.93
56.29 ⫾ 3.07
54.50 ⫾ 9.19
96.31 ⫾ 4.46*
91.40 ⫾ 4.46†
162.14 ⫾ 4.41*
171.50 ⫾ 19.50
62.77 ⫾ 2.97*
59.60 ⫾ 2.23†
Results are presented as means ⫾ SE; n ⫽ 7 rats for experimental steps and 4 rats for time-matched control at 30°C. [Ca2⫹]i, intracellular Ca2⫹ concentration.
*P ⬍ 0.05 vs. baseline temperature (30°C); †P ⬍ 0.05 vs. initial control values (0 h).
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Time-matched control group maintained at 30°C for 2.5 h.
A general decline (⬍12% of initial control values) in the peak
[Ca2⫹]i transient and peak twitch force was found in timematched controls at baseline temperature (30°C), but there
were no significant differences in the peak [Ca2⫹]i transient
and peak twitch force between initial (0 h) and final (2.5 h)
control values (Table 1). Furthermore, there were no significant
changes in control values of time to peak (TTP) and time to
50% decay (TT50%) of [Ca2⫹]i, whereas there were significant
decreases in TTP (⬃20% at 2 and 2.5 h) and TT50% (20% at
0.5 h, 30% at 2 h, and 40% at 2.5 h) of twitch force compared
with the initial control values (0 h; Table 1).
Changes in [Ca2⫹]i transients and twitch force after tissues
were cooled to 15°C (15°C, 0 h). Compared with values at the
baseline temperature (30°C), the following characteristics of
both the peak [Ca2⫹]i transient and twitch force were significantly increased: amplitude, by 26% and 72%, respectively;
TTP, by 184% and 245%, respectively; and TT50%, by 285%
and 277%, respectively (Fig. 2, A and B, and Table 1). These
significant increases in the [Ca2⫹]i transient and/or force dur-
ing tissue cooling to 15°C were comparable with those presented in previous studies (12, 18, 20, 32, 37).
Changes in [Ca2⫹]i transients and twitch force during stable
hypothermia for 1.5 h at 15°C. At the end of 1.5 h at 15°C,
peak [Ca2⫹]i increased by 16%, whereas peak force remained
unchanged, compared with values obtained after tissue was
cooled to 15°C (0 h at 15°C). TTP in peak [Ca2⫹]i transients
remained unchanged, whereas TTP in force was significantly
decreased (6.1%). There were significant reductions in TT50%
in both peak [Ca2⫹]i transients and force (6% and 13%,
respectively) compared with those at 0 h at 15°C (Fig. 2, A and
B, and Table 1).
Changes in [Ca2⫹]i transients and twitch force after tissue
was rewarmed to baseline temperature (30°C). After the tissue
was rewarmed, the peak [Ca2⫹]i transient was increased by
13%, whereas peak force was reduced by 36%, compared with
baseline temperature (30°C). TTP in peak [Ca2⫹]i transients
returned to within prehypothermic control values, whereas
TTP in force decreased by 20%. TT50% in both peak [Ca2⫹]i
transients and force was decreased by 10% and 30%, respectively, compared with baseline (Fig. 2, A and B, and Table 1).
Intact myofilament Ca2⫹ sensitivity. By plotting phase-plane
loops (the relationship between twitch force and [Ca2⫹]i),
which represent a coarse dynamic index of myofilament Ca2⫹
sensitivity during the relaxation phase of twitch force (3, 8,
31), we observed a rightward shift in the relaxation phase
during stable hypothermia (15°C) as well as after rewarming in
intact papillary muscle, indicating a decrease in myofilament
Ca2⫹ sensitivity due to hypothermia and rewarming (Fig. 3).
Force-pCa relationship in permeabilized muscle fibers. Fmax
was significantly decreased in rewarmed muscle fibers (32.1 ⫾
3.2 vs. 45.9 ⫾ 3.9 mN/mm2 in controls; Fig. 4A). In Fig. 4B,
ALTERATIONS IN Ca2⫹ SENSITIVITY AFTER HYPOTHERMIA/REWARMING
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DISCUSSION
the data for Fmax are normalized to maximal values and plotted
against increasing pCa concentrations. In rewarmed muscle
fibers, pCa50 was significantly reduced (5.57 ⫾ 0.03 vs. 5.71 ⫾
0.06 in controls). However, nH remained unchanged (3.19 ⫾
0.13 in rewarmed muscle fibers vs. 3.04 ⫾ 0.27 in controls).
Western blot analysis of cTnI. The bands of cTnI phosphorylated at Ser23/24 residues (top) as well as total cTnI protein
expression (bottom), used as a reference from time-matched
control and rewarmed LV papillary muscles, are shown in
Fig. 5A. In rewarmed papillary muscle, cTnI phosphorylation
was significantly increased compared with that of timematched control (114.33 ⫾ 7.64 vs. 93.02 ⫾ 3.27 arbitrary
units; Fig. 5B).
2-D gel electrophoresis analysis. Figure 6A shows typical
spots of proteins separated by IEF based on the isoelectric
points of specific proteins. Total cTnI phosphorylation in
rewarmed papillary muscles was significantly increased compared with that of time-matched controls (51.63 ⫾ 5.19 vs.
33.96 ⫾ 4.69 arbitrary units; Fig. 6B).
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Fig. 3. Typical phase-plane loops demonstrating the changes in Ca2⫹ sensitivities during cooling at 15°C and after rewarming (RW) to baseline temperature (30°C).
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Fig. 2. Representative traces of twitch forces (A) and intracellular Ca2⫹
concentration ([Ca2⫹]i) transients (B) in intact papillary muscles undergoing
the four experimental steps. Muscles were continuously perfused with KrebsHenseleit solution during an electrical stimulation at 1 Hz at 30°C and 0.5 Hz
at 15°C. Trace a, baseline temperature (30°C); trace b, initial cooling at 15°C
(0 h); trace c, cooling maintained at 15°C (1.5 h); trace d, rewarming to
baseline temperature.
The major findings in the present study are that rewarming
after 1.5 h of stable hypothermia increased PKA-mediated TnI
phosphorylation as well as the total phosphorylation of cardiac
TnI and decreased the Ca2⫹ sensitivity of force in myocardial
contractile tissue. These novel data, in conjunction with a
substantial reduction in contractile force after rewarming, add
to our understanding of the mechanisms underlying hypothermia-induced cardiac contractile dysfunction and also point to a
potential therapeutic target to counteract rewarming-induced
cardiac failure.
Cooling and rewarming alter myocardial contractile function.
After rewarming, papillary muscle twitch force was reduced by
⬃35% despite a ⬃15% increase in the [Ca2⫹]i transient,
indicating that reduced contractile force after rewarming is
related to a decrease in the myofibrillar Ca2⫹ sensitivity of
force rather than an impairment in [Ca2⫹]i regulation. Significant increases in the temporal parameters of contraction and
relaxation in twitch force after rewarming likely reflect a
change in myosin/actin cross-bridge cycling kinetics (Table 1).
Despite the fact that in the present study we did not measure
cross-bridge rate constants or stiffness, these findings support
another explanation for reduced force after hypothermia/rewarming on the basis of a two-state model (5). According to
the two-state model, maximum isometric force is defined as
follows: maximum isometric force ⫽ nF␣fs, where n is the
number of cross-bridges within the half sarcomere, F is the
average force per cross-bridge, and ␣fs is the fraction of
cross-bridges in a strongly bound force-generating state. Furthermore, ␣fs is defined as follows: ␣fs ⫽ fapp/(fapp ⫹ gapp),
where fapp and gapp are the apparent rate constants for crossbridge attachment and detachment, respectively. Thus, the
reduced force development after rewarming may be attributable to the decrease in cross-bridge recruitment (␣fs) resulting
from increased gapp during hypothermia/rewarming. This assumption is supported by our results showing that after tissue
rewarming, there were significant decreases in TT50% of the
[Ca2⫹]i transient as well as twitch force (⬃10% and 30%,
respectively; Table 1).
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ALTERATIONS IN Ca2⫹ SENSITIVITY AFTER HYPOTHERMIA/REWARMING
Fig. 4. Force-pCa relationships in the CTL and RW groups. A: all the data of
Ca2⫹-activated force in absolute values (ordinate) were plotted as a function of
pCa (abscissa). B: normalized force-pCa relationships in the CTL and RW
groups are shown. Data are means ⫾ SE; n ⫽ 21–22 muscle fibers from 8 rats
in the CTL and RW groups, respectively.
Decreased myofilament Ca2⫹ sensitivity as an underlying
mechanism of posthypothermic contractile dysfunction. Ca2⫹
activates cardiac muscle in a graded manner, which makes the
relationship between [Ca2⫹]i and force development of functional importance (2). Using intact papillary muscle, we found
a significant increase in both the twitch force and peak [Ca2⫹]i
transient at 15°C (Table 1 and Fig. 2, A and B). These findings
are comparable with those in previous studies reporting that
contractility is greater at 23–25°C than at normothermia in the
papillary muscle of various species (20). It is known that
temperature change is one of the major factors that alters
cardiac myofilament Ca2⫹ sensitivity (1), but controversy exists whether cardiac myofilament Ca2⫹ sensitivity will increase
or decrease during hypothermia. Stowe et al. (32) reported that
in isolated hearts, mild hypothermia (27°C) increases myofilament Ca2⫹ sensitivity, whereas moderate hypothermia (17°C)
decreases it. These findings support our observations that
twitch force in intact papillary muscle reaches a maximum at
25°C and then declines as temperature is reduced to 15°C.
However, it needs to be emphasized that the significantly
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Fig. 5. Analysis of troponin I (TnI) phosphorylation in the CTL and RW
samples. A: TnI protein expression (bottom) was used as a reference for the
determination of TnI phosphorylation (top). Bands from the Western blot were
quantified using densitometry. B: results were quantified as a ratio of phosphorylated (p-)TnI intensity to TnI intensity. Quantified results are shown in
the bar graph. Data are means ⫾ SE; n ⫽ 5 from the CTL and RW groups,
respectively. au, Arbitrary units. *P ⬍ 0.05 vs. the CTL group (considered as
a significant difference).
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increased peak [Ca2⫹]i transient at 15°C in the present experiments (Table 1) may counteract an expected decrease in twitch
force due to reduced Ca2⫹ sensitivity at this temperature. By
plotting phase-plane loops (the relationship between twitch
force and [Ca2⫹]i), which represent a coarse dynamic index of
myofilament Ca2⫹ sensitivity during the relaxation phase of
twitch force (3, 8, 31), we observed a rightward shift in the
relaxation phase during stable hypothermia (15°C) as well as
after rewarming in intact papillary muscle, indicating a decrease in myofilament Ca2⫹ sensitivity due to hypothermia and
rewarming (Fig. 3). We used skinned papillary muscle fibers to
better describe the Ca2⫹ sensitivity and contractile force after
rewarming and found that both Fmax and myofilament Ca2⫹
sensitivity were significantly decreased in rewarmed cardiac
muscle fibers. These data suggest that hypothermia decreases
cardiac myofilament Ca2⫹ sensitivity, which is not reversed by
rewarming, thus contributing to posthypothermic cardiac contractile dysfunction.
Increased cTnI phosphorylation and cardiac contractile
dysfunction but maintained relaxation characteristics after
tissue rewarming. Numerous studies (6, 7, 19, 24, 33, 44) have
demonstrated that increased PKA-mediated phosphorylation of
the sarcomeric protein cTnI reduces myofilament Ca2⫹ sensitivity and shifts the force-pCa relationship rightward under
normothermic circumstances. To the best of our knowledge,
the present study is the first to show that rewarming after stable
hypothermia simultaneously increases cTnI phosphorylation
and reduces myofilament Ca2⫹ sensitivity and Fmax. In principle, during systole, Ca2⫹ moves into the cytoplasmic space and
binds to the myofilaments, and contraction starts. One way to
ALTERATIONS IN Ca2⫹ SENSITIVITY AFTER HYPOTHERMIA/REWARMING
increase the contractile force of the sarcomere is to increase
cytosolic Ca2⫹; another is to regulate how the sarcomeres
respond to Ca2⫹. The unique position of cTnI to regulate and
modulate cardiac function under the influence of adrenergic
signaling is well documented (30). In more detail, cTnI has a
NH2-terminal extension of some 30 amino acids that houses
Ser23/24, which is phosphorylated by PKA to depress sarcomere sensitivity to Ca2⫹ and increase cross-bridge kinetics
(15). In the present study, we found that after tissues were
rewarmed, there was a significant increase in cTnI phosphorylation at PKA sites (Ser23/24), as shown by Western blot
analysis (Fig. 5A) and 2-D gel (Fig. 6A), which likely included
all phospohorylatable sites on cTnI (Ser23/24, Ser43/45,
Thr144, and Ser149). Therefore, we could not exclude other
possible phospohorylatable sites on cTnI (Ser43/45, Thr144,
and Ser149) that might be phosphorylated by hypothermia/
rewarming, even though Ser23 and Ser24 are known to be the
dominant phospohorylatable sites on cTnI. Those possible sites
on cTnI to be identified in future studies would require the use
of the mass spectrometry technique.
Phosphorylation of cTnI is mediated by several kinases
[PKA, PKC, PKG, and PAK3 (p21-activated kinases)] (19),
but PKA-mediated phosphorylation via ␤-adrenergic stimulation plays a major physiological role in meeting the increased
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circulatory demand in the physiological state (19). Interestingly, unlike other pathological clinical conditions, such as
ischemia-reperfusion injury, hypertrophy, sepsis, and heart
failure (7, 10, 11, 34, 41, 43– 45), where a slower relaxation
rate is a common finding, a faster relaxation rate after rewarming was observed despite a significant reduction of contractile
force in the present experiments. Furthermore, we noticed that
in time-matched controls at baseline temperature, TTP and
TT50% of twitch force were significantly reduced after 2.5 h
(Table 1), whereas TTP and TT50% of [Ca2⫹]i remained
statistically unchanged. Therefore, we speculate that fapp and
gapp may be altered in response to the baseline temperature in
this time-matched control group.
Yet, it is a challenge to understand the exact mechanism of
the direct effects of hypothermia and rewarming on ␤-adrenergic stimulation leading to PKA-mediated cTnI phosphorylation. In general, changes in the cTnI phosphorylation status, as
reported in the failing human heart (7, 10, 11, 34, 41, 43– 45),
are considered a change in the balance between kinase and
phosphatase activities, and the optimal balance is to be found
during normal cardiac function. Thus, hypothermia and rewarming, depending on severity and depth, appear to tip the
balance between kinase and phosphatase acitivities, leading to
alterations in myofilament Ca2⫹ sensitivity and myocardial
contractility.
Considering the similarities in the changes between cardiac
contractile functional variables of in vitro papillary muscle
preparations and those of the whole intact heart after experimental hypothermia and rewarming previously studied by our
group (16, 17, 36, 38, 39), it is tempting to make a direct
comparison between them. However, such a comparison has to
be made with great caution. Nevertheless, previous reports (22,
35) have shown that rewarming intact hearts from stable
hypothermia (25–15°C) resulted in a significant (⬃50%) reduction in systolic function but maintained diastolic cardiac
function. Thus, similarities in cardiac contractile functional
variables between in vitro papillary muscle and the intact heart
may be ascribed to a hypothermia-induced decrease in Ca2⫹
sensitivity that persists after rewarming. PKA-dependent phosphorylation of cTnI, induced by hypothermia and rewarming,
may explain both the reduction in systolic functional characteristics and proper maintenance in diastolic function in the
rewarmed intact heart. Furthermore, we (16) have reported a
lack of effect of pharmacological agents to support cardiac
contractile function mediated via the ␤-receptor-cAMP-PKA
pathway during as well as after rewarming. Likewise, if hypothermia and rewarming per se activate the ␤-receptor-cAMPPKA pathway, one may interpret that an additional pharmacological stimulus under these circumstances of the ␤-receptor
would bring about much less of cardiac inotropic effect. As
already outlined, these assumptions cannot be applied to draw
any conclusions but may be used as a plausible rationale for
future research protocols.
Although rewarming shock is considered a variant of irreversible circulatory shock, cardiac contractile dysfunction after
rewarming may range from cardiac stunning to severe acute
cardiac failure. The present findings may also have relevance
to rewarming hypothermic patients after heart surgery as well
as to therapeutic hypothermia used to treat patients after being
resuscitated after acute cardiac standstill. However, there are
still limitations in extrapolating experimental results using
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Fig. 6. Analysis of the total TnI phosphorylation in the CTL and RW samples
using two-dimensional gel electrophoresis (A). P1, a single phosphorylated
spot of TnI; P0, an unphosphorylated spot of TnI. B: phosphorylated spots of
TnI were quantified as the ratio of P1 to the sum of P1 and P0. Quantified results
are shown in the bar graph. Data are means ⫾ SE; n ⫽ 6 from the CTL and
RW groups, respectively. MW, molecular weight. *P ⬍ 0.05 vs. the CTL
group (considered as a significant difference).
H895
H896
ALTERATIONS IN Ca2⫹ SENSITIVITY AFTER HYPOTHERMIA/REWARMING
ACKNOWLEDGEMENTS
The authors thank Dr. Frank Brozovich and Dr. Ozgur Ogut for helping to
set up the 2-D gel electrophoresis procedure for this study.
GRANTS
This work was supported by the Mayo Foundation and Graduate School
(Rochester, MN).
DISCLOSURES
No conflicts of interest are declared by the author(s).
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