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heart failure
Zuzana Kubalova*, Dmitry Terentyev*, Serge Viatchenko-Karpinski*, Yoshinori Nishijima†, Inna Györke*,
Radmila Terentyeva*, Daise N. Q. da Cuñha†, Arun Sridhar‡, David S. Feldman*§, Robert L. Hamlin†,
Cynthia A. Carnes‡¶, and Sandor Györke*¶储
*Department of Physiology and Cell Biology, ¶Davis Heart and Lung Research Institute, †Department of Veterinary Biosciences, §Department of Internal
Medicine, and ‡College of Pharmacy, Ohio State University, Columbus, OH 43210
Edited by Ramon Latorre, Center for Scientific Studies, Valdivia, Chile, and approved August 4, 2005 (received for review May 24, 2005)
Diminished Ca release from the sarcoplasmic reticulum (SR) is an released from the SR into the cytosol is reduced, accounting for,
important contributor to the impaired contractility of the failing or contributing to, the reduced contractile force generated by the
heart. Despite extensive effort, the underlying causes of abnormal failing heart. In a majority of studies, including those in human
SR Ca release in heart failure (HF) remain unknown. We used a cardiomyocytes, reduced SR Ca release has been shown to be
combination of simultaneous imaging of cytosolic and SR intralu- associated with a decrease in the SR Ca content (2, 13–17).
minal [Ca] in isolated cardiomyocytes and recordings from single- Several explanations have been put forth for the diminished SR
ryanodine receptor (RyR) channels reconstituted into lipid bilayers Ca stores in HF. In a number of reports, the reduction of SR Ca
to investigate alterations in intracellular Ca handling in an exper- content has been attributed to depressed SR Ca-ATPase func-
imental model of chronic HF. We found that diastolic free [Ca] tion and兾or enhanced NCX activity during HF (12, 17, 18).
inside the SR was dramatically reduced because of a Ca leak across These changes are expected to facilitate Ca removal from the cell
the SR membrane, mediated by spontaneous local release events at the expense of its uptake into the SR and result in under-filled
(Ca sparks), in HF myocytes. Additionally, the magnitudes of SR Ca stores in HF. Another potential cause of reduced SR Ca
intrastore Ca depletion signals during global and focal Ca release content is enhanced diastolic leak of Ca via the RyRs (18–20).
events were blunted, and [Ca]SR recovery was slowed after global Although an enhanced RyR-mediated Ca leak could explain the
but not focal Ca release in HF myocytes. At the single-RyR level, the reduced SR Ca content observed in HF, direct experimental
sensitivity of RyRs to activation by luminal Ca was greatly en- support for this possibility in myocytes from failing heart is
hanced, providing a molecular mechanism for the maintained lacking. Thus, further studies are needed to determine the
potentiation of Ca sparks (and increased Ca leak) at reduced
mechanisms of altered SR Ca handing in HF.
intra-SR [Ca] in HF. This work shows that the diminished SR Ca
This report presents an investigation of subcellular and mo-
lecular features of HF in a canine model of chronic tachypacing-
release characteristic of failing myocardium could be explained by
induced HF (21). To investigate the causes of aberrant SR Ca
increased sensitivity of RyRs to luminal Ca, leading to enhanced
function in chronic HF, we performed measurements of myocyte
spark-mediated SR Ca leak and reduced intra-SR [Ca].
cytosolic and SR luminal Ca, and conducted recordings from
single-RyR channels. This work shows that the diminished SR Ca
excitation– contraction coupling 兩 ryanodine receptor 兩 sarcoplasmic
release that is characteristic of failing myocardium could be
reticulum
explained by increased sensitivity of RyRs to luminal Ca, leading
to enhanced spark-mediated SR Ca leakage and reduced
14104 –14109 兩 PNAS 兩 September 27, 2005 兩 vol. 102 兩 no. 39 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0504298102
Fig. 2. HF increases Ca spark frequency and reduces both spark amplitude
and SR Ca content in permeabilized myocytes. (A) Representative line-scan
images of Ca sparks recorded in control and HF myocytes. (B and C) Average
frequency (B) and amplitude (C) of sparks (*, P ⬍ 0.01; **, P ⬍ 0.001; n ⫽ 573
events from 43 control cells and 1,416 sparks from 32 HF cells). (D) Represen-
tative recordings of caffeine-induced Ca transients. Average amplitudes of
caffeine-induced Ca transients (⌬F兾F0, mean ⫾ SE) were 2.8 ⫾ 0.2 (n ⫽ 14) and
2.1 ⫾ 0.2 (n ⫽ 9) for control and HF myocytes (P ⬍ 0.05).
PHYSIOLOGY
Fig. 1. HF reduces ICa-induced Ca transients and SR Ca load. (A) Represen- The SR Ca content was inferred from recordings of Ca transients
tative confocal line-scan images of Ca transients (Top) along with their spatial and Na兾Ca exchange currents recorded on application of caffeine.
averages (Middle) and recordings of ICa (Bottom), induced by a depolarizing Based on these measurements, the SR Ca content was reduced to
step from ⫺50 to 0 mV, in control (Left) and HF (Right) myocytes. The voltage ⬇65% of control in HF myocytes (Fig. 1C). Similar changes in
step is depicted above the images. (B) Averages of Ca transient amplitudes and ICa-induced Ca transients and SR Ca content have been described
peak ICa densities as functions of membrane potential for control and HF
previously in experimental models of hypertrophy and HF in the
myocytes (P ⬍ 0.05; n ⫽ 8 cells from four control hearts; n ⫽ 9 cells from three
HF hearts). (C) Caffeine-induced Ca transients and INCX in control and HF
dog (22, 23). The primary focus of the present study was to
myocytes (Left). The average amplitudes (F兾F0) of caffeine-induced Ca tran- determine the factors responsible for the abnormal Ca releasing
sients (Center) were 2.5 ⫾ 0.2 in control and 1.7 ⫾ 0.3 in HF myocytes. The and storing capacities of SR in chronic HF.
integrals of INCX (Right) were 47 ⫾ 5 and 31 ⫾ 7 pC for control and HF myocytes,
respectively (P ⬍ 0.05). Ca Sparks and SR Ca Content. To further explore the effects of HF
on the SR Ca release mechanism, we performed measurements
of elementary Ca release events, Ca sparks, in saponin-
myocytes), consistent with ventricular hypertrophy. Depolariza- permeabilized myocytes (Fig. 2A). The myocytes were bathed in
tion-induced ICa and spatially resolved intracellular Ca transients a fluo-3-containing internal solution at a buffered [Ca] (⬇50
were measured in cardiomyocytes dialyzed with fluo-3. Fig. 1A nmol/liter Ca兾0.1 mmol/liter EGTA). In control myocytes, spon-
shows representative traces of ICa and confocal line-scan images taneous Ca sparks occurred with an average frequency of ⬇3.0 ⫾
along with the spatial average of Ca transients recorded during 0.3 s⫺1. In myocytes from failing hearts, spark frequency was
a depolarizing step from ⫺50 to 0 mV in myocytes from control increased to 6.6 ⫾ 1.2 (Fig. 2B), whereas event amplitude was
and failing hearts. The amplitude of Ca transients was reduced significantly decreased (Fig. 2C). Additionally, although spark
and their duration prolonged in HF myocytes. Whereas the width was not altered there was a small but significant increase
amplitude of current density (ICa) did not change, the time in event duration in HF myocytes compared with controls. These
constant of ICa inactivation was slowed in HF myocytes, appar- HF-related changes in Ca sparks were confirmed in intact
ently because of diminished Ca-dependent inactivation of the myocytes loaded with fluo-3 acetoxymethyl ester (Fig. 8 and
L-type Ca channels (Table 1, which is published as supporting Tables 2 and 3, which are published as supporting information
information on the PNAS web site). on the PNAS web site).
Averaged Ca transient and ICa amplitudes are plotted as a Ca spark-mediated leak of Ca from the SR plays an important
function of voltage in Fig. 1B. The observed decreases in Ca role in setting the SR Ca content (11). The observed increase in
transient amplitude in HF myocytes with respect to control were frequency of sparks would be expected to result in reduced
significant through the whole range of membrane potentials sequestration of Ca in the SR in HF myocytes, unless the SR Ca
tested (from ⫺40 to 60 mV). At the same time, ICa density was uptake rate was also increased. The total SR Ca content in the
preserved, suggesting that the reduced SR Ca release is not same two groups of cells was estimated by application of caffeine
simply due to a reduced Ca trigger in myocytes from failing (10 mmol兾liter). As shown in Fig. 2D, the amplitude of caffeine-
hearts. induced Ca transients was decreased to ⬇75% of control in HF
Kubalova et al. PNAS 兩 September 27, 2005 兩 vol. 102 兩 no. 39 兩 14105
is published as supporting information on the PNAS web site),
consistent with the blunted depolarization- and caffeine-induced
Ca transients in HF myocytes. Additionally, the velocity of Ca
wave propagation was reduced in HF myocytes as indexed by the
prolonged slope of the Ca wave front on the myocyte line-scan
images (Fig. 4A). Similar HF-induced changes in properties of
Ca waves were observed in intact myocytes loaded with fluo-3
acetoxymethyl ester (Fig. 8 and Table 5, which are published as
supporting information on the PNAS web site).
Strikingly, the SR-entrapped fluo-5N revealed a dramatic
reduction in the basal [Ca]SR in HF myocytes compared with
controls (Fig. 4 A–C and Table 6, which is published as sup-
porting information on the PNAS web site). Additionally, the
amplitudes of both the global and focal Ca depletion signals (i.e.,
during Ca waves and sparks, respectively) were diminished in HF
cells compared with controls (Figs. 4 A and B and 5 A and B).
The amplitude of the Ca wave depletion signal constituted
⬇45% of the total reduction of fluo-5N fluorescence in caffeine,
indicative of partial SR depletion during Ca wave-associated Ca
release in both control and HF myocytes (10 mmol兾liter, FCaf;
Fig. 4C). Recovery kinetics of [Ca]SR for Ca wave-associated Ca
release were significantly slower in HF myocytes, consistent with
a compromised ability of the SR to sequester and retain Ca in
HF myocytes (Fig. 4C). However, [Ca]SR recovery after focal Ca
release was similar in HF and control myocytes (Fig. 5B). The
differential effects of HF on [Ca]SR recovery after large scale vs.
spatially limited SR Ca release suggests different SR refilling
mechanisms after global and localized Ca release events (24).
Collectively, these results showed that SR luminal [Ca] is re-
duced in HF myocytes. They also demonstrated that generation
of spontaneous Ca waves and sparks in HF myocytes occurs at
Fig. 3. HF reduces both local and averaged [Ca] in SR stores. (A) Images of abnormally low intra-SR Ca levels, thereby accounting for, or
control and HF permeabilized myocytes with SR-entrapped fluo-5N. (B) En- contributing to, the reduced sequestration of Ca in the SR in HF
larged contour images of subcellular regions illustrating reductions in overall myocytes. The increased occurrence of spontaneous release
and spatially localized fluo-5N fluorescence in HF myocytes. (C) Plots of events at reduced SR Ca content in HF myocytes is reminiscent
fluorescence profiles in B at locations indicated by the arrows for control of the effects of the RyR-sensitizing agent caffeine (11, 25) and
(black line) and HF (red line) myocytes. Dotted lines represent mean fluores- consistent with potential changes in the functional activity of
cence for control (black) and HF (red) myocytes. RyRs in failing hearts.
PHYSIOLOGY
(Right) myocytes. (C) Representative time-dependent profiles of intra-SR Ca waves followed by caffeine-induced intra-SR Ca transients in control and HF myocytes
(black and red trace, respectively). (Inset) comparison of time course of [Ca]SR recovery after Ca wave-associated Ca release.
whereas RyR2 content was significantly reduced in homogenates operates to prevent SR Ca overload by releasing excess Ca from
from failing hearts. Therefore, altered stoichiometry between the store (11).
RyR and other proteins of the Ca release channel complex could Our finding that, in HF, RyRs become hypersensitive to
contribute to the abnormal Ca handling in HF. luminal Ca helps to reconcile some apparently contradictory
results and improves our understanding of abnormal Ca handling
Discussion in the failing heart. Previous studies in normal myocytes have
We investigated alterations in intracellular Ca handling in shown that the frequency of Ca sparks and the incidence of Ca
chronic HF using a combination of simultaneous imaging of waves are increased when the SR Ca load is elevated, whereas
cytosolic and SR intraluminal [Ca] in isolated cardiomyocytes reduced SR Ca content is accompanied by reduced Ca spark and
and recordings from single-RyR channels reconstituted into Ca wave occurrence. Yet in HF, an increased frequency of sparks
lipid bilayers. We found that diastolic [Ca] inside the SR was is associated with reduced SR Ca content (ref. 23 and this work).
dramatically reduced because of increased Ca spark-mediated Our demonstration of enhanced RyR activity at a given luminal
Ca leak across the SR membrane in HF myocytes. At the [Ca] provides a logical explanation for these seemingly discrep-
single-RyR level, the sensitivity of RyRs to activation by luminal ant results. Moreover, our finding may explain the paradoxically
increased incidence of arrhythmogenic Ca waves and delayed
Ca was greatly enhanced, providing a molecular mechanism for
after depolarizations in various models of HF, despite the
the maintained potentiation of Ca sparks (and increased Ca
reduced SR Ca content (28–30). Of note, we did observe
leak) despite a reduced intra-SR [Ca] in HF. These results show
premature ventricular depolarizations in some of the HF dogs,
that defective intrastore Ca signaling is involved in the patho- consistent with an arrhythmogenic response to impaired calcium
genesis of HF. regulation during HF (Supporting Materials and Methods).
Defective RyR Luminal Ca Regulation as a Cause of Abnormal Ca Comparison with Previous HF Models. Whereas the observed re-
Handling in HF. [Ca] inside the SR is an important determinant of duction and prolongation of SR Ca release are hallmarks of
the functional activity of the RyR Ca release channels (5, 26). failing myocardium (2, 14–16), the effects of HF on other EC
Normally, RyR luminal Ca regulation appears to play a stabi- parameters, including ICa and SR Ca load, vary widely between
lizing role by countering the intrinsic positive feedback of different species and models. Our finding that alterations of SR
Ca-induced Ca release during the release process. Specifically, Ca release occurred in the absence of significant changes in ICa
dissociation of Ca from the luminal sensing sites is involved in density but in parallel with a decrease in SR Ca content is
termination of SR Ca release, permitting cardiac relaxation (9, consistent with results obtained previously in shorter term (3–6
10, 27). Additionally, RyRs sensitive to [Ca]SR provide a luminal weeks) canine models of tachypacing-induced HF (22, 31).
Ca-dependent leak pathway and play a role in regulating SR Ca Similar to a recent report by Song et al. (23) in which a canine
content in cardiac muscle (11, 12). Presumably, this mechanism model of left ventricular hypertrophy due to chronic pressure
Kubalova et al. PNAS 兩 September 27, 2005 兩 vol. 102 兩 no. 39 兩 14107
Fig. 5. Simultaneous measurements of focal cytosolic and intra-SR Ca during
spontaneous Ca sparks. (A) Representative line-scan images of rhod-2 (Upper)
and fluo-5N (Lower) fluorescence in control (Left) and HF (Right) myocytes. (B)
Time courses of the local cytosolic and intra-SR Ca signals. (Inset) Comparison
of time course of [Ca]SR recovery after spark-associated Ca release in control
(black) and HF (red) myocytes. Fig. 6. RyR sensitivity to luminal [Ca] is increased in failing hearts. (A) Repre-
sentative traces of current recordings in RyR channels from control (Left) and HF
(Right) samples at different trans Ca concentrations (as indicated above the
overload was used, our experiments showed an increase in the traces). Single-channel activities were determined at a ⫹40-mV holding poten-
frequency of sparks in HF myocytes. In the study by Song et al., tial. Experimental solutions contained 350 mmol兾liter CsCH3SO3, 0.07 mmol兾liter
the increase in the Ca spark rate was rationalized by proposing CaCl2, 3 mmol兾liter MgATP, and 20 mmol兾liter Hepes (pH 7.4) on the cytosolic (cis)
that local regions with elevated SR Ca content exist in HF side of the bilayer, and 20 mmol兾liter CsCH3SO3, 0.0001–2 mmol兾liter CaCl2, 0 –1
myocytes that escape detection by the caffeine method used to mmol兾liter EGTA, and 20 mmol兾liter Hepes (pH 7.4) on the luminal (trans) side of
measure total SR Ca content. However, our direct [Ca]SR the bilayer. Channel openings are shown as upward deflections from the closed
level. (B) Open probability (Po) of RyR channels from control and failing hearts as
measurements demonstrated that [Ca] was lowered throughout
a function of trans [Ca]. Data are presented as means ⫾ SEM for three to eight
the entire SR network, and that the increased Ca spark rate was control and two to four failing hearts. Continuous lines were obtained by fitting
a result of enhanced sensitivity of RyRs to luminal Ca in the data with sigmoidal functions.
myocytes from failing hearts. It is unknown whether calcium
dysregulation and the underlying mechanisms differ between left
ventricular hypertrophy and HF. RyR modulation by luminal Ca appears to involve auxiliary
In previous studies measuring RyR function in lipid bilayers,
proteins such as CASQ2, triadin, and junctin that are removed
no differences in channel Po between preparations from control
by RyR purification (5) (Fig. 9, which is published as supporting
and failing canine hearts were reported (31). This result is in
contrast to the altered RyR function that we observed. We
suggest that the substantially longer duration of HF in our study
may contribute to this difference. Alternatively, it is possible that
the experimental conditions may underlie this discrepancy. We
note that in our experiments, differences in channel behavior
were observed only in the presence of cytosolic MgATP (un-
published results). This observation would be consistent with
previous reports that the effects of luminal Ca on RyR activity
rely on the presence of an allosteric activating ligand (ATP,
caffeine, sulmazole) (3, 4).
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