Download Heart Failure

Heart Failure
Intracellular Protein Aggregation Is a Proximal Trigger of
Cardiomyocyte Autophagy
Paul Tannous, PhD; Hongxin Zhu, PhD; Andriy Nemchenko, PhD; Jeff M. Berry, MD;
Janet L. Johnstone, BS; John M. Shelton, BS; Francis J. Miller, Jr, MD;
Beverly A. Rothermel, PhD; Joseph A. Hill, MD, PhD
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Background—Recent reports demonstrate that multiple forms of cardiovascular stress, including pressure overload,
chronic ischemia, and infarction-reperfusion injury, provoke an increase in autophagic activity in cardiomyocytes.
However, nothing is known regarding molecular events that stimulate autophagic activity in stressed myocardium.
Because autophagy is a highly conserved process through which damaged proteins and organelles can be degraded, we
hypothesized that stress-induced protein aggregation is a proximal trigger of cardiomyocyte autophagy.
Methods and Results—Here, we report that pressure overload promotes accumulation of ubiquitinated protein aggregates
in the left ventricle, development of aggresome-like structures, and a corresponding induction of autophagy. To test for
causal links, we induced protein accumulation in cultured cardiomyocytes by inhibiting proteasome activity, finding that
aggregation of polyubiquitinated proteins was sufficient to induce cardiomyocyte autophagy. Furthermore, attenuation
of autophagic activity dramatically enhanced both aggresome size and abundance, consistent with a role for autophagic
activity in protein aggregate clearance.
Conclusions—We conclude that protein aggregation is a proximal trigger of cardiomyocyte autophagy and that autophagic
activity functions to attenuate aggregate/aggresome formation in heart. Findings reported here are the first to
demonstrate that protein aggregation occurs in response to hemodynamic stress, situating pressure-overload heart
disease in the category of proteinopathies. (Circulation. 2008;117:3070-3078.)
Key Words: autophagy 䡲 heart failure 䡲 hypertrophy 䡲 protein aggregation 䡲 remodeling
U
nder conditions of stress, the heart undergoes a compensatory hypertrophic growth response that serves to
normalize wall stress and to diminish myocardial oxygen
demand.1 In the chronic state, cardiac hypertrophy is an
independent risk factor for heart failure and lethal arrhythmia,
leading causes of morbidity and mortality in Western society.
Numerous pathways have been causally implicated in the
transition from myocyte hypertrophy to failure,2 including
programmed cell death, cellular atrophy, and very recently,
autophagy.3,4 However, mechanisms governing the transition
from compensated hypertrophy to heart failure are incompletely characterized.
cytoplasmic contents without clearly defined substrate specificity.6 As the autophagosome matures, it fuses with a
lysosome to form an autolysosome, leading to proteolysis of
engulfed materials.
Autophagic activity is associated with the pathogenesis of
various diseases, including neurodegenerative disorders, skeletal myopathy, cancer, and microbial infection.7 Recent
reports demonstrate that multiple forms of cardiovascular
stress, including pressure overload, chronic ischemia, ischemia-reperfusion, and diphtheria toxin–induced injury, provoke an increase in autophagic activity in cardiomyocytes.4
Our group demonstrated recently that in pressure-overload
heart failure, a common form of clinical heart failure,
induction of autophagic activity is maladaptive.3
A prominent feature among neurodegenerative diseases
associated with autophagy, including Huntington’s disease,
amyotrophic lateral sclerosis, parkinsonism, and Alzheimer
disease, is deposition of proteins within intracellular aggregates.8 The prevailing notion is that autophagic pathways
serve a salutary function by facilitating removal of aggregates
Clinical Perspective p 3078
Autophagy is a highly conserved process of protein degradation involved in turnover of mitochondria and peroxisomes and nonselective degradation of cytoplasmic components during periods of starvation or stress.5 The autophagic
reaction begins with formation of the autophagosome, a
double-membrane structure of unknown origin that engulfs
Received January 19, 2007; accepted March 20, 2008.
From the Departments of Internal Medicine (Cardiology) (P.T., H.Z., A.N., J.M.B., J.L.J., J.M.S., B.A.R., J.A.H.) and Molecular Biology (J.A.H.),
University of Texas Southwestern Medical Center, Dallas, and the Department of Internal Medicine (F.J.M.), University of Iowa, Iowa City.
The online Data Supplement can be found with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.107.763870/DC1.
Correspondence to Joseph A. Hill, MD, PhD, UT Southwestern Medical Center, NB11.200, 6000 Harry Hines Blvd, Dallas, TX 75390 – 8573. E-mail
[email protected]
© 2008 American Heart Association, Inc.
Circulation is available at http://circ.ahajournals.org
DOI: 10.1161/CIRCULATIONAHA.107.763870
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Tannous et al
too large for efficient proteasome-mediated clearance.9 In
fact, in several neurodegenerative diseases, including Huntington’s disease10 and amyotrophic lateral sclerosis,11 a
strong association exists between induction of autophagy and
the presence of protein aggregates. In the absence of basal
levels of autophagic activity in brain, abnormal aggregates of
intracellular proteins develop,12,13 and pharmacological or
genetic induction of autophagy is sufficient to reduce
polyglutamine-induced cytotoxicity in animal models of
Huntington’s disease.14 Taken together, these data suggest
that intracellular protein aggregates are capable of stimulating
autophagic activity, which serves, in turn, to facilitate clearance of the aggregates.
Methods
Pressure-Overload Hypertrophy
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Male C57BL6 mice (6 to 8 weeks old) were subjected to pressure
overload by severe thoracic aortic banding (sTAB), a model of
pressure-overload heart failure.15
Primary Culture of Neonatal Rat
Ventricular Myocytes
Cardiomyocytes were isolated from the ventricles of 1- to 2-day-old
Sprague-Dawley rat pups and plated as described16 at a density of
1250 cells per 1 mm in medium containing 10% FCS with 100
␮mol/L bromodeoxyuridine. Forty-eight hours after plating, cells
were transferred to medium supplemented with 1% FBS and 1
␮mol/L bromodeoxyuridine, at which point treatment began.
Immunohistochemistry
We performed antigen retrieval by microwave heat-induced epitope
retrieval using 1⫻ Biogenex Citra (Biogenex, San Ramon, Calif; 10
minutes at 95°C). Primary antibody dilutions were as follows: 1:30
␣-B-crystallin (CryAB; Vector Laboratories, Burlingame, Calif;
VP-A103), 1:50 anti–MAP-LC3 (Santa Cruz Biotechnology, Inc,
Santa Cruz, Calif; sc-16756), 1:50 anti-vimentin (Santa Cruz; sc5565), 1:50 anti-ubiquitin (Santa Cruz; sc-9133), 1:1000 antiubiquitin (Abcam; ab7254), or 1:50 ␥-tubulin (Santa Cruz;
sc-10732).
Immunocytochemistry
Cultured cardiomyocytes were washed 3 times in PBS supplemented
with calcium (CaCl2 0.1g/L) and magnesium (MgCl2-6H2O 0.1g/L).
Cells were then fixed in 4% paraformaldehyde, permeabilized for 2
minutes in 0.1% Triton-X 100, and then blocked for 15 minutes in
PBS with 3% normal goat serum and 1% BSA.
Transient Transfection of
Cultured Cardiomyocytes
Twenty-four hours after plating, neonatal rat ventricular myocytes
(NRVMs) were transfected with a green fluorescent protein (GFP)–
tagged LC3 construct as previously described.3
Chymotrypsin-Like Activity
Cells were harvested in buffer H (20 mmol/L Tris-HCl, 20 mmol/L
NaCl, 1 mmol/L EDTA, 5 mmol/L bromomercaptoethanol, pH 7.6 at
4°C) and sheared by repeated passage through a 27-gauge needle,
and the supernatant was collected after sedimentation of insoluble
material (centrifugation at 16 000g for 10 minutes). Then, 10 ␮g
supernatant protein was added to 300 ␮L activity assay buffer
(50 mmol/L Tris-HCl, 5 mmol/L bromomercaptoethanol, 50 ␮mol/L
Suc-LLVY-AMC, pH 8.0 at 37°C).
Statistical Methods
Averaged data are reported as mean⫾SEM. Statistical significance
was analyzed (StatView) with Student’s unpaired, 2-tailed t test
Aggresomes and Cardiac Autophagy
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(comparison of 2 groups) or 1-way ANOVA (comparison of ⱖ2
independent groups), followed by Bonferroni’s method for post hoc
pairwise multiple comparisons.
The authors had full access to and take full responsibility for the
integrity of the data. All authors have read and agree to the
manuscript as written.
Results
Pressure Overload Induces Proteotoxic Stress in
Ventricular Myocytes
We have shown previously that cardiomyocyte autophagy
contributes to pathological remodeling of the left ventricle
(LV) induced by severe afterload stress.3 To define proximal
events that trigger autophagy, we subjected mice to sTAB, a
procedure that induces pressure-overload heart failure.15 As
reported previously,15 7 days of sTAB was sufficient to
induce clinical heart failure as demonstrated by development
of robust cardiac hypertrophy, pulmonary edema, and diminished systolic function (Figure 1).
Elevated afterload imposes oxidative, biomechanical, and
neurohumoral stress on the heart, each of which is capable of
eliciting proteotoxic injury. Given this, we examined pressure-overload–stressed LV for accumulation of damaged,
misfolded proteins. Damaged proteins are covalently coupled
to ubiquitin, which targets the tagged substrate for proteolysis
by the proteasome. If accumulation of ubiquitinated proteins
outpaces proteasomal degradation, buildup of intracellular
ubiquitinated protein occurs, resulting in formation of protein
aggregates, large heterogeneous complexes that are poor
substrates for proteasome-mediated proteolysis.17
To test for increases in ubiquitinated proteins and protein
aggregates, we immunostained sections of pressure-stressed
heart for ubiquitin. Here, we detected dramatic accumulation
of ubiquitin-positive inclusion bodies distributed throughout
the LV 72 hours after sTAB (Figure 2A). No significant
ubiquitin-like immunoreactivity was detected in regions not
subjected to biomechanical stress such as right ventricle or
either atria. Of interest, we detected a preponderance of
ubiquitin staining in the basal septum, a region previously
reported to be a “hot spot” for load-induced autophagic
activity.3 Western blot analysis confirmed the robust induction of ubiquitinated protein, detected as diffuse, highmolecular-weight bands on immunoblot, in pressure-stressed
LV (Figure 2B).
In the context of cellular stress, the abundance of chaperone proteins, molecules that play a critical role in protein
targeting and stability, increases as an adaptive response.
Consistent with this notion, we detected significant increases
in the abundance of multiple heat shock proteins (HSPs) in
pressure-stressed myocardium 7 days after surgery (Figure
2C). Steady-state abundances of CryAB, HSP25, and
GADD45 were modestly increased and that of BiP was
dramatically increased. Together, these findings are consistent with a model in which the intracellular milieu within
load-stressed cardiomyocytes is conducive to protein damage
and misfolding.
Accumulation of ubiquitinated proteins is generally believed to occur as a consequence of 1 of 3 mechanisms: an
increase in protein ubiquitination, a decrease in deubiquitina-
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Figure 2. Proteotoxic stress in load-induced heart failure. A,
Representative immunohistochemical staining revealing accumulation of ubiquitin-conjugated proteins in basal interventricular septum 72 hours after sTAB (scale bar⫽200 ␮m). B, Representative immunoblot probed for ubiquitin demonstrating
induction of high-molecular-weight, ubiquitinated protein in
pressure-stressed LV. C, Representative immunoblot of LV
lysates 72 hours after sTAB or sham operation and probed for
multiple protein chaperones, including CryAB, HSP25, BiP, and
GADD45. D, Proteasome activity assayed 1 week after sTAB is
increased (1.35⫾0.2; n⫽4) relative to sham-operated controls
(1.0⫾0.3; n⫽4). RV indicates right ventricle; AV, aortic valve;
and BS, basal septum. *P⬍0.05.
tion of exogenous ATP (data not shown). Together, these data
suggest that accumulation of ubiquitinated protein is not a
consequence of diminished proteasome activity but rather is
due to increased flux that exceeds proteasome capacity.
Autophagic Activity Increases in
Pressure-Stressed Ventricle
Figure 1. sTAB induces clinical heart failure. A, Representative
4-chamber sections of mouse hearts 7 days after sham or sTAB
surgery. Scale bar⫽1 mm. B, Seven days after sTAB, animals
demonstrated signs of heart failure with significant increases in
heart mass, lung mass (indicative of pulmonary edema), and
impairment in systolic performance. Mean data from sham (n⫽5)
and sTAB (n⫽5) mice subjected to surgery as listed. HW/BW
indicates heart weight/body weight; LWeight, lung weight.
*P⬍0.05.
tion, or a decrease in proteasome-mediated clearance. To
determine whether the accumulation of ubiquitinated proteins
in pressure-stressed LV was due to a decrease in proteasome
activity, we assayed ventricular lysates for chymotrypsin-like
activity (CTL-A). One week after sTAB, basal septum
CTL-A was increased 35⫾2% compared with sham-operated
controls (P⬍0.05; n⫽4; each lysate run in triplicate; Figure
2D). To test for a component of ATP-dependent proteasome
activity, CTL-A was measured in lysates (n⫽3) supplemented with ATP. In these experiments, CTL-A was not
significantly different from that measured without the addi-
Autophagic activity was monitored by morphological and
biochemical means. In sTAB hearts, we detected an increased
abundance of multilamellar autophagosomes by 72 hours
after surgery (Figure 3A), findings indicative of autophagic
activity. Additionally, during the autophagic response, LC3
(microtubule-associated protein 1 light chain 3), an 18-kDa
homologue of Atg8 in yeast, is processed and lipid conjugated.18 The resulting 16-kDa active isoform migrates from the
cytoplasm to isolation membranes and autophagosomes. Recently, intracellular migration of LC3 to vesicular membranes
has emerged as a reliable marker of autophagic activity.3,19
Consistent with increased autophagic activity, we detected robust increases in autophagosome-localized LC3 in pressurestressed cardiomyocytes (Figure 3B). These findings were corroborated by immunoblot analysis that demonstrated increases
in LC3 processing and elevated levels of Beclin 1 protein, 2
markers of autophagic activity (Figure 3C, data not shown).
Intracellular Aggresome Formation in
Pressure-Stressed Ventricle
In certain contexts, protein aggregates coalesce as perinuclear
structures called aggresomes.8,20 Aggresomes are cytoplasmic
inclusion bodies located in the perinuclear region near the
microtubule-organizing center that result from active, dynein-
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Figure 4. Aggresome formation in pressure-stressed LV. A,
Representative immunohistochemical sections 7 days after
sTAB immunostained for vimentin and CryAB (A) or ubiquitin
and CryAB (B) (scale bar⫽25 ␮m; n⫽4 each group). Confocal
imaging revealed perinuclear colocalization consistent with
aggresome formation (arrow).
Figure 3. Severe pressure stress induces cardiomyocyte autophagy. A, Representative electron micrograph revealing a multilamellar autophagosome in basal septum of LV 48 hours after
sTAB (scale bar⫽500 nm). B, Anti-LC3 immunofluorescence
reveals redistribution of LC3 into punctate dots, indicative of
autophagosome accumulation (scale bar⫽10 ␮m). C, Representative immunoblot demonstrating that severe pressure stress
triggers significant LC3 processing.
dependent transport of small aggregates from other parts of
the cell. Given our observations of robust increases in
chaperone protein levels in the setting of accumulation of
ubiquitin-positive high-molecular-weight proteins, we hypothesized that aggresome formation was occurring. To test
for their presence, we costained pressure-stressed LV tissues
for CryAB and vimentin, a structural component of the
aggresome.9 After 7 days of severe pressure-overload stress,
we detected juxtanuclear organization of CryAB-associated
proteins with an adjacent vimentin-containing shell (Figure
4A, arrow), a finding consistent with aggresome formation.
Further evidence of aggresome formation was found when we
coimmunostained for CryAB and ubiquitin. Again, after 7
days of pressure stress, we observed an increase in both
ubiquitin and CryAB staining, with colocalization of signal in
the perinuclear region (Figure 4B, arrow). Thus, ubiquitinlike immunoreactivity in pressure-stressed cardiac myocytes
accumulated as large perinuclear aggregates that colocalize
with CryAB and vimentin (additional images in Figure I of
the online Data Supplement). From these findings, we con-
clude that severe pressure overload triggers protein damage,
protein ubiquitination, and formation of perinuclear
aggresomes.
Protein Aggregation Triggers Autophagy
These findings led us to hypothesize that accumulation of
ubiquitinated proteins is a proximal step leading to protein
aggregation and subsequent activation of autophagic clearance mechanisms. To test this, we treated NRVMs with a
proteasome inhibitor (MG-132, 5 ␮mol/L), isolated protein
from the insoluble fraction, and probed for ubiquitin. In these
studies, we found that insoluble, ubiquitin-tagged protein
accumulation was detectable as early as 4 hours after proteasome inhibition (Figure 5A). At subsequent time points,
accumulation of ubiquitin-tagged protein within the insoluble
fraction was progressive and robust. No evidence of cytotoxicity was detected out to 16 hours (Figure 5B), although a
modest degree of cell death was detected on exposure to
MG-132 for ⱖ24 hours. These findings suggest that the
cardiomyocyte cytosol has a limited capacity to retain ubiquitinated protein in the soluble fraction, with excessive
accumulation resulting in protein precipitation as an insoluble
matrix.
Next, we set out to confirm that autophagy is activated in
cardiac myocytes under conditions of protein aggregation. To
do this, we induced protein aggregation in NRVMs by
proteasome inhibition (MG-132 or epoxomicin); autophagic
capacity was reduced pharmacologically (3-methyladenine
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Figure 5. In vitro aggresome formation is secondary to accumulation of insoluble ubiquitinated protein. A, NRVMs were loaded
with polyubiquitinated protein by selective proteasome inhibition
(MG-132, 5 ␮mol/L). Representative immunoblot demonstrating
that ubiquitinated protein accumulates in the insoluble fraction
beginning at 4 hours. B, Exposure to rapamycin, 3-MA,
MG-132, or epoxomycin did not induce significant cell death
(MTS assay). C, Representative immunoblot demonstrating that
LC3-II levels, indicative of autophagic activation, are increased
by rapamycin and diminished by 3-MA. Proteasome inhibition
by MG-132 or epoxomycin elicited modest increases in LC3
processing. D, Mean data from 3 independent experiments. E,
Representative immunocytochemical staining after 16 hours of
proteasome inhibition revealing perinuclear coalescence of
ubiquitin-tagged protein (E2). Inhibition of autophagy by 3-MA
(5 mmol/L) amplified aggresome formation (E4) (scale bar⫽10
␮m). Ctrl indicates control.
[3-MA]). 3-MA inhibits autophagy at the stage of sequestration in which a double-membrane structure forms around a
portion of the cytosol and sequesters it from the rest of the
cytoplasm to form an autophagosome.21 In these experiments,
we observed increased LC3 processing in response to proteasome inhibition, consistent with an increase in autophagic
activity (Figure 5C and 5D). Suppression of autophagy
(3-MA) elicited decreases in LC3 processing, as expected,
and a robust accumulation of ubiquitinated substrates. Rapamycin, an activator of autophagy, triggered modest increases
in LC3 processing above that elicited by proteasome inhibition (Figure 5C and 5D). Together, these data are consistent
with a model in which protein aggregation is sufficient to
induce cardiomyocyte autophagy, which then functions as an
aggregate clearance mechanism.
To determine the subcellular localization of ubiquitinated
proteins that accumulate in response to proteasome inhibition—and to test further for aggresome formation—we studied NRVMs exposed to prolonged inhibition of the proteasome (16 hours). Here, we detected perinuclear coalescence
of ubiquitin-conjugated proteins (Figure 5E) similar to that
seen in pressure-stressed LV in vivo. To test whether autophagic activity can reduce the abundance of aggregated
proteins, we cultured cells in medium supplemented with
3-MA (5 mmol/L). As expected, we detected no change in
ubiquitin levels or subcellular organization of aggregates in
control cells treated with a short course of 3-MA alone
(Figure 5E, image 3). However, in cells in which proteasome
activity was inhibited (MG-132) and autophagy was blocked
(3-MA), we observed a dramatic increase in the size of
perinuclear aggresomes (Figure 5E, image 4). These data
suggest that autophagy antagonizes aggresome formation by
providing an alternative clearance pathway.
This model was further tested by suppressing autophagy by
siRNA-mediated knockdown of Beclin 1. First, robust knockdown of Beclin 1, a protein required for recruitment of
Atg12-Atg5 conjugates to preautophagosomal membranes,22
was confirmed in control experiments (supplemental Figure
II). Knockdown of Beclin 1 led to significant decreases in
constitutive autophagic activation in NRVMs, and it was not
associated with cytotoxicity (supplemental Figure II). Next,
we tested the effects of Beclin 1 knockdown on autophagic
activity. First, it is important to note that Beclin 1 is not
required for LC3-I to LC3-II conversion (ie, lipidation) but is
required for LC3 association with membranes.23 Given this,
tracking LC3 processing is not a reliable means of quantifying autophagy in the setting of Beclin 1 knockdown. Thus, we
tracked p62 abundance because this protein is degraded in
autophagosomes.24 As expected, Beclin 1 knockdown at both
24 hours (Figure 6) and 48 hours (not shown) elicited
increases in the abundance of p62. Proteasome inhibition with
MG-132 decreased p62 levels, consistent with degradation of
p62 by activated autophagy (Figure 6). Thus, these data lend
further credence to the notion that protein aggregation is
sufficient to induce cardiomyocyte autophagy.
Parallel Activation of Proteasomal and Autophagic
Clearance Pathways
In neurons, protein aggregates, similar to those reported here,
are capable of inducing autophagy.25 Thus, we tested whether
the presence of aggregates in cardiomyocytes is sufficient to
trigger autophagy. To do this, we monitored autophagic
activity in vitro by tracking the localization of GFP-tagged
LC3 in transiently transfected cardiac myocytes. With this
approach, autophagic activity can be detected as punctate,
autophagosome-localized GFP signal, as opposed to the
diffuse, cytosolic distribution seen under resting conditions.3,19 In cells treated with a proteasome inhibitor (MG132, 5 ␮mol/L for 16 hours), we detected an abundant
increase in autophagosome-localized LC3 (Figure 7A). In
these experiments, autophagic activity, quantified as the
number of cells with autophagic vacuoles divided by the total
number of transfected cells, was increased 1.8-fold (P⬍0.05;
Figure 7B). Of note, induction of cardiomyocyte autophagy
in response to proteasome inhibition was similar in magnitude to that triggered by rapamycin (10 nmol/L), a powerful
activator of autophagy (Figure 7B). Together, these data are
consistent with the induction of autophagy in response to
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Figure 6. Levels of p62, an autophagy substrate, decline with
proteasome inhibition. A, Representative immunoblot demonstrating that p62, a protein component of the autophagosome,
increases with Beclin 1 knockdown–induced suppression of
autophagy. Proteasome inhibition (MG-132, 5 ␮mol/L for 16
hours) antagonizes these increases in p62, consistent with activation of autophagy by protein aggregate accumulation. B,
Mean data from 2 independent experiments. *P⬍0.05 vs
control.
accumulation of protein aggregates and argue strongly
against nonspecific effects resulting from MG-132 toxicity.
Given our in vivo findings demonstrating that both proteasomal and autophagic clearance pathways are activated in
response to pressure-overload stress, combined with our in
vitro data demonstrating that inhibition of the proteasome
leads to an increase in autophagic activity, we set out to
determine whether the converse pertains, ie, whether inhibition of autophagy results in an increase in proteasome
activity. NRVMs were cultured for 7 days in the presence of
3-MA or vehicle, with proteasome activity determined by
measuring CTL-A. In these experiments, we observed a
73⫾17% increase (n⫽3; each lysate run in triplicate;
P⬍0.05) in CTL-A after 7 days of 3-MA treatment relative to
control (Figure 7C). These results are consistent with our
finding of coordinated regulation of proteasomal and autophagic mechanisms in pressure-stressed heart. Furthermore,
they lend support to a model in which autophagic and
proteasomal clearance mechanisms function in parallel in
cardiac myocytes.
Discussion
Recent reports reveal that cardiomyocyte autophagy is
activated in response to multiple forms of cardiac injury.4
However, proximal triggers of autophagic activity in the
heart are unknown. The major findings of this study are
Figure 7. Aggresomes are a proximal trigger of cardiomyocyte
autophagy. A, NRVMs were transfected with GFP-tagged LC3
and subjected to proteasome inhibition (16 hours), resulting in
redistribution of LC3 in an autophagosome-specific punctate
pattern. Images of representative cells are depicted (scale
bar⫽10 ␮m). B, Quantification of the numbers of cells manifesting autophagic activity (*P⬍0.05 relative to control). More than
300 cells were evaluated in each treatment condition in 2 independent experiments. C, Cells treated with 3-MA for 7 days
were assayed for proteasome activity, revealing a robust
increase in activity. *P⬍0.05.
that (1) pressure-overload hemodynamic stress elicits
changes in the cardiomyocyte milieu conducive to protein
damage and misfolding with consequent accumulation of
polyubiquitinated proteins and increases in chaperone
protein levels; (2) protein aggregation takes place, leading
to formation of perinuclear aggresomes; (3) clearance is
activated via both autophagic and proteasomal protein
quality control pathways; (4) protein aggregate and aggresome formation are sufficient to trigger cardiomyocyte
autophagy, and (5) autophagic activity in the cardiomyocyte participates in clearance of aggregated proteins.
Finally, the findings reported here are the first to demonstrate that protein aggregation occurs in response to
hemodynamic stress, situating pressure-overload heart disease in the category of proteinopathies.
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Stress-Induced Cardiomyocyte Proteinopathy
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Proteinopathy, toxic aggregations of misfolded proteins, is a
growing family of human disorders that includes Alzheimer
disease, parkinsonism, amyotrophic lateral sclerosis, and both
polyglutamine and polyalanine expansion disorders.26,27 In
heart disease, abnormal protein aggregation and accumulation
of ubiquitinated proteins have been detected in human hearts
with idiopathic or ischemic cardiomyopathies.28,29 In both
brain and heart, however, relatively little is known regarding
whether these intracellular inclusions are toxic themselves or
whether they represent a compensatory mechanism that
sequesters harmful, soluble proteins within the cytoplasm.
Insoluble protein aggregates are processed by pathways
that are just now being deciphered. First, misfolded proteins
are delivered to the microtubule-organizing center by dyneindependent retrograde transport along microtubules. When the
degradative capacity of the proteasome is exceeded, protein
aggregates accumulate in perinuclear inclusions called aggresomes,20 organized structures surrounded by vimentin filaments that recruit chaperones, ubiquitin, proteasomes, and
mitochondria. In pressure-stressed myocardium, we have
detected an abundance of large perinuclear inclusions marked
by ubiquitin that colocalize with chaperone proteins and
vimentin, fulfilling the definition of aggresomes. In liver,
aggresomes (Mallory bodies) form in response to a variety of
stresses, including proteasome inhibition.30 Aggresomes also
have been detected in a model of desmin-related cardiomyopathy, a disorder characterized by abnormal amyloid deposition owing to dysfunctional chaperone protein function.31
The findings reported here, however, are the first to describe
aggresome formation in a common cardiovascular disorder,
ie, load-induced heart disease.
Cardiomyocyte Autophagy Contributes to Protein
Quality Control
Autophagy occurs in a wide range of eukaryotic cells and is
the sole pathway for organelle turnover. Autophagy, the basic
mechanisms of which are largely conserved from yeast to
mammals,5 is a vital pathway for degrading normal and
aggregated proteins, particularly under stress or injury conditions. Autophagy can be induced by diverse stimuli, including starvation, hypoxia, intracellular stress, and developmental signals. It accomplishes a host of functions critical to
normal homeostasis and development. In the context of
nutrient deprivation, autophagic activity is adaptive in that
degradation of cytosolic components releases substrates for
intermediary metabolism. Autophagy is also a mechanism for
eliminating damaged proteins and organelles that might
otherwise be toxic or trigger apoptotic death. For example,
targeted inhibition of Atg7, a gene required for autophagic
activity, leads to accumulation of ubiquitin-positive aggregates.32 During development, autophagy is a means of remodeling tissues or removing unneeded cells.33 In other settings,
however, autophagic activity is associated with the pathogenesis of disease, and unrestrained autophagic activity can cause
cell death.7
In the present study, we establish a link between protein
aggregation and induction of cardiomyocyte autophagy. In
vivo, we describe the accumulation of ubiquitinated proteins
in a spatial and temporal pattern consistent with our recent
report of pressure overload–induced autophagy.3 We further
characterize the organization of damaged proteins into
vimentin-associated, perinuclear, aggresome-like structures.
Because aggresome formation is a general response of cells
that occurs when proteasome capacity is exceeded by the
production of aggregation-prone misfolded proteins,20 we
inhibited the proteasome in cultured cardiomyocytes to test
directly for a causal link between aggregate formation and
cardiomyocyte autophagy. In these experiments, we found
that accumulation and aggregation of ubiquitinated protein is
a powerful inducer of autophagy, capable of robust activation
of autophagy to levels comparable to pharmacological induction. Together, these findings are consistent with a model in
which autophagic activity is a general response to protein
aggregation in the heart and point to a potential role for
autophagy in cardiomyopathies of diverse origin.
Interestingly, we and others do not detect inclusions that
are membrane bound. The inclusions we detect are much
larger than mammalian autophagosomes. Together, these
findings suggest that autophagy serves to clear monomeric
and oligomeric precursors of aggregates rather than the large
inclusions themselves.
To test the involvement of proteasomal activity, we measured chymotryptic activity acting on a small fluorogenic
peptide substrate Suc-LLVY-AMC. Owing to its small size,
this peptide diffuses easily into the proteolytic chamber of the
20S proteasome34; hence, it provides a readout of 20S
proteasome activity. Consistent with this, the addition of
exogenous ATP to stimulate 26S proteasome activity had no
significant effect on CTL-A (⬍10%). In these experiments,
we find that total proteasome function is increased in heart
failure, consistent with similar findings in a model of desminrelated cardiomyopathy,35 suggesting increased flux through
proteasomal degradation pathways. Our data demonstrate that
proteasomal and autophagic activities increase in parallel.
␣-1-Antitrypsin deficiency is another proteinopathy in
which autophagy is activated as a clearance mechanism. In
this disease, proteolytic destruction of elastic connection
tissue matrix occurs in lung because of unrestrained proteolysis. In liver, however, toxicity resulting from the accumulation of misfolded mutant ␣-1-antitrypsin leads to chronic
liver inflammation and hepatocellular carcinoma. Recent
studies have revealed dramatic increases in autophagic activity in liver, which serves to prevent toxic accumulation of
mutant ␣-1-antitrypsin by selectively targeting insoluble
aggregates.36
Thus, a growing body of evidence implicates autophagy as
a protective response in genetic diseases associated with
cytoplasmic aggregation-prone proteins. Here, we extend this
to disease triggered by environmental stress. Importantly,
recent studies demonstrating that pharmacological upregulation of autophagy is protective in a wide variety of disease
models associated with intracellular protein aggregation raise
the exciting prospect of autophagic activation as a novel
therapeutic strategy.37 Our findings here extend this prospect
to the myocardium.
Tannous et al
Role in Heart Failure Progression
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The presence of increased autophagic activity in animal
models of heart failure and in samples from human failing
hearts provides no insight into whether cardiomyocyte autophagy is beneficial or pathological. Indeed, it is possible that
autophagic activity carries out different functions, depending
on disease stage and severity or the nature of the components
being degraded. For example, we have shown previously that
genetic amplification of the autophagic response to acuteonset pressure stress leads to increased hypertrophic growth
of the heart.3 On the other hand, if the protein aggregates
themselves are toxic, increased autophagic removal could be
beneficial. Furthermore, recent evidence strengthens the assertion that dysregulated autophagic activity induces a
caspase-independent programmed cell death program (type II
programmed cell death38 – 40). Nakai et al41 reported that
cardiac-specific inactivation of atg5 early in cardiogenesis
was not associated with an abnormal cardiac phenotype.
However, subjecting these animals to pressure stress during
adulthood triggered rapid and dramatic declines in cardiac
function. Intriguingly, inactivation of atg5 in the heart after
the mice had reached maturity led rapidly to cardiac hypertrophy, LV dilation, contractile dysfunction, and a syndrome
consistent with heart failure.41 Clearly, to exploit autophagy
as a therapeutic target, it is essential to identify when it is
protective and when it contributes to the pathogenesis of
disease.4
Evidence presented here indicates that autophagy is activated to eliminate damaged proteins that accumulate within
the cardiomyocyte. Several reports point to functional defects
in the ubiquitin-proteasome system in heart failure, resulting
in accumulation of misfolded proteins.27 Consistent with this,
we have observed increased ubiquitin staining in pressurestressed ventricle, which is most prevalent in the basal
septum. Therefore, activation of autophagy at early stages of
the disease may be a protective mechanism to scavenge and
eliminate misfolded, polyubiquitinated protein aggregates
that have overwhelmed the degradative capacity of the
proteasomal system.
Perspective
Cardiac hypertrophy is a major predictor of heart failure, a
prevalent disorder with high mortality, and there is urgent
need for novel therapies to prevent or reverse pathological
cardiac remodeling. Our data point to a progression of protein
damage, aggregation, and coalescence into aggresomes as a
previously unrecognized mechanism of disease. Furthermore,
our findings suggest that autophagic activity in cardiomyocytes is a general response to diverse stressors and contributes
to disease pathogenesis in multiple contexts. As new advances in deciphering molecular regulation of autophagy
emerge, it may soon be possible to enhance or inhibit
autophagic activity selectively to affect heart disease meaningfully. In addition, given that some drugs already in clinical
use alter the process of autophagy and organ systems in
which increased autophagic activity is associated with disease, advances in this field are all the more urgent.
Aggresomes and Cardiac Autophagy
3077
Acknowledgment
We gratefully acknowledge Dr Beth Levine (UT Southwestern) for
helpful advice.
Sources of Funding
This work was supported by grants from the National Institutes of
Health (HL-075173, HL-006296, HL-080144 to Dr Hill; HL-072016
to Dr Rothermel) and American Heart Association (0640084N to Dr
Hill, 0655202Y to Dr Rothermel).
Disclosures
None.
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CLINICAL PERSPECTIVE
Chronic hemodynamic stress such as poorly controlled hypertension induces hypertrophic growth of the heart. This
pathological growth response, with time, leads to ventricular dilation, systolic dysfunction, and clinical heart failure, which
is a leading cause of morbidity and mortality in Western society. Whereas numerous signaling pathways have been
implicated in the stress response of the myocardium, mechanisms governing the transition from compensated hypertrophy
to heart failure are poorly understood. In this study, we report that pressure overload promotes accumulation of
ubiquitinated protein aggregates in left ventricular myocytes that are processed into structures called aggresomes. We also
demonstrate that these protein aggregates activate an evolutionarily conserved process of protein sequestration and removal
called autophagy. We go on to show that attenuation of autophagic activity dramatically enhances both aggresome size and
abundance, consistent with a role for autophagic activity in protein aggregate clearance. From these data, we conclude that
pressure overload–induced protein aggregation is a proximal trigger of cardiomyocyte autophagy and that autophagic
activity functions to attenuate aggregate/aggresome formation in heart. Findings reported here are the first to demonstrate
that protein aggregation occurs in response to hemodynamic stress, situating load-induced heart disease in the expanding
category of proteinopathic diseases.
Intracellular Protein Aggregation Is a Proximal Trigger of Cardiomyocyte Autophagy
Paul Tannous, Hongxin Zhu, Andriy Nemchenko, Jeff M. Berry, Janet L. Johnstone, John M.
Shelton, Francis J. Miller, Jr, Beverly A. Rothermel and Joseph A. Hill
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
Circulation. 2008;117:3070-3078; originally published online June 9, 2008;
doi: 10.1161/CIRCULATIONAHA.107.763870
Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2008 American Heart Association, Inc. All rights reserved.
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