Download Localization, Macromolecular Associations, and Function of the

Localization, Macromolecular Associations, and Function of
the Small Heat Shock–Related Protein HSP20 in Rat Heart
Walter Pipkin, MD; John A. Johnson, PhD; Tony L. Creazzo, PhD; Jarrett Burch, BS;
Padmini Komalavilas, PhD; Colleen Brophy, MD
Downloaded from http://circ.ahajournals.org/ by guest on June 16, 2017
Background—The small heat shock proteins HSP20, HSP25, ␣B-crystallin, and myotonic dystrophy kinase binding
protein (MKBP) may regulate dynamic changes in the cytoskeleton. For example, the phosphorylation of HSP20 has
been associated with relaxation of vascular smooth muscle. This study examined the function of HSP20 in heart muscle.
Methods and Results—Western blotting identified immunoreactive HSP20, ␣B-crystallin, and MKBP in rat heart
homogenates. Subcellular fractionation demonstrated that HSP20, ␣B-crystallin, and MKBP were predominantly in
cytosolic fractions. Chromatography with molecular sieving columns revealed that HSP20 and ␣B-crystallin were
associated in an aggregate of ⬇200 kDa, and ␣B-crystallin coimmunoprecipitated with HSP20. Immunofluorescence
microscopy demonstrated that the pattern of HSP20, ␣B-crystallin, and actin staining was predominantly in transverse
bands. Treatment with sodium nitroprusside led to increases in the phosphorylation of HSP20, as determined with
2-dimensional immunoblots. Incubation of transiently permeabilized myocytes with phosphopeptide analogues of
HSP20 led to an increase in the rate of shortening. The increased shortening rate was associated with an increase in the
rate of lengthening and a more rapid decay of the calcium transient.
Conclusions—HSP20 is associated with ␣B-crystallin, possibly at the level of the actin sarcomere. Phosphorylated HSP20
increases myocyte shortening rate through increases in calcium uptake and more rapid lengthening. (Circulation. 2003;
107:●●●-●●●.)
Key Words: myocytes 䡲 muscle, smooth 䡲 contractility
S
everal recent studies have demonstrated that nitric oxide
(NO) has direct effects on myocardial contractile function. Exogenous NO donors characteristically enhance relaxation without major effects on peak systolic function.1 This
selective relaxant action has been observed in intact hearts
and cardiac myocytes, and the effects have been attributed to
a direct action of NO on the heart.1,2 Increases in cGMP and
activation of cGMP-dependent protein kinase have been
implicated as the cellular mechanism.2 However, the specific
protein that is phosphorylated by cGMP-dependent protein
kinase that produces the relaxant effect in the heart has not
been identified.
We have recently determined that cyclic nucleotide–
dependent relaxation of vascular smooth muscle is associated
with an increase in the phosphorylation of the small heat
shock–related protein HSP20.3 These results have been independently confirmed by two other laboratories.4,5 HSP20 is
highly and constitutively expressed in muscle tissues and can
be phosphorylated in vitro by cGMP-dependent protein kinase.6,7 A more direct role for HSP20 in mediating smooth
muscle relaxation has been suggested, as follows: (1) Physiological release of NO, induced by acetylcholine or increased
flow, leads to measurable increases in the phosphorylation of
HSP208; (2) HSP20 is not phosphorylated in muscles that are
refractory to cGMP-dependent relaxation9; (3) pretreatment
of smooth muscles with cyclic nucleotide activators leads to
increases in the phosphorylation of HSP20 and inhibition of
contraction responses to agonists10; (4) the introduction of
phosphopeptide analogues of HSP20 into transiently permeabilized vascular smooth muscles inhibits contraction.3 Thus,
cyclic nucleotide-dependent relaxation of smooth muscle
seems to be modulated by increases in the phosphorylation of
HSP20.
The family of small heat shock proteins, HSP20, HSP25,
␣B-crystallin, and myotonic dystrophy kinase binding protein
(MKBP), are expressed in muscle tissues and share considerable sequence homology, primarily at the c-terminus.11
These proteins are often associated in large macromolecular
aggregates.12,13 Heat shock proteins have been implicated in
modulating the cellular response to many stressors (heat,
Received September 26, 2002; accepted September 30, 2002.
From the Departments of Surgery (W.P.), Medicine (J.A.J.) (Institute of Molecular Medicine and Genetics), and Pharmacology and Toxicology (J.A.J.),
Medical College of Georgia, Augusta; Pediatrics/Neonatology Division, Neonatal/Perinatal Research Institute (T.L.C., J.B.), Duke University Medical
Center, Durham, NC; Carl T. Hayden, Phoenix VAMC (P.K., C.B.), Phoenix, Ariz; and the Department of Bioengineering (P.K., C.B.), Arizona State
University, Tempe.
Correspondence to Colleen Brophy, MD, Research Professor of Bioengineering, Arizona State University, Vascular Surgeon, Phoenix VAMC CS112,
650 E Indian School Rd, Phoenix, AZ 85012. E-mail [email protected]
© 2002 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
DOI: 10.1161/01.CIR.0000044386.27444.5A
1
2
Circulation
January 28, 2003
chemical, osmotic, oxidative, and mechanical) and in modulating protein–protein interactions. An additional recently
described function of heat shock proteins is to modulate the
activity of kinases. MKBP activates myotonic dystrophy
protein kinase,11 and HSP90 modulates the activity of NO
synthase.14 Three of the small heat shock proteins, HSP25,
␣B-crystallin, and HSP20, are likely involved in cytoskeletal
dynamics. HSP25 (the homologous human protein is referred
to as HSP27) is an actin-associated protein and modulates
actin filament dynamics.15–17 HSP20 is also an actinassociated protein.18,19 HSP25 and ␣B-crystallin both associate with intermediate filament proteins and modulate the
interactions that occur between filaments in cellular networks.20 The small heat shock proteins may function to
preserve myofibrillar integrity in response to stress and may
serve a physiological function in the dynamic cytoskeletal
changes associated with contraction and relaxation.
Downloaded from http://circ.ahajournals.org/ by guest on June 16, 2017
Methods
Materials
Sodium dodecylsulfate (SDS), glycine, tris-(hydroxymethyl) aminomethane (Tris), and dithiothreitol (DTT) were from Research
Organics. Coomassie brilliant blue was from ICN Biomedicals Inc.
Recombinant HSP27 was from StressGen. Recombinant HSP20 was
produced as previously described.18 Piperazine diacrylamide and
other electrophoresis reagents were from BioRad. 3-[(3Cholamidopropyl dimethylammonio]-1-propanesulfonate (CHAPS),
ethylene glycol bis (␤-aminomethyl ether)-N,N,N⬘,N⬘-tetra acetic
acid (EGTA), ethylene diaminetetraacetic acid (EDTA),
polyoxyethylene-sorbitan monolaurate (Tween-20), purified bovine
␣B-crystallin, sodium nitroprusside, and all other reagent-grade
chemicals were from Sigma. Protease inhibitor cocktail [2 ␮mol/L
4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), 130 ␮mol/L
Bestain, 14 ␮ mol/L trans-epoxysuccinyl- L -leucylamido(4guanidino)butane (E-64), 1 ␮mol/L leupeptin, and 0.3 ␮mol/L
aprotinin, final concentrations] from Sigma was added to all homogenization buffers. Molecular weight standards were from Pharmacia.
Immobilon was from Millipore. Antibodies against sarcomeric actin
were from Sigma (St Louis, Mo); HSP20 and ␣B-crystallin were
from Dr Kanefusa Kato (Aichi, Japan)6; HSP25 was from Dr
Michael Welsh (University of Michigan, Ann Arbor, Mich)21; and
Accurate, StressGen, and myotonic dystrophy kinase binding protein
were from Dr Atshushi Suzuki (Yokohama, Japan).11 Goat antimouse and anti-rabbit secondary antibodies were from Jackson
Immunochemical (West Grove, Pa). Protein concentrations were
determined with the Coomassie Plus Protein Assay Reagent (Pierce).
Isolation of Rat Heart Tissue
Adult rats (2 to 3 months of age) were killed with CO2 inhalation, and
the hearts were dissected free from the thoracic cavity and placed in
PBS (10 mmol/L phosphate, pH 7.5, 0.15 mol/L NaCl, 4°C). The
atria were dissected free and the ventricle was cut into small strips
and homogenized for biochemical analyses. For immunohistochemical analyses, the thoracic aorta was cannulated and perfused with
PBS followed by 4% formalin in PBS at 100 mm Hg. Ventricular
myocytes were isolated from the hearts of 2-day-old SpragueDawley rats by gentle trypsinization and placed in culture as
previously described.22 Animal use was approved by the Institutional
Animal Care and Use Committee.
SDS-PAGE gels and transferred to Immobilon (100 mAmp for 12
hours). The blots were air dried and subsequently blocked with
tris-buffered saline (TBS, 10 mmol/L Tris, 150 mmol/L NaCl, pH
7.4) and 5% milk for 1 hour. The blots were then incubated with
anti-HSP20 (1/1000), anti–␣B-crystallin (1/1000), anti-HSP25 (1/
1000), or anti-MKBP (1/1000) antibodies in TBS/milk for 1 hour at
room temperature. The blots were washed 3 times (5 minutes each)
in TBS/Tween-20 (0.5%). The blots were then placed in goat
anti-rabbit secondary antibody diluted in TBS/milk (1/2000) for 1
hour at room temperature. The blots were then washed 6 times (5
minutes each) in TBS/Tween-20. Immunoreactive protein was determined with enhanced chemiluminescence (DuPont NEN) exposed
on x-ray film.
Subcellular Fractionation
Tissues were homogenized in homogenization buffer (HEPES
buffer, 25 mmol/L HEPES, 150 mmol/L NaCl, 10 mmol/L EDTA,
1 mmol/L DTT, 2 mmol/L benzamidine, pH 7.4, and protease
inhibitor cocktail [2 ␮mol/L 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), 130 ␮ mol/L Bestain, 14 ␮ mol/L transepoxysuccinyl-L-leucylamido(4-guanidino)butane (E-64), 1 ␮mol/L
leupeptin, and 0.3 ␮mol/L aprotinin, final concentrations] from
Sigma) (0.5 g tissue/1 mL buffer) at 4°C. The homogenate was
centrifuged at 3000g to remove debris and the nuclear pellet. The
supernatants were diluted to 5 ␮g of protein/␮L, and 250 ␮L of
supernatant was centrifuged at 10 000g for 10 minutes. The pellet
(P1) was resuspended in 125 ␮L of homogenization buffer and 125
␮L of 2⫻ sample buffer (6.25 mmol/L Tris, pH 6.8, 2% SDS, 5%
2-␤ mercaptoethanol, 10% glycerol, 0.025% bromophenol blue). To
125 ␮L of the 10 000g supernatant (S1), 125 ␮L of 2⫻ sample buffer
was added. The remaining 125 ␮L of 10 000g supernatant was
centrifuged at 100 000g. The 100 000g pellet (P2) was resuspended
in 125 ␮L of homogenization buffer and 125 ␮L of 2⫻ sample
buffer. A total of 125 ␮L of 2⫻ sample buffer was added to the
100 000g supernatant (S2). The samples were boiled for 8 minutes
after the addition of sample buffer, separated on 15% SDS-PAGE
gels, transferred to Immobilon membranes, and probed with antiactin antibodies (1/2000 dilution), anti-HSP20 antibodies (1/2000
dilution), anti–␣B-crystallin antibodies (1/2000 dilution), and antiMKBP antibodies (1/2000 dilution).
Gel Filtration
Gel filtration was performed as previously described.13 In brief, the
tissues were homogenized in HEPES buffer (0.5 g tissue/1 mL
buffer) at 4°C. The homogenate was centrifuged at 100 000g for 30
minutes at 4°C. 200 ␮L of supernatant (containing 200 ␮g total
protein) was applied to a Superose-6 HR 10/30 fast protein liquid
chromatography column (Pharmacia), eluted with column buffer,
and 0.5-mL fractions collected. For calibration, 100 ␮L of catalase
(232 kDa, 5 mg/mL) and BSA (67 kDa, 8 mg/mL) was applied to the
column.
Dot Blotting
One hundred microliters of each fraction from the column was
dot-blotted onto nitrocellulose. The blots were fixed with 20%
methanol, dried, blocked with TBS, 5% milk, for 1 hour, washed 3
times with TBS, and then probed with anti-HSP20, anti-MKBP, and
anti-␣B crystallin antibodies (1/2000 dilution in TBS, 5% milk) for
1 hour. Goat anti-rabbit secondary antibodies (1/2000 dilution) were
added to the blots for 1 hour. The blots were then washed 6 times
with TBS/0.5%Tween-20. Immunoreactive proteins were visualized
as described above with enhanced chemiluminescence, and densitometric analysis was performed with UN-SCAN-IT automated digitizing software (Silk Scientific Corporation).
Immunoblotting
Tissues were homogenized in 10 mmol/L EGTA, 2 mmol/L EDTA,
10 mmol/L ␤-mercaptoethanol, 1% glycerol, and 4% SDS in
60 mmol/L Tris, pH 7.0, with the use of a polytron homogenizer
(Brinkman Instruments). The homogenate was centrifuged (10 000g)
to remove insoluble material. Proteins were separated on 15%
Immunoprecipitation
Tissue was homogenized in TBS (0.5 g tissue per mL of buffer), and
then the samples were centrifuged at 10 000g for 15 minutes. The
soluble proteins were then diluted 10-fold with TBS. The antiHSP20 antiserum was added to the supernatants (1:50 dilution). The
Pipkin et al
samples were shaken gently for 14 hours at 4°C. Protein
A-Sepharose beads (1/10 volume) were added, and the samples were
incubated for an additional 3 hours at 4°C. The beads were washed
6 times with TBS, 0.5% Tween-20. A final wash of 10 mmol/L Tris
pH 7.4 was then done. The proteins were separated on 15%
SDS-PAGE gels, transferred to Immobilon, and probed with antiHSP20, anti–␣B-crystallin, or anti-MKBP antibodies as described
above.
Immunofluorescence Microscopy
Downloaded from http://circ.ahajournals.org/ by guest on June 16, 2017
Perfusion-fixed ventricular tissue was embedded in paraffin. Fivemicron cross-sections were mounted on polylysine slides. The slides
were deparaffinized with xylene and graded dilutions of ethanol.
Neonatal cardiac myocytes were grown on glass coverslips and fixed
with 4% paraformaldehyde. The sections were rinsed in PBS and
blocked with donkey serum (Jackson Immunoresearch, West Grove,
Pa) for 30 minutes. The slides were then incubated with anti-HSP20
(1/100), anti–␣B-crystallin (1/100), anti-sarcomeric actin (1/100), or
anti-MKBP (1/100) overnight at 4°C. The slides were then washed
with PBS, 4 times (15 minutes each), at room temperature. Antimouse and anti-goat Cy3 secondary antibodies (1/100) were used to
detect immunoreactive protein. The sections were imaged on a Zeiss
Axiophot microscope interfaced with a SPOT camera (Diagnostic
Instruments) and a Gateway computer.
Dimensional Gels
Tissues were snap-frozen in liquid nitrogen and ground to a fine
powder by mortar and pestle. The proteins were solubilized in
100 mmol/L DTT, 6 mol/L urea, 2% CHAPS overnight. Protein 30
␮g was loaded onto 12⫻15-cm-slab isofocusing gels consisting of
4% acrylamide, 0.1% piperazine diacrylamide, 9 mol/L urea, 5%
ampholines (5 parts 6 to 8, 3 parts 5 to 7, and 2 parts 3 to 10), and
2% CHAPS. The cathode buffer consisted of 20 mmol/L sodium
hydroxide and the anode buffer 10 mmol/L phosphoric acid. The
proteins were focused for 10 000 volt hours. The gels were fixed in
10% trichloroacetic acid and stained overnight with Neuhoff’s
Coomassie stain. The lanes of stained proteins were cut from the
isofocusing slab gels and equilibrated in 10 mmol/L Tris (pH 6.8),
3% SDS, 19% ethanol, 4% ␤-mercaptoethanol, and 0.004% bromophenol blue for 10 minutes. The proteins were then separated on 12%
acrylamide SDS gels and transferred to Immobilon 100 mAmp for 12
hours. The blots were probed for HSP20 as described above. The
isoelectric focusing gradient was determined with BioRad IEF
standards.
Peptide Synthesis
The peptides, HSP20 phosphoserine analogue N-WLRRASphos
APLPGLK (HSP20-PS), a scrambled phosphorylated peptide
N-PRKSphosLWALGRPLA (HSP20-SC), and a nonphosphorylated
peptide N-WLRRAAAPLPGLK (HSP20-NP), were synthesized on
a Procise (Applied Biosystems, Model 492) instrument according to
standard protocols. The peptides were purified with high-pressure
liquid chromatography, and purity was assured with mass spectrometry, as previously described.3
Permeabilization of Cardiac Myocytes
Permeabilization of cardiac myocytes was performed as described.22
Briefly, cells were slowly cooled by sequential 2-minute incubations
with room temperature PBS and then with 4°C PBS in an ice bath for
2 minutes. The PBS was discarded, and the cells were incubated with
ATP (30 ␮L of 200 mmol/L ATP, pH 7.4) followed immediately by
permeabilization buffer (20 mmol/L HEPES, pH 7.4, 10 mmol/L
EGTA, 140 mmol/L KCl, 50 ␮g/mL saponin, 5 mmol/L oxalic acid
dipotassium salt) containing the peptides (10 ␮mol/L) for 10 minutes
in an ice bath. The cells were then washed 4 times on ice with chilled
PBS. The cells were then returned to 37°C by incubations with room
temperature PBS and 37°C PBS. The original cell media was then
added at 37°C.
HSP20 in Rat Heart
3
Measurement of Cardiac Myocyte Shortening
Rate/Lengthening Rate
The culture dishes containing the myocytes were placed on a
Harvard Apparatus temperature-regulation device positioned on the
stage of an inverted microscope (Carl Zeiss Inc) and maintained at
37°C with a jacketed water bath. The microscope was outfitted with
a digital camera and Video Savant software. To determine the effects
of HSP20 peptide analogues on shortening rate, individual cells were
monitored before and after permeabilization. Shortening rates were
determined every 2 minutes (for a 15-second period) for a total of 10
minutes, as previously described.23 Images of individual cells were
captured at 30 frames/second.
Calcium Transients
Ca2⫹ transients were measured as previously described in detail
elsewhere with only minor modifications.24 For these experiments,
myocytes were cultured on 25-mm glass coverslips. After permeabilization (as described above), each coverslip was mounted in a
leak-proof circular holding chamber (Medical Systems Inc) and
gently washed several times with a Ringer solution containing
(in mmol/L) NaCl 142.5, KCl 4.0, MgCl2 1.8, CaCl2 1.8, N-2hydroxyethypiperazine-N-2-ethanesulfonic acid 5.0 (HEPES, pH
7.0), and glucose 5.0. After washing, the myocytes were incubated in
1 mL Ringer solution with 1 ␮mol/L fura-2 AM for 10 minutes at
37°C in a rotating water bath. The myocytes were subsequently
washed several times with dye-free Ringer solution and allowed to
stand covered at room temperature for 30 minutes to facilitate
deesterification.
Fluorescence measurements in individual rhythmically beating
myocytes were carried out at 37°C with the use of a DeltaScan
microspectrofluorometer (Photon Technology International) coupled
with an Olympus IX70 microscope equipped with an Olympus
UApo/340⫻40 oil immersion objective with a numerical aperture of
1.35. The fura-2 transients reported are the ratio of Ca2⫹ fluorescence
transients measured at excitation wavelengths 340 and 380 nm. The
myocytes were only illuminated at short intervals, and each sample
preparation was used for ⬍1 hour to reduce the possibility of
photobleaching or fura-2 leakage.
Statistical Analysis
Values are reported as mean⫾SEM, and n refers to the number of
animals examined. The statistical differences between 2 groups were
determined by Student’s t test and between multiple groups by 1-way
repeated-measures ANOVA with Sigma Stat software (Jandel Scientific). P⬍0.05 was considered significant.
Results
Specificity of Small HSP Antibodies
The small heat shock proteins are highly homologous11,25;
thus, to determine the specificity of the antibodies, immunoblots of recombinant HSP20, recombinant HSP25, bovine
lense ␣B-crystallin, and homogenized rat heart were probed.
The HSP20, ␣B-crystallin, and MKBP antibodies recognized
the corresponding purified or recombinant protein (Figure 1).
The anti-HSP20 antibodies recognized a major band at 20
kDa with a smaller band at a slightly lower relative mobility
in heart homogenate proteins (Figure 1, HSP20). This additional lower band may represent cross-reactivity with other
proteins or may represent HSP20 that has been posttranslationally modified (eg, phosphorylated and nonphosphorylated
HSP20). The anti–␣B-crystallin antibodies recognized a single band with a relative mobility of 20 kDa in heart
homogenates (Figure 1, Crystallin). The immunoreactive
band above 20 kDa in the lane containing purified ␣Bcrystallin may represent dimerized ␣B-crystallin. The anti-
4
Circulation
January 28, 2003
Downloaded from http://circ.ahajournals.org/ by guest on June 16, 2017
Figure 1. Specificity of the small heat shock protein antibodies.
Rat heart tissue homogenates (HH, 100 ␮g), bovine ␣B-crystallin
(crystalline, 1 ␮g), recombinant HSP25 (HSP25, 1 ␮g), and
recombinant HSP20 (HSP20, 1 ␮g) were separated by SDSPAGE and transferred to Immobilon as described in Methods.
The blots were then probed with anti-HSP20 antibodies (HSP20,
1:1000 dilution), anti–␣B-crystallin antibodies (crystalline, 1:1000
dilution), or anti-MKBP antibodies (MKBP, 1:1000 dilution). The
relative mobility of molecular weight standards is indicated on
the left of each panel. Arrows depict the relative immunoreactive
proteins. This blot is representative of 3 separate experiments.
MKBP antibodies recognized a band with a relative mobility
of 20 kDa and another band with a higher relative mobility
(⬇35 kDa) in heart homogenates (Figure 1, MKBP). The
additional band may represent cross-reactivity with other
proteins or dimerized MKBP. There were very low levels of
immunoreactive HSP25 detected in adult rat heart with an
affinity-purified mouse monoclonal antibody21 or commercially available antibodies (data not shown). It has been
previously reported that HSP25 is highly expressed in neonatal cardiac tissue but is markedly downregulated in adult
heart tissues.26 Only the associations between HSP20, ␣Bcrystallin, and MKBP were examined in subsequent studies.
Subcellular Localization and Macromolecular
Associations of the Small HSPs
To determine the intracellular distribution of the small heat
shock proteins in cardiac myocytes, subcellular fractionation
was performed as described in Methods. HSP20 and MKBP
were in the cytosolic fraction in rat heart homogenates
(Figure 2, HSP20 and MKBP). ␣B-crystallin was predominantly in the cytosolic fraction, but there was also a minor
component of immunoreactive ␣B-crystallin in the particulate fraction (Figure 2, crystallin).
The small heat shock proteins have been shown to be
associated with each other in macromolecular aggregates.12,13
Figure 2. Subcellular fractionation of rat heart tissues. Rat heart
tissues were homogenized as described in Methods. Proteins
from the 10 000g supernatant (S1), 10 000g pellet in (P1),
100 000g supernatant (S2), and 100 000g pellet (P2) were
probed with anti-HSP20 antibodies (HSP20, 1:1000 dilution),
anti–␣B-crystallin antibodies (crystallin, 1:1000 dilution), or antiMKBP antibodies (MKBP, 1:1000 dilution). The relative mobility
of molecular weight standards is indicated on the left of each
panel, and arrows depict the relative immunoreactive proteins.
These results are representative of 3 separate experiments.
Because the small HSPs were predominantly cytosolic proteins, gel filtration was performed on the cytosolic fractions
of heart muscle homogenates with superose 6 columns.13
Fractions from the column were dot-blotted with antibodies
against HSP20, ␣B-crystallin, and MKBP. HSP20 and ␣Bcrystallin were found in similar fractions that eluted from the
column after the catalase standard (MW 232, Figure 3A).
MKBP eluted from the column in the same fraction as the
BSA standard (MW 67 kDa, Figure 3A).
To confirm the associations of the small heat shock protein
in rat heart, we performed coimmunoprecipitations. Immunoprecipitation was performed with antibodies against HSP20,
the proteins separated and probed with antibodies against
HSP20, ␣B-crystallin, MKBP, and sarcomeric actin. ␣Bcrystallin coimmunoprecipitated with HSP20 (Figure 3B).
Neither MKBP nor sarcomeric actin coimmunoprecipitated
with HSP20 from rat heart homogenates (data not shown).
The secondary antibodies also recognized the light chains of
the rabbit polyclonal antibodies used to perform the immunoprecipitations (Figure 3B, 50-kDa band). HSP20, ␣Bcrystallin, and MKBP all have a relative mobility of 20 kDa;
thus, they could readily be detected at a relative mobility
lower on the gel than the rabbit polyclonal antibodies used in
the immunoprecipitations. The antibodies used to probe for
sarcomeric actin were mouse monoclonal. Thus, a signal
from the immunoprecipitating antibody would not interfere
with the actin probe. There was no nonspecific immunoreactive protein when heart homogenates were immunoprecipitated with preimmune rabbit serum (data not shown). The
Pipkin et al
Downloaded from http://circ.ahajournals.org/ by guest on June 16, 2017
Figure 3. A, Macromolecular associations of HSP20,
␣B-crystallin, and MKBP in rat heart. Cytosolic supernatants
were separated on a molecular sieving column and immunoreactive HSP20 (䢇), ␣B-crystallin (䡩) or MKBP (‘) was measured
in each of the column fractions (fraction number). The amount of
immunoreactive protein in each fraction was determined with
densitometry (densitometric units). The fraction of the catalase
standard (232 kDa) and the BSA standard (67 kDa) is indicated
on the top of the figure. These results are representative of 3
separate experiments. B, Coimmunoprecipitation of ␣B-crystallin
and HSP20 in adult rat heart. Proteins were immunoprecipitated
with anti-HSP20 antibodies as described in Methods. The blots
were probed with anti-HSP20 antibodies (HSP20, 1:1000 dilution) or anti–␣B-crystallin antibodies (CRY, 1:1000 dilution). The
relative mobility of molecular weight standards is indicated on
the left of each panel, and the relative immunoreactive proteins
are depicted with arrows. The upper bands represent crossreactivity with the antibody light chains. These blots are representative of 3 separate experiments.
antibodies used to detect ␣B-crystallin did not immunoprecipitate ␣B-crystallin; thus, experiments to immunoprecipitate ␣B-crystallin and detect immunoreactive HSP20 were
not performed.
HSP20 in Rat Heart
5
Figure 4. Immunofluorescence localization of HSP20,
␣B-crystallin, and MKBP in rat heart. Rat hearts were perfusion
fixed, and sections were stained with anti-sarcomeric actin antibodies (1:100 dilution, A), anti–␣B-crystallin antibodies (1:100
dilution, B), or anti-HSP20 antibodies (1:100 dilution, C; magnification ⫻40). Neonatal rat cardiac myocytes were stained with
anti-sarcomeric actin antibodies (1:100 dilution, D) or antiHSP20 antibodies (1:100 dilution, E; magnification, ⫻100). Cy3conjugated secondary antibodies were used, and the slides
were imaged with a fluorescence microscope. These images are
representative of staining 5 different rat hearts.
(Figure 4A). Immunoreactive ␣B-crystallin was also present
in transverse bands, but the staining was more punctate
(Figure 4B). Immunoreactive HSP20 was present in transverse bands, and there was also staining of the cell membranes (Figure 4C). MKBP was not included because of the
limited specificity of the MKBP antibodies. Neonatal cardiac
myocytes were also stained with anti-actin and anti-HSP20
antibodies, and similar staining patterns were observed (Figures 4D and 4E). There was no specific pattern of immunoreactivity using preimmune serum or no primary antibody
(data not shown).
Cellular Localization of the Small HSPs With
Immunofluorescence Microscopy
Activation of Cyclic Nucleotide Signaling Pathways
Leads to an Increase in the Phosphorylation of
HSP20 in Cardiac Myocytes
To additionally localize the small HSPs in rat heart, immunofluorescence microscopy was performed. Immunoreactive
sarcomeric actin was present in distinct transverse bands
To determine if activation of cyclic nucleotide signaling
pathways leads to increases in the phosphorylation of HSP20,
two-dimensional immunoblotting was performed. Increases
6
Circulation
January 28, 2003
Downloaded from http://circ.ahajournals.org/ by guest on June 16, 2017
Figure 5. HSP20 is phosphorylated by activation of cyclic nucleotide signaling pathways in neonatal ventricular myocytes.
Cardiac myocytes were treated with PBS (A) or sodium nitroprusside (10 ␮mol/L for 10 minutes) (B). The cells were then homogenized, solubilized, and separated by 2-dimensional electrophoresis. The proteins were then transferred to Immobilon and
probed with anti-HSP20 antibodies (1:1000 dilution). The relative
mobility of molecular weight standards is indicated to the left of
each panel, and the mobility of IEF standards is on the top of
panel B. The arrows represent immunoreactive HSP20. This blot
is representative of 3 separate experiments.
in the phosphorylation of HSP20 in vascular smooth muscle
leads to a shift of HSP20 from a basic to more acidic
isoforms.7 Rat cardiac myocytes were equilibrated in bicarbonate buffer for 1 hour. The myocytes were then treated with
buffer alone or with the NO donor sodium nitroprusside (10
␮mol/L, 10 minutes). The cells were homogenized and
separated by two-dimensional electrophoresis, transferred to
Immobilon, and probed with anti-HSP20 antibodies. Blots
from cells treated with buffer alone demonstrated a single
point of immunoreactive protein with a relative mobility of
20 kDa and an isoelectric focusing point of 6.5 (Figure 5A).
Blots from strips treated with sodium nitroprusside demonstrated a single point of immunoreactive protein with a
relative mobility of 20 kDa and an isoelectric focusing point
of 5.6 (Figure 5B). These isoelectric values are consistent
with nonphosphorylated and phosphorylated HSP20,
respectively.5,7
Phosphorylated Peptide Analogues of HSP20
Leads to an Increase in the Rate of Shortening in
Cardiac Myocytes
To determine if HSP20 has a direct role in myocyte function,
a model of transient permeabilization of cultured myocytes
was used.22 The rate of shortening and lengthening in
unloaded isolated cells was used as a measure of contraction.
Introduction of the phosphorylated peptide analogue of
HSP20 resulted in a significant increase in the rate of
shortening (37.6⫾2.4%, Figure 6A). Similar increases in the
rate of shortening occurred after treatment with sodium
nitroprusside (10 ␮mol/L, 33.5⫾5.3%). In experiments
where the myocytes were permeabilized in the absence of
peptide (sham, 1.1⫾0.5% increase, Figure 6A) or in the
presence of scrambled phosphorylated peptide (5.9⫾1.9%
Figure 6. The effect of phosphopeptide analogues of HSP20 on
myocyte function. The basal rate of shortening and lengthening
of unloaded isolated cardiac myocytes was determined by
computer-assisted digital microscopy imaging techniques. The
cells were then transiently permeabilized and incubated with no
peptide (sham, SM); N-PRKSphosLWALGRPLA (SC, a scrambled
phosphorylated peptide); N-WLRRAAAPLPGLK (NP, a nonphosphorylated peptide); or N-WLRRASphosAPLPGLK (PS, the phosphoHSP20 peptide analogue). After permeabilization, the cells
were placed back on the microscope stage and the rate of
shortening and lengthening was again determined. The data
represent the percent increase in rate of change over basal rate
(percent increase in rate, A; n⫽25 cells per group from 5 different experiments; *P⬍0.05). With the use of digital camera and
Video Savant software, images of individual cells were captured
at 30 frames per second before (PRE) and after (POST) permeabilization and introduction of the PS peptide analogue. The
time from contraction (maximal shortening) to relaxation (maximal lengthening) was measured (relaxation time in seconds; B,
n⫽16 cells from 5 different experiments; *P⬍0.05).
increase, Figure 6A) or nonphosphorylated peptide
(5.5⫾2.4%, Figure 6A), there was no significant change in
the shortening rate.
Because phosphorylated HSP20 is associated with relaxation of vascular smooth muscle, we hypothesized that
phosphopeptide analogues of HSP20 increased shortening
rate by increasing the rate of relaxation of the cardiac
myocytes. To determine the rate of relaxation of the cardiac
myocytes, contractile properties were analyzed with the use
of a digital camera and Video Savant software. The times for
contraction and relaxation were measured before and after the
transient permeabilization in the presence of peptide analogues. There was a significant decrease in the time of
relaxation in the cells exposed to the phosphopeptide analogue of HSP20 (Figure 6B). There were no significant
changes in the relaxation rate in myocytes permeabilized in
the absence of peptide (0.058⫾0.005 seconds) or incubated in
the presence of the scrambled phosphorylated peptide
(0.055⫾0.006 seconds).
Calcium Transients
Because myocyte contractility at the level of the sarcomere is
regulated by Ca2⫹ transients, we next examined Ca2⫹ fluxes in
the transiently permeabilized myocytes with the Ca2⫹ fluoroprobe, fura-2. For these experiments, cultured myocytes were
permeabilized before loading with fura-2. The results demonstrated a significant decrease in the time constant for
exponential (␶) decay of the Ca2⫹ transients in the group that
was permeabilized in the presence of the phosphopeptide
analogues of HSP20 (0.104⫾0.005 seconds, n⫽16; Figure
7B) compared with the group treated with the scrambled
phosphopeptide (0.236⫾0.011 seconds, n⫽18; Figure 7B) or
Pipkin et al
HSP20 in Rat Heart
7
transients. The diastolic Ca2⫹ level was not significantly
different in any of the 3 groups.
Discussion
Downloaded from http://circ.ahajournals.org/ by guest on June 16, 2017
Figure 7. The effect of phosphopeptide analogues of HSP20 on
Ca2⫹ transients. Myocytes cultured on glass coverslips were
permeabilized with HSP20 or scrambled phosphopeptides or
permeabilized in the absence of peptides just before loading
with fura-2 AM. Data were collected from individual rhythmically
beating myocytes (A; ratio of light emitted at 510 nm when
alternately excited at 340 and 380 nm). For each myocyte, data
were collected for 20 to 25 seconds and used only if the basal
level and the peak magnitudes of the Ca2⫹ transients remained
stable over this period. The phosphorylated HSP20 analogue
resulted in a significantly faster rate of decline of Ca2⫹ transient
when determined by fitting the declining phase of the transient
by a first-order exponential (B). For each data set, the time constants for the decay (␶) were determined for 5 consecutive Ca2⫹
transients and averaged. The data were collected from 16 to 23
myocytes per group in 3 separate experiments (see text;
*P⬍0.05).
the myocytes permeabilized in the absence of any phosphopeptide (0.242⫾0.021 seconds, n⫽23). There were no
differences in the peak systolic Ca2⫹ transients in the myocytes incubated with the HSP20 phosphopeptide analogue
compared with the scrambled phosphopeptide analogues
(P⬎0.05; Figure 7A). However, the permeabilization with
either of the phosphopeptides resulted in a decrease in the
peak magnitude of the Ca2⫹ transient that was evident when
comparing myocytes permeabilized with scrambled phosphopeptide (1.05⫾0.02 340/380 ratio units) or phosphopeptide HSP20 analogue (1.01⫾0.02) to permeabilized myocytes
in the absence of peptide (1.16⫾0.02; P⬍0.05). These data
indicated that permeabilization with phosphopeptides resulted in a nonspecific decrease in the magnitude of the
The small heat shock proteins share considerable homology
and are often found in association with other small heat shock
proteins. Despite the sequence homology, the antibodies used
in these studies were specific for the individual heat shock
protein (Figure 1). The small HSPs HSP20, ␣B-crystallin,
and MKBP were predominantly cytosolic proteins, although
some ␣B-crystallin was in a particulate fraction (Figure 2).
Molecular sieving columns suggested that ␣B-crystallin and
HSP20 coexist in a macromolecular aggregate of ⬇200 kDa
(Figure 3A). MKBP was in a much smaller aggregate (60 to
70 kDa). This association was confirmed in that ␣B-crystallin
coimmunoprecipitated with HSP20 (Figure 3B). Immunofluorescence microscopy demonstrated that ␣B-crystallin and
HSP20 had a staining pattern of distinct transverse bands
similar to the pattern observed after staining for sarcomeric
actin (Figure 4). Thus, both biochemical analyses and immunofluorescence microscopy suggest that HSP20 and ␣
B-crystallin are associated proteins in adult rat hearts. Other
studies have suggested that HSP20 is an actin-associated
protein in vascular smooth muscle.18,19 The localization of the
small heat shock proteins to specific cytoskeletal domains
suggests a role for these proteins in regulating cytoskeletal
dynamics.
HSP25, HSP20, and ␣B-crystallin can be posttranslationally modified by phosphorylation. HSP20 is phosphorylated
by cAMP-dependent protein kinase (PKA) and cGMPdependent protein kinase (PKG).7 Increases in the phosphorylation of HSP20 are associated with relaxation of vascular
and skeletal muscle.4,5,7 In the present investigation, we
demonstrate that treatment of neonatal ventricular tissue with
sodium nitroprusside leads to increases in the rate of shortening and increases in the phosphorylation of HSP20 (Figure
5). The addition of phosphopeptide analogues of HSP20 into
transiently permeabilized cardiac myocytes led to increases in
the rate of shortening of the myocytes (Figure 6). These
results are consistent with previously reported findings demonstrating that the NO-cGMP pathway increases beat rate by
stimulating the hyperpolarization-activated pacemaker current, If.27 The present results suggest that the mechanism of
NO stimulation of heart rate via If involves phosphorylation
of HSP20 and that phosphorylated HSP20 may have a direct
effect on If channels. The increased rate of shortening was also
related to increases in the rate of relaxation of the myocytes
(Figure 6). Although there was no change in the magnitude of
the Ca2⫹ transient after treatment with the phosphopeptide
analogues of HSP20, there was an increase in the rate of decline
of the Ca2⫹ transient (Figure 7). This suggests that the phosphopeptide analogue of HSP20 facilitates the increased rate by
stimulating a more rapid uptake of Ca2⫹ by the sarcoplasmic
reticulum. Taken together, these data suggest that HSP20 may
have a direct role in modulating the lusitropic actions of NO and
NO donors.
In summary, HSP20 is biochemically associated with
␣B-crystallin and localizes to distinct transverse bands in a
similar pattern as ␣B-crystallin and sarcomeric actin. The
8
Circulation
January 28, 2003
possible association of HSP20 and ␣B-crystallin with actin
suggests that these two small heat shock proteins may be
involved in modulating cytoskeletal or contractile dynamics
of cardiac myocytes. Specifically, our results show that the
addition of phosphorylated peptide analogues of HSP20 leads
to increases in the rate of shortening, which was associated
with a more rapid uptake of Ca2⫹ in cardiac myocytes. This
suggests that HSP20 may have a physiological role in cardiac
myocyte function.
Acknowledgments
This work was supported by a VA Merit Review Award and NIH
grant RO1 HL58027 to Dr Brophy and NIH grants RO1 HL 58861
and PO1 HL 36059 (Project 2) to Dr Creazzo. Dr Pipkin was the
recipient of the Society of University Surgeons-Ethicon Scholarship.
References
Downloaded from http://circ.ahajournals.org/ by guest on June 16, 2017
1. Muller-Strahl G, Kottenberg K, Zimmer HG, et al. Inhibition of nitric
oxide synthase augments the positive inotropic effect of nitric oxide
donors in the rat heart. J Physiol. 2000;522(pt 2):311–320.
2. Layland J, Li JM, Shah AM. Role of cyclic GMP-dependent protein
kinase in the contractile response to exogenous nitric oxide in rat cardiac
myocytes. J Physiol. 2002;540(pt 2):457– 467.
3. Beall A, Bagwell D, Woodrum DA, et al. The small heat shock-related
protein, HSP20, is phosphorylated on serine 16 during cyclic nucleotidedependent relaxation. J Biol Chem. 1999;274:11344 –11351.
4. Rembold CM, Foster DB, Strauss JD, et al. cGMP-mediated phosphorylation of heat shock protein 20 may cause smooth muscle relaxation
without myosin light chain dephosphorylation in swine carotid artery.
J Physiol. 2000;524(pt 3):865– 878.
5. Wang Y, Xu A, Cooper GJS. Phosphorylation of P20 is associated with
the actions of insulin in rat skeletal and smooth muscle. Biochem J.
1999;344:971–976.
6. Kato K, Goto G, Inaguma Y, et al. Purification and characterization of a
20-kDa protein that is highly homologous to ␣B-crystallin. J Biol Chem.
1994;269:15302–15309.
7. Beall AC, Kato K, Goldenring JR, et al. Cyclic nucleotide-dependent
vasorelaxation is associated with the phosphorylation of a small heat
shock-related protein. J Biol Chem. 1997;272:11283–11287.
8. Jerius H, Karolyi DR, Mondy JS, et al. Endothelial-dependent vasodilation is associated with increases in the phosphorylation of a small heat
shock protein (HSP20). J Vasc Surg. 1999;29:678 – 684.
9. Brophy CM, Beall A, Lamb S, et al. Small heat shock proteins and
vasospasm in human umbilical artery smooth muscle. Biol Reprod. 1997;
57:1354 –1359.
10. Woodrum D, Brophy CM, Wingard CJ, et al. The phosphorylation events
associated with cyclic nucleotide-dependent inhibition of smooth muscle
contraction. Am J Physiol. 1999;277:H931–H939.
11. Suzuki A, Sugiyama Y, Hayashi Y, et al. MKBP, a novel member of the
small heat shock protein family, binds and activates the myotonic dystrophy protein kinase. J Cell Biol. 1998;140:1113–1124.
12. Kato K, Hasegawa K, Goto S, et al. Dissociation as a result of phosphorylation of an aggregated form of the small stress protein, hsp27. J Biol
Chem. 1994;269:11274 –11278.
13. Brophy CM, Dickinson M, Woodrum D. Phosphorylation of the small
heat shock-related protein, HSP20, in vascular smooth muscles is associated with changes in the macromolecular associations of HSP20. J Biol
Chem. 1999;274:6324 – 6329.
14. Shah V, Wiest R, Garcia-Cardena G, et al. Hsp90 regulation of endothelial nitric oxide synthase contributes to vascular control in hypertension.
Am J Physiol. 1999;277:G463–G468.
15. Miron T, Vancompernoll K, Vanderkerckhove J, et al. A 25-kD inhibitor
of actin polymerization is a low molecular mass heat shock protein. J Cell
Biol. 1991;114:255–261.
16. Lavoie JN, Lambert H, Hickey E, et al. Modulation of cellular thermoresistance and actin filament stability accompanies phosphorylationinduced changes in the oligomeric structure of heat shock protein 27. Mol
Cell Biol. 1995;15:505–516.
17. Guay J, Lambert H, Gingras-Breton G, et al. Regulation of actin filament
dynamics by p38 map kinase-mediated phosphorylation of heat shock
protein 27. J Cell Sci. 1997;110(pt 3):357–368.
18. Brophy CM, Lamb S, Graham A. The small heat shock-related protein-20
is an actin-associated protein. J Vasc Surg. 1999;29:326 –333.
19. Rembold CM, Zhang E. Localization of heat shock protein 20 in swine
carotid artery. BMC Physiol. 2001;1:10.
20. Perng MD, Cairns L, van den IJssel PR, et al. Intermediate filament
interactions can be altered by HSP27 and ␣B-crystallin. J Cell Sci.,
1999;112:2099 –2112.
21. Welsh MJ, Wu W, Parvinen M, et al. Variation in expression of hsp27
messenger ribonucleic acid during the cycle of the seminiferous epithelium and co-localization of hsp27 and microfilaments in Sertoli cells
of the rat. Biol Reprod. 1996;55:141–151.
22. Johnson JA, Gray MO, Karliner JS, et al. An improved permeabilization
protocol for the introduction of peptides into cardiac myocytes: application to protein kinase C research. Circ Res. 1996;79:1086 –1099.
23. Johnson JA, Mochly-Rosen D. Inhibition of the spontaneous rate of
contraction of neonatal cardiac myocytes by protein kinase C isozymes:
a putative role for the epsilon isozyme. Circ Res. 1995;76:654 – 663.
24. Brotto MA, Creazzo TL. Ca2⫹ transients in embryonic chick heart: contributions from Ca2⫹ channels and the sarcoplasmic reticulum. Am J
Physiol. 1996;270(2 pt 2):H518 –H25.
25. Welsh MJ, Gaestel M. Small heat-shock protein family: function in health
and disease. Ann N Y Acad Sci. 1998;851:28 –35.
26. Lutsch G, Vetter R, Offhauss U, et al. Abundance and location of the
small heat shock proteins HSP25 and ␣B-crystallin in rat and human
heart. Circulation. 1997;96:3466 –3476.
27. Musialek P, Lei M, Brown HF, et al. Nitric oxide can increase heart rate
by stimulating the hyperpolarization-activated inward current, If. Circ
Res. 1997;81:60 – 68.
Localization, Macromolecular Associations, and Function of the Small Heat Shock-Related
Protein HSP20 in Rat Heart
Walter Pipkin, John A. Johnson, Tony L. Creazzo, Jarrett Burch, Padmini Komalavilas and
Colleen Brophy
Downloaded from http://circ.ahajournals.org/ by guest on June 16, 2017
Circulation. published online December 16, 2002;
Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2002 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7322. Online ISSN: 1524-4539
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://circ.ahajournals.org/content/early/2002/12/16/01.CIR.0000044386.27444.5A.citation
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published
in Circulation can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial
Office. Once the online version of the published article for which permission is being requested is located,
click Request Permissions in the middle column of the Web page under Services. Further information about
this process is available in the Permissions and Rights Question and Answer document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Circulation is online at:
http://circ.ahajournals.org//subscriptions/