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Cardiovascular Researchdoi:10.1093/cvr/cvn148

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Identification and physiological activity of survival
factor released from cardiomyocytes during ischaemia
and reperfusion

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Yoichi Mizukami1*, Kyoichi Ono2, Cheng-Kun Du3, Toshihiko Aki1, Naoya Hatano4, Yosuke Okamoto2,
Yasuhiro Ikeda5, Hiroshi Ito6, Kimikazu Hamano6, and Sachio Morimoto3
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Center for Gene Research, Yamaguchi University, Yamaguchi 755-8505, Japan; 2Department of Physiology, Akita University
School of Medicine, Akita 010-8543, Japan; 3Department of Clinical Pharmacology, Kyushu University Graduate School of
Medicine, Fukuoka 812-8582, Japan; 4Department of Cell Physiology, Faculty of Medicine, Kagawa University, Kagawa
761-0793, Japan; 5Department of Molecular Cardiovascular Biology, Yamaguchi University School of Medicine, Yamaguchi
755-8505, Japan; and 6Department of Surgery and Clinical Science, Yamaguchi University School of Medicine, Yamaguchi
755-8505, Japan

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Received 20 August 2007; revised 30 May 2008; accepted 2 June 200885
Time for primary review: 25 days
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KEYWORDS30

M-LDH;
Survival factor;
Cardiomyocyte;
Secretion;
Ischaemia

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Aims We carried out a screening of survival factors released from cells exposed to simulated ischaemia
and reperfusion (sI/R) using the embryonic rat heart-derived cell line, H9c2 cells, and examined thephysiological role of the identified factor.
Method and results The culture medium supernatant of H9c2 cells exposed to sI/R was separated bycolumn chromatography and the fractions examined for survival activity. The protein with survival
activity was identified by mass spectrometry, and its physiological role was examined in the models
of ischaemia. Cell survival activity was detected in at least three fractions of the cell supernatant collected during sI/R and subjected to a series of column chromatographic steps. Among the proteinsmeasured by mass spectrometry and western blotting, a p36 protein identified as a glycolytic
enzyme, lactate dehydrogenase muscle subunit (M-LDH), showed strong survival activity. H2O2induced intracellular calcium overload in H9c2 cells and irregular Ca2þ transients in adult rat cardiomyocytes were both found to be inhibited by pretreatment with M-LDH. M-LDH also lowered the frequency
and amplitude of early afterdepolarizations induced by H2O2 in adult rat cardiomyocytes and suppressed
the ischaemia–reperfusion-induced reduction of cardiac output from mouse working heart preparations.
M-LDH was found to increase the phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2),
which plays a role in H9c2 cell survival.
Conclusion M-LDH released from cardiomyocytes after hypoxia and reoxygenation has a role in protecting the heart from oxidative stress-induced injury through an intracellular signal transduction pathway
involving ERK1/2.

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1. Introduction
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Ischaemia–reperfusion of the heart is a major disease statusarising from acute myocardial infarction, cardiopulmonary
bypass surgery, and heart transplantation. After the recanalization of an infarct-damaged coronary artery, an expansion of
the ischaemic region is observed as acute dilation and thinning of the infarcted area, hypertrophy of the remaining myocardium, induction of apoptotic cell death in the surrounding* Corresponding author. The Center for Gene Research, Yamaguchi
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University School of Medicine, 1-1-1 Minami-Kogushi, Ube, Yamaguchi
755-8505, Japan. Tel: þ81 836 22 2183; fax: þ81 836 22 2185.
E-mail address: mizukami@yamaguchi-u.ac.jp

region of ischaemia, and an increase in cardiac fibrosis in theinfarcted region.1 These processes are referred to as remodelling and have been observed not only in cardiomyocytes
directly exposed to ischaemia–reperfusion, but also incells surrounding the ischaemic region. This suggests thatcardiomyocytes exposed to ischaemia–reperfusion release avariety of factors that transmit some signal(s) to thesurrounding cells.
It is well known that the heart functions as an endocrine
tissue by secreting hormonal factors in addition to its role
as a pump to propel blood to other tissues of the body.2,3
Molecules released from heart, such as nitric oxide, prostaglandins, adenosine, and endothelin, are involved in the

Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2008.
For permissions please email: journals.permissions@oxfordjournals.org.

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regulation of the cardiovascular system after myocardialischaemia.4–8 In addition to these small molecules, inflammatory cytokines are also released from cardiomyocytes
after ischaemia and reperfusion.9 For example, tumour
necrosis factor-a is produced in the ischaemic heart andinvolved in cardiac contractile depression through the inhibition of the sarcoplasmic ryanodine receptor.10 The inflammatory cytokines interleukin (IL)-1b and IL-6 are also
released from the myocardium after ischaemia and reperfusion and are secreted to the blood. These factors generated
in the ischaemic heart not only aggravate cardiac contractility but also lead to dysfunction in intact cardiomyocytes.11–
13
Growth factors are reported to act as the survival factors
released during myocardial ischaemia.14–18 Most growth
factors are involved in angiogenesis to the infarcted area
as well as in inhibiting apoptotic cell death, thus leading
to the protection of the myocardium from ischaemic injury
through the activation of signal transduction molecules.19
However, these factors have the ability to stimulate the proliferation of various cell types, including fibroblasts, hepatocytes, and lymphocytes, which may be a risk in heartdisease.20 During oxidative stress caused by ischaemia andreperfusion, we have demonstrated that the activation of
extracellular signal-regulated kinase1/2 (ERK1/2) maintains
contractility and survival in rat cardiomyocytes through the
up-regulation of glycolytic a-enolase,21–23 strongly
suggesting that the activation of ERK1/2 might be required
to restore heart function following ischaemic injury.
Although Urocortin has been demonstrated as a substancethat induces ERK1/2 activation and improves myocardial
dysfunction during oxidative stress, a novel cardioprotective
factor would be beneficial for patients with ischaemic heartdisease.24
In the present studies, we identified a novel cardioprotective factor released from a rat heart-derived cell line, H9c2
cells, during simulated ischaemia (oxygen and glucose deprivation) and simulated reperfusion (oxygen and glucose
addition) (sI/R). This factor was purified from polypeptides
released from cells as a p36 protein having survival activity
and identified as the lactate dehydrogenase muscle subunit(M-LDH). We found that the application of M-LDH to cardiomyocytes reduces the H2O2-induced early afterdepolarizations (EADs) closely related to arrhythmia by inhibiting
intracellular Ca2þ ([Ca2þ]i) overload. M-LDH was found toactivate ERK1/2 and suppress the reduction of cardiac
output by ischaemia–reperfusion in the isolated mouseheart. These findings suggest that M-LDH is released fromcardiomyocytes during ischaemia–reperfusion, and that it
plays a cardioprotective role against myocardial injury
arising from oxidative stress in the ischaemic heart.

Y. Mizukami et al.
concentrations of H2O2 were decided by measuring the activity
after the addition of H2O2 in control cells. All other chemicals
were commercially available.

2.2 Cell culture and ischaemic cell model(simulated ischaemia and reperfusion)

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Cell culture and simulated ischaemia were achieved as described
previously.22,23,25,26 Briefly, an embryonic rat heart-derived cellline, H9c2 cells, was plated in dishes. After an incubation in Dulbecco’s modified Eagle’s medium (DMEM) with 10% foetal bovine serum(FBS), the cells were cultured in serum-free DMEM and incubated in 200
slightly hypotonic Hanks’ balanced saline solution (1.3 mM CaCl2,
5 mM KCl, 0.3 mM KH2PO4, 0.5 mM MgCl2, 0.4 mM MgSO4, 69 mM
NaCl, 4 mM NaHCO3, and 0.3 mM NaH2PO4) without glucose andserum for 2 h at 378C. Hypoxia was achieved using an air-tight incu- Q1bator from which the oxygen was reduced to 1% by replacementwith nitrogen. After incubation under conditions of simulated 205
ischaemia, the cells were incubated in DMEM without FBS under normoxic conditions at 378C.

2.3 Measurement of survival activity
H9c2 cells were grown in DMEM containing 10% FBS, and the medium
was changed to DMEM without 10% FBS, and each of the eluate fractions was added to a well. The plates were then incubated for 24 h
in a CO2 incubator. Cell number was determined by a modified MTT
assay as described previously (Dojindo Chemicals, Kumamoto,Japan).27

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2.4 Purification of survival factors
Twenty litres of the supernatant of H9c2 cells (2  1010 cells)
exposed to simulated ischaemia for 2 h and simulated reperfusion
for 24 h were used for the purification of survival factors.
Ammonium sulfate was added to the supernatant to give a final concentration of 80% and the mixture was stirred for 24 h at 48C. The
sample was centrifuged for 30 min at 48C (18 000 g), the supernatant was removed, and the pellet was stored at –808C for the
purification of survival factors. The pellet was dissolved and dialysed against 10 mM Tris–HCl (pH 7.0) for 24 h at 48C. The sample
was loaded onto a column of HiTrap heparin Sepharose (5 mL) (GEHealthcare UK Ltd, Buckinghamshire, UK) equilibrated with thesame buffer. Bound proteins were eluted with a linear NaCl gradientfrom 0 to 1000 mM for 30 min at 1 mL/min. A part of the pooled
fractions was applied to gel filtration on Micro Bio-Spin 6 (Bio-Rad,
Richmond, CA, USA) to determine the survival activity and to
detect the proteins by SDS–PAGE and silver staining. After theactive fractions were pooled and the salt was removed by gel filtration, the sample was applied to a Resourse Q Column (GE Healthcare, Milwaukee, WI, USA) equilibrated with 10 mM Tris–HCl, pH
8.0. The column was eluted with a stepwise NaCl gradient asshown in Figure 1B over 30 min at 1 mL/min, and 1 mL fractions
were collected. The active fractions were desalted and concentrated to 0.05 mL by passing through a centrifugal filter device(Millipore, Bedford, MA, USA).

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2.1 Materials

2.5 Identification of survival factors bymatrix-assisted laser desorption
ionization-time-of-flight mass spectrometry

Anti-M-LDH,
anti-glyceraldehyde-3-phosphate
dehydrogenase(G3PDH), anti-phosphofructokinase (PFK), anti-phospho-tyrosine
antibodies, M-LDH protein, taurin, creatine, 2,3-butanedione monoxime, insulin, and transferrin were purchased from Sigma (St Louis,
MO, USA). Anti-PKCd isoform antibody was purchased from Cell Signalling Technology Inc. (Beverly, MA, USA). DAPI nucleic acid stain(40 ,6-diamino-2-phenylindol) was purchased from Molecular Probes
Inc. (Eugene, OR, USA). Collagenases B and D and laminin A werepurchased from Roche Diagnostics (Mannheim, Germany). The

The sample was subjected to SDS–PAGE and stained with a mass
spectrometry-compatible silver stain according to the instruction
manual (Invitrogen, Carlsbad, CA, USA). In-gel digestion withtrypsin was performed as described previously.28,29 The tryptic peptides from the proteins of interest were dissolved in 0.1% trifluoroacetate and desalted using a Zip Tip C18 (Millipore, Bedford, MA,
USA) according to the manufacturer’s protocol. Peptides were
eluted with matrix (a-cyano-4-hydroxycinnamic acid) prepared in
50% acetonitrile, 0.1% trifluoroacetate, and spotted onto a stainless

2. Methods
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Page 3 of 11Mode, none; Min. number of peptides required to match, 4; Mass
Tolerance, 25 ppm.

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2.6 Electrophoresis and immunoblotting
Electrophoresis and immunoblotting were carried out as described
previously.21,30,31

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2.7 Immunofluorescent staining
Immunofluorescentpreviously.22,23

staining

wascarriedout

asdescribed

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2.8 Preparation of adult cardiomyocytes

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Figure 1 Identification of polypeptides released from H9c2 cells exposed tosimulated ischaemia and reperfusion A–D, H9c2 cell supernatant (20 L) wasconcentrated by ammonium sulfate precipitation, and separated by heparin(A) and anion exchange column chromatographies (B). The survival activity
and absorbance at 280 nm of each fraction were measured (A and B). The proteins in peak A1 of panel B were subjected to SDS–PAGE and silver staining(C ). Purified p36 was digested with trypsin after the protocol described in‘Methods’. A portion of the resulting digest supernatant was used for matrixassisted laser desorption ionization-time of flight mass spectrometry analysis.
The peptides were eluted from the C18 material (Zip Tips) with matrix(a-cyano 4-hydro cinnamic acid) prepared in 50% acetonitrile and 0.1% trifluoroacetate. The observed m/z values were submitted to ProFound (proteomics software package) for peptide mass fingerprint search (D). (E) H9c2 cells
were grown for 2–3 days in 96-well plates in Dulbecco’s modified Eagle’s
medium containing 10% foetal bovine serum, and then were treated
without or with lactate dehydrogenase muscle subunit (M-LDH) at the indicated concentrations for 12 h. The cells were incubated for an additional
24 h in the presence of 200 mM H2O2. Cell number was determined by a modified MTT assay. Data represent the means + SE of eight independent experiments. Statistical significance was determined by ANOVA followed by post
hoc Dunnett’s test. #P , 0.05 vs. 0 mM LDH. F–H, cell supernatants of H9c2
cells exposed to 2 h simulated ischaemia followed by 24–48 h simulated
reperfusion (sI/R) or just exposed to 0–48 h serum starvation under normoxic
conditions (serum (2)), broken cells were removed by centrifugation, andthe supernatant was concentrated by ammonium sulfate precipitation. The
samples were subjected to immunoblotting with anti-M-LDH antibody (F),anti-glyceraldehyde-3-phosphate dehydrogenase antibody (G), and antiphosphofructokinase antibody (H ). The blots show representative dataobtained from three independent experiments. The protein levels were
determined from the immunoblots by densitometric analysis. Data represent
the means + SE. Statistical significance was determined by ANOVA followedby post hoc Tukey’s test. *P , 0.05 vs. 0 h serum (2) (control), #P , 0.05
vs. 24 h serum (2), þP , 0.05 vs. 48 h serum (2).

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steel target plate. The protein digests were analysed by matrixassisted laser desorption ionization-time of flight mass spectrometry(MALDI-TOF-MS) using a Voyager DE-STR instrument (Applied Biosystems, Foster City, CA, USA). Calibration was performed using the
autodigestion peaks of trypsin (1045.5642 and 2211.1046). The
observed m/z values were submitted to MS-Fit (available at
http://prospector.ucsf.edu/ucsfhtml4.0/msfit.htm) for mass fingerprint search. Search parameters were set as follows: Database,
NCBInr; Digest, Trypsin; maximum number of missed cleavages, 1;
Cys modified by, carbamidomethylation; Possible Modifications

Ventricular cardiomyocytes were isolated from adult rat hearts as
described previously.32 In brief, hearts were perfused with perfusion
buffer [MEM, pH 7.3 containing taurin (5 mM), 2,3-butanedione monoxime (10 mM), creatine (50 mg/100 mL), and insulin (2 U/100 mL)].
Enzymatic digestion was initiated by the addition of collagenase
type B (0.5 mg/mL), collagenase type D (0.5 mg/mL), and proteasetype XIV (0.02 mg/mL) to the perfusion buffer. After 3 min of digestion, 50 mM CaCl2 was added and the solution was incubated for7 min. The ventricle was removed, cut into several pieces, anddigested in a shaker (60–70 rpm) for 10 min at 378C. The supernatantcontaining the cardiomyocytes was placed in a sterilized tube andgently centrifuged at 500 rpm for 1 min. The pellet was re-suspended
in the perfusion buffer at a volume equal to that of the incubationbuffer [MEM containing bovine serum albumin (500 mg/100 mL) and
CaCl2 (150 mM)] to stop the enzymatic reaction.

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2.9 Digital imaging of [Ca21]i345[Ca2þ]i transient in adult cardiomyocytes was measured by fura-2 fluorescence ratio digital imaging. Cardiomyocytes on glass-based
dishes precoated with lamin B were loaded with 0.1 mM fura-2 AM(Dojin Chemical Co.) for 30 min at 378C, placed on the stage of an
inverted microscope (Nikon, Eclipse TS2000, Tokyo, Japan), andstimulated with 25 mA at a frequency of 0.5 Hz using a pair of platinum wires connected to a stimulator (IonOptix, Milton, MA, USA) inthe presence of 40 mM H2O2. Digital imaging of the fura-2 fluorescence emitted at 510 nm was carried out during alternative excitation at 340 and 380 nm with a digital image system (Aquacosmos,
Hamamatsu Photonics, Co., Hamamatsu, Japan). A pair of digital
images was successively obtained at specific time intervals (21.0 ms
for cardiomyocytes) and stored. The ratio of each image pair was
calculated offline with background correction. H9c2 cells were alsomeasured similarly without stimulation.

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2.10 Measurement of action potentials in adultcardiomyocytes
Action potentials were recorded in isolated ventricular cells from
Wistar rats (body weight, 200–300 g) by the Perforated Patch-Clamp
Technique.33 Pipettes had resistances in the range 1.5–3 MV when
filled with the following solution (in mM) K-aspartate 110, KCl 30,
NaCl 10, CaCl2 1.0 and HEPES 5.0 (pH 7.2 with KOH). Amphotericin
B (Sigma), dissolved in dimethylsulfoxide as a stock solution(60 mg/mL), was added to the pipette solution to give a final concentration of 0.2 mg/mL. The bath solution was the control Tyrode solution containing (in mM) NaCl 136.9, KCl 5.4, MgCl2 0.53, CaCl2 1.8,
NaH2PO4 0.33, HEPES 5.0, and glucose 5.5 (pH 7.4). The bath temperature was maintained at 36 + 0.58C. Action potentials were elicited by the brief (1–3 ms) current injections applied at 0.2 Hz.
After stabilization of the resting potential and action potentials,
selected concentrations of H2O2 were added to the cells. Voltage
signals were stored in a microcomputer using PCLAMP software(Molecular Devices, Sunnyvale, CA, USA).

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Figure 1 (Continued).

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2.11 Working heart analysis
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Hearts were excised from female ICR mice (8.5–12 weeks of age;weight 30 g) after heparinization (15 U i.v.) under anaesthesia
with 2,2,2-tribromoethanol (5 mg i.p.). After all pulmonary veinsin a heart with lung preparation were tied with silk threads, thelung was trimmed away, and 18-gauge cannulae were tied onto
the aortic stump and left auricle. The heart was mounted in an
isolated heart perfusion apparatus (Model IPH-W2; Primetech,Japan). After several minutes of perfusion in Langendorff modewith a Krebs–Henseleit solution (118 mM NaCl, 4.7 mM KCl,
1.2 mM MgSO4, 1.2 mM KH2PO4, 25 mM NaHCO3, 0.5 mM EDTA-Na2,
10 mM HEPES, and 11 mM D-glucose) oxygenated with 95% O2 and
5% CO2 at 378C, an anterograde perfusion was achieved by applying
a preload of 22 cm H2O on the left atrium. Preload was then changed
from 26 to 6 cm H2O at a rate of 2 cm H2O per 2 min at a constantafterload of 70 mmHg. The cardiac output was determined as thesum of the aortic and coronary flow during 1 min of perfusion justbefore changing the preload. The left ventricular (LV) pressure
was simultaneously monitored with a blood pressure transducer
DTXPlus (Nihon Koden, Japan) using a 26-gauge catheter inserted
into the LV chamber from the apex.34

2.12 Measurement of the activation of signaltransduction molecules by flow cytometry
The activation of signal transduction molecules was measured bycytometric bead array (CBA) using flow cytometry. Briefly, H9c2
cells were incubated in DMEM without FBS for 24 h and were stimulated with M-LDH for 15 min. After the stimulation, a denaturing
buffer was added to stop the reaction, and the phosphorylationsof ERK1/2 (T202/Y2004), c-Jnu N-terminal kinase (JNK1/2)(T183/Y185), p38 mitogen-activated protein kinase (MAPK) (T180/
Y182), and phospholipase Cg (Y783) were measured according
to the instruction manual (BD Bioscience, San Diego, CA, USA).
Standard curves based on the recombinant proteins were prepared
before the measurement.

2.13 Determination of isozymes of lactate
dehydrogenase activity in subjects
LDH isozyme assay was determined using LDH isozymes Test Wako
(Wako Pure Chemical, Tokyo, Japan) according to the instruction
manual.

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2.14 Patients, anaesthesia, cardiopulmonary
bypass, and myocardial protection
A total of five patients ranging from 54 to 75 years who were scheduled to undergo open heart surgery (coronary artery bypass grafting,two; aortic valve replacement, one; mitral valve replacement, one;double valve replacement, one) were included in this study with theapproval by the local Ethics Committee. During surgery, anaesthetic
induction and maintenance were achieved with a high dose of fentanyl. The extracorporeal circuit consisted of biopump (Bio-Console,
Bio-Medics, Minneapolis, MI, USA), a cardiotomy reservoir, and amembrane oxgenator (Capiox-E, Terumo, Tokyo, Japan). The circuit
was primed with 150–200 mL of Ringer’s lactate. Heparin, 400 IU/
kg body weight, was infused to maintain an activated clotting timeof more than 400 s during bypass, and beta-methasone (4 mg/kg)
was added to the priming solution. A flow rate of 2.4 L/min/m2 wasused to maintain a systemic perfusion pressure of 60–80 mmHg.
After aortic cross-clamping, rapid blood cardioplegic solution(10 mL/kg body weight) was administered antegradely every 20 min
at a pressure of 60–80 mmHg for myocardial protection. The cardioplegic solution consisted of the following: Na 85.3 mmol/L, K
25.0 mmol/L, Cl 85.5 mmol/L, Mg 10.0 mmol/L, and glucose
25 g/L; pH 7.38 (378C), and osmolarity was 360 mOsm/L. One
patient with OPCAB was not used extracorporeal circuit and aortic
cross-clamping.

3. Results
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3.1 Purification of survival factors released during
simulated ischaemia and reperfusion
To purify the survival factors released from cardiomyocytes,
the supernatant of cultured cells exposed to sI/R was loaded
onto a heparin Sepharose column. The proteins bound to theaffinity column were eluted with a linear salt gradient, withthe survival activity of the eluate being monitored by cellnumber after serum removal from culture medium(Figure 1A). Strong survival activities to inhibit cell deathcaused by the removal of serum were detected in two
peaks (A and B), and the fractions showing stronger survival
activity (peak A) were subjected to further purification byanion exchange column chromatography (Figure 1B). The
bound proteins were eluted with a step-wise salt gradient,and strong survival activities were detected in two peaks(A1 and A2). The survival activity profile of peak A1 wasalmost the same as the absorbance profile at 280 nm(Figure 1B), and active fractions of peak A1 (fraction
numbers 14 and 15) contained a single protein with a molecular mass of 36 kDa (p36) on SDS–PAGE (Figure 1C).
Peak A2 is currently under investigation.

3.2 Survival factor released during simulated
ischaemia and reperfusion is lactate
dehydrogenase muscle subunit
The p36 protein was treated with trypsin under the conditions described in ‘Methods’, and subjected to peptide
mass fingerprinting by MALDI-TOF-MS. The molecularweights of the peptide fragments derived from the p36
protein were MHþ: 912.579, 942.574, 1117.556, 1143.574,
1180.681, 1245.653, 1249.646, 1674.791, 1829.860, and1979.058, which are almost identical to the theoretical molecular weights of the peptide fragments of mouse M-LDH(also designated as LDH-A) digested with trypsin(Figure 1D). The calculated molecular mass of M-LDH fromits amino acid sequence is 36 367, which is in approximate

Page 5 of 11agreement with the apparent molecular mass of p36 estimated by SDS–PAGE. Furthermore, an anti-M-LDH antibody
recognized p36 (data not shown). Purified M-LDH inhibited
cell death induced by the addition of 200 mM H2O2 in a dosedependent manner (0.025–0.2 mM) when added to cell line
H9c2 derived from the embryonic heart (Figure 1E). Taken
together, we conclude that the p36 protein is M-LDH.

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3.3 Lactate dehydrogenase muscle subunit
is released during simulated ischaemia
and reperfusion
To confirm the release of M-LDH from cultured cells duringsI/R, we carried out immunoblotting with an anti-M-LDH
antibody using the supernatant of cells exposed to sI/R.
The amount of M-LDH protein in the supernatant increased
after 24 h of simulated reperfusion following simulated
ischaemia for 2 h (Figure 1F); exposure to medium without
serum for a long-time damages the cells so that no significant difference could not be detected between 48 h serum(2) and sI/R. In contrast, the releases of G3PDH and PFK,
other glycolytic enzymes in the cytosol, during sI/R, werereduced compared with the control cells, indicating that
M-LDH is released relatively specifically during sI/R
(Figure 1G and H ).

3.4 Lactate dehydrogenase muscle subunit
has survival activity by protecting cardiomyocytes
from oxidative stress-induced damage
Since oxidative stress induced by ischaemia and reperfusion
plays an important role in heart injury, we examined theeffects of H2O2 on adult rat cardiomyocytes in the presenceof M-LDH. After 20 min of stimulation with 40 mM H2O2 in theabsence of M-LDH, the cardiomyocytes changed to a roundshape and lost the ability to contract in response to electrical stimulation (Figure 2A, B, and D). The rounded cells
bound to an initial apoptosis marker annexin V-enhanced
green fluorescence protein after 60 min of stimulation with
H2O2 (data not shown). Pretreatment with M-LDH significantly reduced the morphological change of the cardiomyocytes to a round shape (Figure 2A, C, and D) and preserved
cardiomyocyte contractility in response to electric stimulation (data not shown), indicating that M-LDH protects
cardiomyocytes from oxidative stress-induced damage.

3.5 Lactate dehydrogenase muscle subunit
inhibits [Ca21]i overload induced byan oxidative stress
To explore the mechanism involved in the survival activity of
M-LDH, we examined the effects of M-LDH and H2O2 on[Ca2þ]i using H9c2 cells loaded with fura-2. Because theexcess contraction of cardiomyocytes by H2O2 was inhibitedby M-LDH treatment, Ca2þ influx might be involved in theexcess contraction. Acute exposure to H2O2 caused a rapid
increase in [Ca2þ]i within 4 min followed by a gradualincrease in [Ca2þ]i up to 40 min after stimulation (see Supplementary material online, Figure S1). Pretreatment with
M-LDH significantly delayed the initial rise in [Ca2þ]i of
H9c2 cells exposed to H2O2 up to 20 min after stimulation
and reduced the final level of the [Ca2þ]i increase (see Supplementary material online, Figure S1). We also examinedthe effects of M-LDH and H2O2 on [Ca2þ]i transients evoked

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EADs increased up to 10 and 8 min after treatment, respectively, and then decreased with the elapse of time (Figure 4B
and C). M-LDH inhibited the frequency and amplitude of
EADs induced by H2O2 significantly (Figure 4F–H ). H2O2
also prolonged the action potential duration; however,
M-LDH had no effect on the prolongation of action potential
duration by H2O2 (Figure 4D and I ).

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3.7 Lactate dehydrogenase muscle subunit
suppresses oxidative stress- or
ischaemia–reperfusion-induced decrease incontractility of isolated working heart

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Figure 2 Effect of lactate dehydrogenase muscle subunit (M-LDH) on morphological changes that take place in adult cardiomyocytes in response to
H2O2. Adult cardiomyocytes were prepared from rat heart, and the cells
were treated without (A and B) or with (C) M-LDH (final 0.5 mM) for 12 h. Morphological changes to the cells were observed under bright field microscopy
at 0 min (A) and 40 min (B and C ) after the addition of H2O2 (final 40 mM),
and the length and breadth of the cells were analysed by Metamorph
Imaging Software (D). Data represent the means + SE determined for cells
prepared from the six independent experiments (control, n ¼ 42 cells;
H2O2, n ¼ 152 cells; H2O2 þM-LDH, n ¼ 173 cells). Statistical significancewas determined by ANOVA followed by post hoc Tukey’s test. *P , 0.05 vs.
control. Scale bar ¼ 100 mm.

The effects of M-LDH on the intact heart were examinedusing isolated working heart preparations (Figure 5). H2O2
reduced the cardiac output from the working heart preparation significantly and M-LDH suppressed the reductionof cardiac output caused by H2O2; there were no significant
differences in the cardiac output between hearts treatedwith H2O2 in the presence of M-LDH and controls(Figure 5A). A brief ischaemia–reperfusion reduced the
cardiac output and systolic function (dP/dtmax) significantly
and M-LDH suppressed the reduction of cardiac output andsystolic function caused by this brief ischaemia–reperfusion;
there were no significant differences in the cardiac output
and systolic function between hearts exposed to ischaemia–reperfusion with M-LDH and controls (Figure 5B and
C). M-LDH had no effects on the function of the heart
exposed to no ischaemia (data not shown). The release of
M-LDH was increased in the perfusion medium of intact
heart exposed to ischaemia and reperfusion as comparedwith that of the control (Figure 5D). These observations indicated that M-LDH was involved in the recovery of the heart
function from ischaemia.

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by the electric stimulation of adult cardiomyocytes. Under
control conditions, cardiomyocytes underwent normal contractions with a transient increase in [Ca2þ]i in response toelectrical stimulation (Figure 3A and C; Supplementarymaterial online, Movie 1A and C). Treatment of cardiomyocytes with H2O2 for 20 min resulted in making the cell
shape round, with Ca2þ transients being irregular and finallydisappearing (Figure 3B and Supplementary material online,
Movie 1B). However, pretreatment of cardiomyocytes with
M-LDH preserved their normal morphology and contractility
with regular Ca2þ transients even after 20 min of treatment
with H2O2 (Figure 3D and Supplementary material online,
Movie 1D). These findings strongly suggest that M-LDH suppresses the [Ca2þ]i overload caused by oxidative stress allowing cardiomyocytes to survive and maintain their contractile
function intact even under severe oxidative conditions.

3.6 Lactate dehydrogenase muscle subunit
suppresses early afterdepolarizations induced byoxidative stress in cardiomyocytes[Ca2þ]i overload and a spontaneous rise in intracellular-free
Ca2þ levels are implicated in EADs and ventricular tachycardia as mechanisms that trigger the long QT syndrome.35–37
We observed the action potential of cardiomyocytes
treated with H2O2 in the presence of M-LDH. H2O2 induced
EADs in ventricular cardiomyocytes within 6 min after treatment (Figure 4A–C). The frequency and amplitude of the

3.8 Lactate dehydrogenase muscle subunit
alters the phosphorylation levels of signaltransduction molecules
To examine whether the survival activity of M-LDH involves
any intracellular signalling pathways, the phosphorylationlevels of signal transduction molecules ERK1/2, JNK1/2,
p38MAPK, and PLCg were determined by CBA using flow
cytometry (Figure 6A–D). CBA is an assay that carries out
particle-based immunoassay using flow cytometry, andbeads with different positions can be combined to create amultiplex assay. Incubation of H9c2 cells with M-LDH
increased the phosphorylation of ERK1/2, but the phosphorylation levels of JNK1/2, p38MAPK, and PLCg remained
unchanged. ERK1/2 activity was dependent on the M-LDH
concentration (0.025–1.0 mM) in H9c2 cells (Figure 6E), consistent with western blotting data obtained using ananti-phospho-ERK1/2 antibody (data not shown). M-LDH
inactivated by boiling did not induce ERK activity in H9c2
cells. These results suggest that M-LDH exhibits survival
activity through some membrane receptors involving signaltransduction molecules ERK1/2.

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3.9 Lactate dehydrogenase muscle subunit isreleased into blood of patients exposed toischaemia–reperfusion
We examined the release of M-LDH in patients exposed toischaemia by pump-oxygenator during open heart surgery.

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Figure 3 Effect of lactate dehydrogenase muscle subunit (M-LDH) on [Ca2þ]i transients in rat adult cardiomyocytes stimulated with H2O2. Rat adult cardiomyocytes were incubated without (A, B) or with (C, D) M-LDH (final 0.5 mM) for 12 h, and then loaded with fura-2AM (final 0.1 mM) for an additional 30 min.
The cells were stimulated with 25 mA at a frequency of 0.5 Hz using the pairs of platinum wires connected to a stimulator in the absence (A and C ) or presenceof H2O2 (final 40 mM) (B and D), and pairs of digital images of fura-2 fluorescence at 510 nm with excitation at 340 and 380 nm were successively obtained at timeintervals of 21.0 ms. The figure shows representative data obtained from three independent experiments. Scale bar ¼ 20 mm.

Total LDH activity in serum was significantly increased inpatients subjected to the surgery with pump-oxygenator at
30 min after reperfusion and continued to increase for 72 h(data not shown). Next, two LDH isozymes, the activitiesof M-LDH and LDH heart-type subunit (H-LDH) were

determined in the serum of patients, because H-LDH was
also known to be released from the heart in response tovarious injuries. H-LDH was rapidly released into the blood
at 0.5 h after reperfusion, and the release of H-LDH continued to increase at least for 72 h after reperfusion (see

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Figure 4 Effect of lactate dehydrogenase muscle subunit (M-LDH) on themembrane action potential of rat adult cardiomyocytes in the presence of
H2O2. Rat adult cardiomyocytes were incubated without (A–E) or with
(F–J ) M-LDH (final 0.05 mM) for 6 h, and the current and action potential
were determined using a clamp amplifier for the indicated times (A and F )after H2O2 stimulation (final 50 mM) as described in Experimental Procedures.
The figure shows representative data obtained from independent experiments (A and F) (control; n ¼ 13, M-LDH; n ¼ 6). Early afterdepolarizations(EADs) frequency (B and G), EADs amplitude (E and J ), APD90 (D and I ), andVm (E and J ) were determined from the membrane potential data.

Supplementary material online, Figure S2). In contrast,
much less amount of M-LDH was rapidly released intoblood at 0.5 h after reperfusion and the release wasdecreased within 48 h after reperfusion (see Supplementarymaterial online, Figure S2), suggesting that M-LDH rather
than H-LDH may play an important role in the recovery
from ischaemia of the human heart.

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4. Discussion
945

As shown in this study, many polypeptides are released into
the supernatant of cells exposed to sI/R, a cell model ofY. Mizukami et al.

ischaemia. Among these polypeptides, at least three havesurvival activities for cells exposed to the stress by serum
removal. Strong survival activities were found in an eluate
containing a nearly pure single protein at the second stepof purification by anion exchange column chromatography,
suggesting that the major protein in this eluate is responsible for the survival activity. The protein was identified as
M-LDH by molecular peptide mass fingerprinting and a
specific antibody.
Pretreatment with M-LDH significantly inhibited the morphological change of cardiomyocytes to a round shapeinduced by H2O2, suggesting that M-LDH inhibits [Ca2þ]ioverload caused by oxidative stress. Cardiomyocytes contract through a myosin–actin interaction in response to anincrease in [Ca2þ]i, and a transient overload of [Ca2þ]i isthought to induce excess irreversible cell shortening,leading to the morphological change to a round shape.
Indeed, pretreatment with M-LDH efficiently blocked theabnormal increase in [Ca2þ]i caused by H2O2. H2O2 hasbeen reported to cause a biphasic pattern of [Ca2þ]i overload in a variety of cells including insulin-secreting cells38
and smooth muscle cells,39 consistent with the results
obtained using the cardiac myocyte-derived cell line asshown in Supplementary material online, Figure S1. The
spontaneous release of Ca2þ from the sarcoplasmic reticulum (SR) triggers EADs, oscillations in the membrane potential of the cells, which are generally thought to be a cause
for arrhythmia associated with Q–T interval prolongation,
including Torsades de Pointes.40–43 M-LDH inhibits theincrease in [Ca2þ]i in H9c2 cells exposed to H2O2 and theEADs induced by H2O2 in rat adult cardiomyocytes,
suggesting that M-LDH may block [Ca2þ]i overload due tospontaneous Ca2þ release from SR. Consistent with these
data, pretreatment with M-LDH significantly improves the
cardiac pump function of the isolated mouse heart
exposed to H2O2 or ischaemia–reperfusion, indicating that
M-LDH has survival activity that protects the heart fromoxidative stress and ischaemic injury.
It is well known that M-LDH reversibly controls theformation of lactate according to the intracellularenvironment. Under anaerobic conditions such as ischaemia, pyruvate, NAD, and Hþ are not further metabolized,
leading to LDH-catalysed lactate accumulation. Intracellular lactate is reported to open sarcolemmal ATP-sensitive
K channels in cells despite the presence of millimolar concentrations of ATP, and protect cells against Ca2þ overload
and cell death.44 Crawford et al. showed that M-LDH binds
to ATP-sensitive K channels, leading to an effective inhibition of the harmful effects of [Ca2þ]i through the metabolic product lactate.45 However, it is unlikely that
lactate, a metabolic product of M-LDH, was acting as asurvival factor in the present study, because the additionof lactate had no effect on the Ca2þ overload induced byoxidative stress (data not shown). H-LDH, a heartsubunit of M-LDH, also catalyses the production of
lactate, and large amounts of H-LDH are released fromcardiomyocytes exposed to ischaemia and reperfusion;
however, no cell survival activity was detected in the fractions containing H-LDH. In addition, the coenzyme NADH
was not required for the survival activity of M-LDH under
our experimental conditions, indicating that M-LDH does
not exert its survival activity as an enzyme lactate
dehydrogenase.

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Figure 5 Effects of lactate dehydrogenase muscle subunit (M-LDH) on the heart function during H2O2 perfusion or ischaemia/reperfusion of isolated workperforming heart A, Cardiac outputs from working heart preparations were determined 10 min after perfusion with H2O2 (final 50 mM) (open circle and open triangle) or without H2O2 (closed circle), and the effect of M-LDH was determined in the presence of 25 nM M-LDH throughout the experiment (open triangle). Data
were obtained in four independent experiments in each group, and the results are expressed as means + SE. (*P , 0.01 vs. control; #P , 0.05 vs. þH2O2-M-LDH,
Tukey’s Multiple Comparison Test). (B–C ) After working heart preparations were exposed to ischaemia for 5 min followed by reperfusion for 10 min at a preload of
22 cm H2O, cardiac output (B) and the maximum and minimum rates of LV pressure change (dP/dtmax and dP/dtmin, respectively) (C ) were determined simultaneously. The effects of M-LDH on the heart function during ischaemia–reperfusion were determined by adding 0.025 mM M-LDH into the perfusate from
5 min before exposure to ischaemia. Control data were obtained from hearts neither pretreated with M-LDH nor exposed to ischaemia–reperfusion. Data
were obtained in four independent experiments in each group, and the results are expressed as means + SE (*P , 0.05, **P , 0.01 vs. control, Dunnett’s Multiple
Comparison Test, I/R, ischaemia–reperfusion). (D) Perfusates of mouse hearts exposed to ischaemia–reperfusion were collected during reperfusion, and wereconcentrated by size-exclusion columns. The samples were subjected to immunoblotting with anti-M-LDH antibody. The figure shows representative blots
obtained from three independent experiments.

In this study, the mechanism of M-LDH release during sI/R
remains to be clarified, but we could speculate that amechanism other than release by a damaged membrane
is involved, because if M-LDH was released from thedamaged membrane, G3PDH and PFK, also present in thecytosol, should also be detected during sI/R. However,
the amounts of G3PDH and PFK released into theextracellular spaces decreased during sI/R. In addition,
the rate of cell death among cells exposed to sI/R was
10% or less, under our experimental conditions, and was
almost equal to that of control cells, suggesting that cell
injury was not very severe (data not shown). LDH activity
is often determined as a biomarker of cell damage.
However, we found that more than 90% of the activity
released into the blood in a human ischaemia heartmodel is due to the H-LDH isoform (see Supplementarymaterial online, Figure S2), suggesting that LDH activity

measured in cell death assay might not be necessarily corresponding to the M-LDH activity.
In conclusion, the present study provides strong evidence
for a novel role of M-LDH in the recovery of heart function
following ischaemia. Two lines of evidence indicate that
M-LDH acts through an intracellular signal transduction
system: (i) pretreatment with M-LDH inhibits H2O2-induced[Ca2þ]i overload and death of cardiomyocytes; however,the simultaneous application of M-LDH and H2O2 does notinhibit them sufficiently strongly suggesting that M-LDH
does not exert its survival activity through direct action,such as ion channel damage, on the membrane protein;(ii) M-LDH increases the phosphorylation of ERK1/2, whichis involved in cell survival,23 without changing the phosphorylation of JNK1/2, which is involved in apoptosis pathways. These observations suggest that a novel receptor for
M-LDH may be present in cardiomyocytes. Further studies

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Page 10 of 11

Y. Mizukami et al.

ischaemia–reperfusion in vivo could activate these signaling
pathways and protect surrounding cells.

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Supplementary material
Supplementary Material is available at Cardiovascular
Research Online.

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Acknowledgements
1145

We thank S. Fujisawa (Akita University School of Medicine) for technical assistance with the measurements of membrane action
potential.

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Conflict of interest: none declared.
1150

Funding
This work was supported in part by grants from the Ministry of Education, Science and Culture of Japan, Takeda Science Foundation,
Japan Heart Foundation Research Grant, NOVARTIS Foundation(Japan) for the Promotion of Science, Research Program for theSpecial Promotion of the Venture Business Laboratory, Yamaguchi
University, The Old Age Research Foundation of Yamaguchi, Yamaguchi Industrial Promotion Foundation. C.-K.D. is a recipient of the
JPSP Postdoctoral Fellowship for Foreign Researchers.

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References
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Figure 6 Phosphorylation of signal transduction molecules in H9c2 cells
after treatment with lactate dehydrogenase muscle subunit (M-LDH) Cellextracts was prepared from H9c2 cells incubated without or with M-LDH(final 0.5 mM for A–D and the indicated concentrations for E) for 15 min,and subjected to cytometric beads array using anti-phospho antibodies for
signal transduction molecules (ERK1/2, JNK1/2, p38MAPK, and PLCg). Phosphorylation levels of ERK1/2 (A and E), JNK1/2 (B), p38MAPK (C ), and PLCg
(D) were determined from standard curves based on recombinant proteins.
Data were obtained from three independent experiments in each group.
(*P , 0.05 vs. control, Student’s t-test, means + SE).

are required to clarify the detailed molecular mechanism
involved in the intracellular signal transduction system andpotential membrane receptors through which M-LDH exhibits its survival activity against ischaemia–reperfusion of
the heart. The present study demonstrated that the protective effects of M-LDH was observed at very low concentrations (25–50 nM), strongly suggesting that even a small
amount of M-LDH released from the cells damaged by

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