Download Expression of calcium regulatory proteins in short

Cardiovascular Research 37 Ž1998. 606–617
Expression of calcium regulatory proteins in short-term hibernation and
stunning in the in situ porcine heart 1
Hartmut Luss
¨
a
a,)
a
, Peter Boknık
, Joachim Neumann a ,
´ a, Gerd Heusch b, Frank Ulrich Muller
¨
a
b
Wilhelm Schmitz , Rainer Schulz
Institut fur
Wilhelms-UniÕersitat
Domagkstr. 12, D-48129 Munster,
Germany
¨ Pharmakologie und Toxikologie, Westfalische
¨
¨ Munster,
¨
¨
b
Abteilung fur
Essen, D-45122 Essen , Germany
¨ Pathophysiologie, Zentrum fur
¨ Innere Medizin des UniÕersitatsklinikums
¨
Received 21 April 1997; accepted 11 September 1997
Abstract
Background: Myocardial hibernation and stunning are characterised by a reversible contractile dysfunction during and after
ischaemia, respectively. Calcium homeostasis might be disturbed in hibernation and stunning due to altered expression of cardiac proteins
involved in calcium handling. Methods: In enflurane-anaesthetised swine the coronary blood flow through the left anterior descending
coronary artery was decreased to reduce regional contractile function Žmicrosonometry. by ; 50%. In transmural biopsies obtained
during ischaemia and reperfusion creatine phosphate as well as the expression of sarcoplasmic reticulum calcium ATPase ŽSERCA.,
phospholamban ŽPLB., calsequestrin ŽCSQ., and troponin inhibitor ŽTnI. were determined. Results: During ischaemia creatine phosphate,
after an initial reduction, recovered back to control values, and necrosis was absent Žhibernation.. After 90 min of ischaemia the
myocardium was reperfused for 120 min but regional contractile function continued to be depressed Žstunning.. PLB, SERCA, CSQ, and
TnI proteins were unchanged during ischaemia as well as reperfusion. Likewise, levels of PLB and SERCA mRNAs were unchanged.
Conclusion: It is concluded that other mechanisms than altered expression of these regulating proteins underlie the contractile
dysfunction observed during acute ischaemia, short-term hibernation and stunning. q 1998 Elsevier Science B.V.
Keywords: Calsequestrin; Short-term hibernation; Ischaemia; Phospholamban; Reperfusion; Sarcoplasmic reticulum calcium ATPase; Swine; Troponin
inhibitor
1. Introduction
Short-term myocardial hibernation is characterised by
perfusion-contraction matching, recovery of metabolic
markers, persistence of inotropic reserve and absence of
necrosis w1–9x. Stunned myocardium is characterised by
prolonged, but reversible postischaemic contractile dysfunction despite restoration of blood flow by reperfusion
w10,11x. Calcium responsiveness is decreased in both
short-term hibernating and stunned myocardium w4x. The
precise mechanisms that underlie hibernation and stunning
remain to be elucidated.
)
Corresponding author. Tel. Žq49-251. 835 5510; Fax Žq49-251.
835 5501.
1
Dedicated to Professor Dr. Hasso Scholz, Hamburg, on the occasion
of his 60th birthday.
0008-6363r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved.
PII S 0 0 0 8 - 6 3 6 3 Ž 9 7 . 0 0 2 3 8 - 1
There is evidence that stunning is accompanied by
cytosolic calcium overload Žfor review see w10,12x.. The
reason for such calcium overload could reside in part in
enhanced inflow of calcium through the sarcolemma or
altered SR function. It is conceivable that calcium release
from the SR Žthrough calcium release channels. is stimulated or that calcium uptake into the SR is depressed.
Calcium is transported into the SR by the sarcoplasmic
reticulum calcium ATPase ŽSERCA. w13,14x and stored by
e.g. calseqestrin ŽCSQ. w14x. Depressed function of SERCA
could result from reduced protein levels or enhanced expression of its repressor protein phospholamban ŽPLB.
w13,15–17x. However, the protein levels of these genes
have not yet been reported in stunning.
Altered responsiveness of myofilaments to calcium
could result from altered expression of contractile proteins.
Time for primary review 31 days.
H. Luss
¨ et al.r CardioÕascular Research 37 (1998) 606–617
Indeed degradation of the inhibitory subunit of troponin
ŽTnI., a component of the myofilament, was observed in a
model of global ischaemia w18x and this finding led others
to speculate that degradation of TnI could be responsible
for the decreased contractility in stunning w19x. Data on
TnI levels in stunning are however lacking.
Likewise, the mechanisms that underlie hibernation are
speculative. For instance one could surmise that increased
levels of plasma catecholamines could lead to a downregulation of adrenoceptors. However, b-adrenoceptor density
was unchanged during short-term hibernation w9x. Moreover, neither adenosine nor ATP-dependent potassium
channels were involved in the development of short-term
hibernation in anaesthetised swine w20,21x. In isolated perfused hearts w22x calcium levels were reduced during lowflow ischaemia, and such reduced calcium levels in the
myocytes might contribute to short-term hibernation. Currently, data on the expression of calcium handling proteins
in the myofilaments Že.g. TnI., or in the SR ŽPLB, SERCA,
CSQ. are not available.
Therefore, the aim of the present study was to gain
more insight into the mechanisms that cause contractile
dysfunction in short-term hibernation and stunning. PLB
and SERCA are important proteins for cardiac contractility
and were reported to be decreased in human end-stage
heart failure w23–25x. However, there is evidence that
protein and mRNA levels of PLB and SERCA do not
always correlate w25,26x. Like others before, we hypothesised that alterations in gene expression of cardiac regulatory proteins might be responsible for the reduction in
contractile function in regional short-term hibernating and
stunned myocardium.
We used an established model of short-term hibernation
with subsequent stunning in an anaesthetised pig preparation with controlled coronary perfusion w7–9x. Biopsies
were taken at defined time-points during ischaemia and
reperfusion and analysed for the expression of calcium
regulatory proteins.
2. Methods
The present study has been approved by the local
authorities of the district of Dusseldorf
and experimental
¨
protocols were in accordance with the guiding principles
of the American Physiological Society.
2.1. Experimental model
Seven Gottinger
miniswine of either sex Žweight: 30–40
¨
.
kg were used for the study. These swine had an age of 6
to 9 months and thus could be viewed as adult pigs. The
animals were sedated with ketamine hydrochloride Ž1 g
im. and then anaesthetised with thiopental Ž500 mg iv..
The trachea was intubated through a midline cervical
607
incision and anaesthesia was then maintained by enflurane
Ž1 to 1.5%. with an oxygenrnitrous oxide mixture
Ž40:60%..
Both common carotid arteries and internal jugular veins
were cannulated with polyethylene catheters: one artery for
the measurement of arterial pressure and the other to
supply blood to the extracorporal circuit. Volume was
replaced through the jugular veins with warmed 0.9%
NaCl and by returning blood to the animal from the
coronary venous line Žsee below.. Rectal temperature was
measured and body temperature was kept above 378C
using a heated surgical table and drapes. Arterial blood
gases were monitored periodically throughout the study
ŽRadiometer, Copenhagen, Denmark..
A left lateral thoracotomy was performed in the fourth
intercostal space and the pericardium was opened. Through
the apex a micromanometer ŽP7, Konigsberg Instruments,
Pasadena, CA, USA. was positioned in the left ventricle
together with a saline-filled polyethylene catheter to calibrate the manometer in situ. Ultrasonic dimension gauges
ŽSystem 6, Triton Technologies, San Diego, CA, USA.
were implanted in the left ventricular myocardium to
measure the thickness of anterior and posterior Žcontrol.
walls. Swine were anticoagulated with 20 000 IU sodium
heparin, additional doses Ž10 000 IU. were given hourly.
The proximal left anterior descending coronary artery
ŽLAD. was dissected over a distance of 1.5 cm, ligated,
cannulated, and the distal LAD was perfused from the
extracorporal circuit. This system included a roller pump, a
windkessel, one side arm of a T-connector with an external
transducer to measure coronary arterial pressure, and one
side port for the injection of radiolabeled microspheres.
The large epicardial vein parallel to the LAD was dissected and cannulated such that the coronary venous blood
was drained to an unpressurised reservoir and then returned to the jugular vein through a second roller pump.
Heart rate was controlled throughout the experiment by
left atrial pacing ŽHugo Sachs Elektronik type 215rT,
Hugstetten, Germany.. The preparation was allowed to
stabilise for at least 30 min before control measurements
were made. During control measurements the flow constant perfusion pump was adjusted to maintain coronary
arterial pressures above 70 mmHg to avoid initial hypoperfusion. Therefore, mean coronary arterial pressure exceeded left ventricular peak pressure ŽLVPP..
2.2. Regional myocardial function
End-diastole was defined as the point when left ventricular d Prdt started its rapid upstroke after crossing the
zero line. Regional end systole was defined as the point of
maximal wall thickness within 20 ms before peak negative
left ventricular d Prdt. Systolic wall thickening was calculated as end-systolic minus end-diastolic wall thickness
divided by the end-diastolic wall thickness. Regional myocardial work was calculated as the sum of the instanta-
H. Luss
¨ et al.r CardioÕascular Research 37 (1998) 606–617
608
neous left ventricular pressure-wall thickness products over
the time of the cardiac cycle with the equation:
ed ny1
WI n s
Ý Ž LVPn .m y LVPn .min . P Ž WTh n .m y WTh n .my1 .
ed n
where ed s end-diastole, n s actual cardiac cycle, m s
sampling point within cardiac cycle n at a sampling frequency of 200 Hz, LVPn.m s instantaneous left ventricular
ŽLV. pressure within cardiac cycle n and at sampling point
m, LVPn.min s minimum LV pressure within cardiac cycle
n, and WTh s wall thickness. The maximal work value
during systole is reported as the work index ŽWI. w4x.
2.3. Regional myocardial blood flow
To determine regional myocardial blood flow and its
distribution throughout the LAD perfusion bed radiolabeled Ž 141 Ce, 114 In, 103 Ru, 95 Nb, or 46 Sc, NENrDuPont,
Bad Homburg, Germany. microspheres Ž15 mm diameter.
were injected into the coronary perfusion circuit Ž1–2 = 10 5
suspended in 1 ml saline.. This procedure for determining
blood flow has been validated extensively w8x. Samples for
blood flow measurements were taken at the end of each
experiment. The heart was removed and sectioned from
base to apex into five transverse slices in a plane parallel
to the atrioventricular groove. Each slice was further subdivided into 8–10 transmural pieces. In the present study,
only the blood flow to the site of the ultrasonic crystal is
reported and this piece of tissue was divided into transmural thirds of approximately equal thickness. The blood flow
to a given myocardium was calculated by counting its
radioactivity content and then multiplying by the ratio of
total blood flow to the LAD Ždigital reading of the roller
pump which was calibrated at the end of each study. and
the total radioactivity content of a given isotope in the
entire LAD perfusion bed Žmeasured by summing the
radioactivity in all isotope-containing myocardial samples..
Thus, in the present study no reference sample was needed.
Samples requiring more than 2 s for acquisition were not
used for this analysis.
Oxygen content was measured in anaerobically sampled
blood drawn simultaneously from the cannulated coronary
vein and an artery using a co-oximeter ŽCavitronrLexO2Con-k, Dr. B.G. Schlag, Bergisch Gladbach, Germany..
Oxygen consumption of the anterior myocardial wall was
calculated by multiplying the coronary arterial-venous oxygen difference by the mean transmural blood flow at the
crystal site.
Lactate was measured in simultaneously drawn coronary venous and arterial blood samples using enzymatic
dehydrogenation and subsequent photometry of NADH.
Lactate consumption was calculated by multiplying the
coronary arterial-venous lactate difference by the mean
transmural blood flow at the crystal site w7x.
2.5. Morphology
At the end of each experiment, following 120 min
reperfusion, the heart was removed and sectioned from
base to apex into five transverse slices in a plane parallel
to the atrioventricular groove. Tissue slices were immersed
in a 0.09 M sodium phosphate buffer ŽpH 7.4. containing
1.0% triphenyl tetrazolium chloride ŽTTC, Sigma, Deisenhofen, Germany. and 8% dextran Žmol wt. 77 800. for 20
min at 378C to identify infarcted tissue w7x.
2.6. Experimental protocol
2.4. Regional myocardial metabolism
The experimental protocol is illustrated in Fig. 1. Only
when systemic haemodynamics and regional myocardial
function remained unaltered for a period of up to 20 min,
the experimental protocol was started Žnormally within 30
min after the cannulation procedure.. In all swine, a period
of controlled hypoperfusion of the LAD for 90 min
Žischaemia. was followed by 120 min of reperfusion.
During ischaemia, blood flow to the LAD was reduced to a
level sufficient to reduce the regional systolic work index
by approximately 50%. This adjustment period lasted 3
min. After 2 more min of steady state ischaemia Ž5 min
The high energy phosphate ŽATP and creatine phosphate, CrP. contents were measured in transmural myocardial biopsies. The analytical procedures Žbioluminescence.
have been described previously in detail w4,7x. Biopsy
specimens Žapproximately 10 mg each. were taken with a
modified dental drill from the LAD perfusion bed. Care
was taken to ensure that the biopsies originated from
within LAD perfusion bed Žusing epicardial arteries as
landmarks., but distal to the sites of the ultrasonic dimension gauges and blood flow measurements. Biopsies were
also taken from the left circumflex coronary artery perfusion bed serving as controls. Biopsies were obtained within
less than 2 s until they were freeze-clamped in liquid
nitrogen and stored at y808C as described before w7x.
Fig. 1. Protocol of ischaemia Ž90 min. and reperfusion Ž120 min..
Biopsies were taken at 5 and 85 min ischaemia and after 30 min of
reperfusion from the left anterior descending coronary artery ŽLAD.
perfusion bed. Control biopsies were obtained in parallel at the 30 min
reperfusion time-point from the left circumflex coronary artery ŽLCX.
perfusion bed.
H. Luss
¨ et al.r CardioÕascular Research 37 (1998) 606–617
from the onset of flow reduction., pairs of arterial and
coronary venous blood samples were simultaneously withdrawn. During the blood sampling, microspheres were
injected into the LAD through the perfusion system for the
measurement of regional myocardial blood flow. Haemodynamic and regional dimension data were recorded. A set
of measurements was obtained within 2 to 3 min. Coronary
arterial pressure was continuously monitored during the
microsphere injection to ensure that it was unaffected by
the injection. Immediately after the microsphere injection,
myocardial biopsies were taken. At 90 min of ischaemia,
measurements were repeated and the myocardium was
reperfused. After 30 min of reperfusion biopsies were
taken from the LAD perfusion territory as well as from the
perfusion bed of the left circumflex coronary artery ŽLCX.
which served as controls.
2.7. Quantification of mRNA by RT-PCR
In order to extract total RNA from the biopsy specimens a modification of the method described by Chomczynski and Sacchi w27x was employed. Biopsies were
homogenised using a microdismembrator ŽBraun Melsungen, Melsungen, Germany. in 1 ml TriStar Reagent e
ŽAGS, Heidelberg, Germany. containing guanidinium thiocyanate and phenol. This homogenate was divided into two
portions; one aliquot was used for quantification of proteins by SDS-PAGE and Western blotting, and from the
other, major portion, total RNA was extracted according to
the manufacturer’s instructions. To 800 ml homogenate
160 ml chloroform were added and the resulting two
phases were separated by centrifugation. The RNA present
in the upper, aqueous, phase was precipitated with approximately the same volume isopropanol and washed twice
with 75% ethanol. After the RNA pellet was dried under
vacuum it was dissolved in diethyl pyrocarbonate treated
water. Aliquots Ž0.5 mg. of each RNA preparation were
visualised using SYBR-Green stain ŽBiozym, Hess. Oldendorf, Germany. on 1% denaturing agarose minigels. First
strand cDNA was reverse transcribed from 1 mg of total
RNA in 10 ml of 50 mM Tris-HCl ŽpH 8.3., 40 mM KCl,
6.0 mM MgCl 2 , 1.0 mM each dNTP ŽPharmacia, Uppsala,
Sweden., 5.0 mM DL-dithiothreitol, 50 mgrml bovine
serum albumin ŽBSA., 10 units of human placental RNAse
inhibitor ŽAGS, Heidelberg, Germany., and 30 units of
TrueScript e reverse transcriptase ŽAGS, Heidelberg, Germany. at 418C for 60 min. To distinguish between PCR
products originated from mRNA or genomic DNA respectively, reverse transcription was also performed in the
absence of reverse transcriptase. Primers based on the pig
PLB w28x and SERCA w29x cDNA sequences were employed to detect respective mRNAs by RT-PCR. The
forward primers for PLB and SERCA were 5X-CCA GCT
AAA CAC CGA TAA GAC-3X and 5X-CTG TCC ATG
TCA CTC CAC TTC-3X respectively. The reverse primers
were 5X-CCC TTC TTC ATG GGA TGA CAG-3X and
609
5X-GGG TAA GGT TTC AGA ACC TCA-3X . Primers were
designed such that they spanned at least one intron. To
avoid cross-hybridisation with mRNA of other SERCA
isoforms SERCA primers were designed such that they
amplify only SERCA class I mRNA encoding the muscle
isoform. All PCR reactions were carried out in a total
volume of 50 ml containing 20 mM Tris-HCl ŽpH 8.55 at
258C., 16 mM ŽNH 4 . 2 SO4 , 200 mM each dNTP, 1.5–2.0
mM MgCl 2 and 1.5 units Taq DNA polymerase ŽAGS,
Heidelberg, Germany.. Each reaction was subjected to
18–24 cycles of denaturation Ž1 min at 948C., annealing Ž2
min at 518C for PLB and 598C for SERCA., and extension
Ž2 min at 728C.. All PCR reactions were performed in a
thermal cycler ŽOmnigene, model TR3 CM220, MWGBiotech, Ebersberg, Germany.. Sizes of PCR products
were compared to DNA size markers ŽMBI Fermentas,
Vilnius, Latvia.. MgCl 2 titration curves were performed
with each pair of primers to optimise amplification specificity. For quantification each PCR reaction was performed
in the presence of 2.0 mCi w a- 32 Px-dCTP ŽNEN DuPont,
Bad Homburg, Germany.. PCR products were visualised
on 2% agarose gels, cut out and counted in a liquid
scintillation counter ŽTri-Carb, Canberra Packard, Dreieich, Germany.. Background values Žin cpm. from corresponding gel slices in the negative control lanes were
subtracted from all data. To define the number of cycles
during which the amount of resulting PCR product increases in an exponential manner, aliquots were taken from
PCR reactions at different cycles. Varying amounts of
RNA were applied in order to establish the linear relationship between RNA included per reaction and the amount
of resulting PCR product. As shown by Ungerer et al. w30x
normalisation of the yields of PCR products to either
internal or external standards did not improve accuracy of
the quantification by RT-PCR. Therefore the results are
given in counts per min Žcpm. of incorporated w a- 32 Px into
the PCR product. This technique has been successfully
used recently by our group to quantitate mRNA levels of
the cAMP response element binding protein in rat heart
w31x.
In order to confirm the identity of the PCR products
Southern blotting was performed. Briefly PCR products
were blotted overnight to nylon membranes ŽAmersham
Buchler, Braunschweig, Germany. by capillary transfer in
20 = SSC. After prehybridisation of the membranes in a
solution containing 50% deionised formamide, 5 =
Denhardt solution, 0.9 M NaCl, 60 mM NaH 2 PO4 , 6.0
mM EDTA, 0.2 mgrml tRNA from yeast, and 0.1%
sodium dodecyl sulfate ŽSDS. at pH 7.4, membranes were
hybridised overnight at 428C in the same buffer containing
the probes, which were labeled with w a- 32 Px-dCTP ŽNEN
DuPont, Bad Homburg, Germany. by random priming
ŽMegaprime-kit, Amersham Buchler, Braunschweig, Germany.. Activity of the probes was 1–2 = 10 6 dpmrml.
Hybridised membranes were washed at a stringency of
0.2 = SSC, 0.1% SDS at 658C and exposed to Phosphor-
610
H. Luss
¨ et al.r CardioÕascular Research 37 (1998) 606–617
Imager screens and visualised in a PhosphorImager ŽMolecular Dynamics, Krefeld, Germany.. A ; 0.8
kb EcoRI fragment of the pig PLB cDNA w28x, and a
; 3.9 kb EcoRI fragment of pig SERCA class I cDNA
w29x were used as probes Žkind gifts from Dr. F. Wuytack..
2.8. SDS-PAGE and Western blotting
In order to extract RNA and protein from the same
biopsy specimen, aliquots of the TriStar homogenates used
for RNA extraction were precipitated with trichloroacetic
acid. Pellets were resuspended in 1.0 M Tris and 5% SDS.
Protein concentrations were determined by the Bio-Rad
Protein Assay ŽBio-Rad, Munchen,
Germany.. Proteins
¨
were separated using 10% polyacrylamide separating gels
with 4% stacking gels. Electrophoresis was initially run at
40 mA per gel until the dye front entered the separating
gel and then the current was increased to 60 mA. Proteins
were electrophoretically transferred to nitrocellulose membranes ŽSchleicher and Schuell, Dassel, Germany. in 50
mM sodium phosphate buffer ŽpH 7.4. 180 min at 1.5 A at
48C as reported w32x. Then membranes were blocked in
Tris buffered saline containing 2.0% BSA for 30 min and
incubated overnight at 48C with antibodies directed against
PLB Žmonoclonal, A1, 1:2500, Phosphoprotein Research,
England., SERCA Žmonoclonal, AB 465, 1:500., calsequestrin Žpolyclonal, 1:5000., and TnI Ž1:3000, kindly
provided by Dr. G.S. Bodor.. Antibodies against SERCA
and calsequestrin were kind gifts from Dr. L.R. Jones,
Indianapolis, USA. In order to visualise proteins binding
the antibodies, membranes were incubated with secondary
antibodies: 125 I-labeled goat-anti-mouse IgG, dilution
1:500, was used to detect PLB, and 125 I-labeled protein A
ŽICN Biomedicals, Meckenheim, Germany. was used for
SERCA, CSQ, and TnI. The blots were incubated with
antibodies for 2 h at room temperature, rinsed several
times and exposed to PhosphorImager screens. Apparent
molecular weights were determined by comparison to the
low molecular weight calibration kit ŽPharmacia, Uppsala,
Sweden. consisting of rabbit muscle phosphorylase b Ž94
kDa., bovine serum albumin Ž67 kDa., egg white ovalbumin Ž43 kDa., bovine erythrocyte carbonic anhydrase Ž30
kDa., soybean trypsin inhibitor Ž20 kDa., and bovine milk
lactalbumin Ž14.4 kDa.. The amount of radioactivity was
quantitated in a PhosphorImager using the ImageQuant
software ŽMolecular Dynamics, Krefeld, Germany.. Background values were substracted from all data.
2.9. Data analysis and statistics
Haemodynamic data were recorded on an 8-channel
recorder ŽGould MK 200A, Cleveland, OH, USA. and
stored directly to the hard disk of an AT-type computer.
Haemodynamic and functional parameters were digitised
and recorded over a 20 s period during each microsphere
injection Žapproximately 33 consecutive beats over at least
Table 1
Haemodynamics, blood flow, regional myocardial function, and metabolism during control conditions, ischaemia and reperfusion
Control
Haemodynamics
HR, beatsPminy1
LVEDP, mmHg
LVPP, mmHg
LVdPrdt ma x , mmHgPsy1
CAP, mmHg
Myocardial function
AWT, %
AWI, mmHg=mm
PWT, %
Myocardial blood flow and metabolism
TMF, mlPminy1Pgy1
Endo, mlPminy1Pgy1
Epi, mlPminy1Pgy1
CrP, mmolPgy1 wet wt
ATP, mmolPgy1 wet wt
MVO 2 , mlPminy1Pgy1
MVLac , mmolPminy1Pgy1
Ischaemia
Reperfusion
5 min
85 min
30 min
105"2
5"1
93"4
1310"125
120"5
105"3
7"1
89"4
1127"221
45"2 )
105"2
8"2
85"5
1054"3641
43"3 )
113"4
7"2
82"5
123"127
116"11a
30"4
260"32
23"4
16"4 )
128"22 )
25"5
14"4 )
101"24 )
22"5
14"3 )
123"33 )
26"5
0.82"0.05
0.88"0.09
0.84"0.07
8.40"0.42
3.92"0.17
88"7
3.66"1.03
0.38"0.03 )
0.30"0.04 )
0.47"0.07 )
2.26"0.35 )
3.16"0.28
53"5 )
y0.89"0.32 )
0.38"0.03 )
0.33"0.04 )
0.47"0.08 )
6.59"0.94a
2.93"0.36
50"5 )
y0.14"0.42 )
1.02"0.14a
1.23"0.32 a
0.90"0.10 a
8.08"1.18
2.75"0.28
59"9
0.88"0.72 )
HR, heart rate; LVEDP, left ventricular end-diastolic pressure; LVPP, left ventricular peak pressure; LVdPrdt ma x , maximum of the first derivative of left
ventricular pressure; CAP, mean coronary arterial pressure; AWT, anterior systolic wall thickening in percent of the end-diastolic wall thickness; PWT,
posterior systolic wall thickening in percent of the end-diastolic wall thickness; AWI, anterior work index; TMF, transmural blood flow; Endo,
subendocardial blood flow; Epi, subepicardial blood flow; CrP, myocardial creatine phosphate content; ATP, myocardial adenosine triphosphate content;
MVO 2 , myocardial oxygen consumption; MVLac , myocardial lactate consumption Žpositive value indicates myocardial uptake.. Calculation of hemodynamic parameters was done on a beat-to-beat basis, and data were then averaged. Values are means"SEM; ns 7, ) : p- 0.05 vs. Control; a: p- 0.05 vs.
preceding value.
H. Luss
¨ et al.r CardioÕascular Research 37 (1998) 606–617
611
When significant differences were detected, individual
mean values were compared using Tukey’s post-hoc tests.
All data are reported as mean values " standard error of
the mean, and a p-value less than 0.05 was accepted as
indicating a significant difference in mean values.
3. Results
For the current series of experiments haemodynamic,
biochemical, and morphological data are indicative of
hibernation and subsequent stunning. Table 1 gives a
summary of the data on haemodynamics, regional myocardial function, blood flow and metabolism during control
conditions, ischaemia and reperfusion.
3.1. Haemodynamics and blood flow
Fig. 2. Phospholamban ŽPLB. and sarcoplasmic reticulum calcium ATPase ŽSERCA. mRNA in porcine heart. A. RT-PCR analysis of total RNA
from normal porcine left ventricle for PLB Žlane 1. and SERCA Žlane 2..
Reverse transcription and PCR reactions were carried out as described in
Methods. Total RNA was reverse transcribed and 40 ng cDNA were
amplified in each reaction. PCR products of the expected sizes of 398 and
481 basepairs Žbp. were detected as compared to DNA size markers Žlane
M. indicating the presence of PLB and SERCA mRNAs respectively. ŽB.
Autoradiography of a Southern blot of PLB and SERCA PCR products
amplified from porcine heart which were hybridised to labeled porcine
PLB and SERCA cDNAs as described in Methods.
two complete respiratory cycles. using CORDAT II software w33x. Haemodynamic parameters reported are heart
rate ŽHR., left ventricular end-diastolic and peak pressure
ŽLVEDP., left ventricular peak pressure ŽLVPP., the maximum of the first derivative of left ventricular pressure ŽLV
d Prdt max ., and mean coronary arterial pressure ŽCAP..
Regional wall function parameters include systolic anterior
wall thickening ŽAWT. and posterior wall thickening
ŽPWT. as well as the anterior work index ŽAWI.. Metabolic
parameters include the myocardial contents of ATP and
CrP, the consumption of oxygen, and lactate Žpositive
value indicates myocardial uptake.. Calculation of all
haemodynamic parameters was done on a beat-to-beat
basis, and data were then averaged.
Statistical analysis was performed using SYSTAT software ŽUrbana, IL, USA.. Haemodynamic, metabolic, and
gene expression data were subjected to a one-way analysis
of variance for repeated measures, accounting for the time
course of the experiment.
The pump blood flow to the LAD perfusion bed during
control conditions averaged 31.4 " 7.2 mlrmin, and was
decreased to 17.6 " 4.0 mlrmin Ž p - 0.05. with ischaemia. At 30 min reperfusion, pump blood flow was
increased above the preischaemic flow Ž47.5 " 18.3
mlrmin, p - 0.05., since coronary arterial pressure was
maintained constant. Following the reduction in coronary
inflow at the beginning of the ischaemic period mean
coronary arterial pressure ŽCAP. fell significantly from
120 " 5 mmHg Žcontrol. to 45 " 2 mmHg Ž5 min ischaemia.. Concomitantly transmural ŽTMF., subepicardial
ŽEpi., and subendocardial blood flows ŽEndo. within the
anterior wall decreased ŽTable 1.. Prolongation of ischaemia to 85 min did not result in any further changes in
these parameters ŽTable 1..
Fig. 3. Quantitative RT-PCR for sarcoplasmic reticulum calcium ATPase
ŽSERCA. — amplification product as a function of cycle number and
amount of RNA. Semilogarithmic plot of w a- 32 Px incorporated into
SERCA amplification products by RT-PCR as described in Methods
using different amounts of total RNA Žinset.. After completion of the
indicated number of cycles Žabscissa. aliquots were removed from the
PCR reactions and analysed as described in Methods. The amount of PCR
product Žordinate: incorporated w a- 32 Px in cpm. increased exponentially
up to cycle 22. Values are means"SEM Ž ns 5–8. of 4 separate experiments.
H. Luss
¨ et al.r CardioÕascular Research 37 (1998) 606–617
612
Table 2
Levels of PLB and SERCA mRNAs in porcine heart biopsies during
ischaemia and reperfusion versus controls
LCX-control
mRNA leÕel (cpm)
PLB
1209"471
SERCA 1647"224
Ischaemia
Reperfusion
5 min
85 min
30 min
1378"449
1424"177
1449"358
1463"195
1445"499
1308"311
PLB, phospholamban; SERCA, sarcoplasmic reticulum calcium ATPase;
cpm, counts per min. Values are means"SEM, ns 7.
Upon reperfusion CAP, transmural and subendocardial
blood flows returned to control values ŽTable 1..
3.2. Regional myocardial function
After 5 min ischaemia, anterior wall thickening ŽAWT.
was decreased from 30 " 4% Žcontrol. to 16 " 4% and
stayed approximately at that level after 85 min of ischaemia. AWT was still reduced after 30 min of reperfusion ŽTable 1.. The anterior work index ŽAWI. fell from
260 " 32 mmHgP mm Žcontrol. to 128 " 22 mmHgP mm
after 5 min of ischaemia and remained approximately at
that level after 85 min of ischaemia and 30 min of
reperfusion.
Posterior wall thickening ŽPWT. was not different from
control measurements at all time points investigated.
3.3. Regional myocardial metabolism
After 5 min of ischaemia, myocardial ATP content was
unchanged but myocardial oxygen consumption ŽMVO 2 .
and CrP content were decreased. Myocardial lactate consumption ŽMVLac . was reversed to net lactate production
ŽTable 1..
When ischaemia was prolonged to 85 min, MVO 2
remained diminished but lactate production tended to be
attenuated ŽTable 1.. After 85 min of ischaemia CrP
content had recovered to values that were not significantly
different from preischaemic values ŽTable 1.. Myocardial
ATP content was not significantly decreased after 85 min
ischaemia.
At 30 min of reperfusion myocardial CrP content had
completely returned to normal. MVO 2 and MVLac tended
to return to normal although MVLac was still significantly
different from control values ŽTable 1..
3.4. Myocardial Õiability and biopsies
Myocardial necrosis ŽTTC-staining. was absent in all
animals after 90 min of ischaemia followed by 120 min
reperfusion.
3.5. Quantification of PLB and SERCA mRNAs
Integrity of isolated RNAs was assessed using 1%
agarose minigels stained with SYBR-Green Ždata not
shown.. The yield of RNA amounted to 2–4 mg RNA per
biopsy. Therefore, mRNA levels were assessed by quantitative PCR. At first total RNA from pig heart was subjected to RT-PCR using primers specific for pig PLB and
pig SERCA. Single 398-bp and 481-bp PCR products of
the expected sizes for PLB and SERCA respectively were
amplified ŽFig. 2, A.. These PCR products were blotted to
Fig. 4. Western blot analysis of porcine heart homogenates for phospholamban ŽPLB. sarcoplasmic reticulum calcium ATPase ŽSERCA. calsequestrin
ŽCSQ. and troponin inhibitor ŽTnI.. Ventricular homogenates were prepared as described in Methods subjected to SDS-PAGE and electrophoretically
transferred to nitrocellulose membranes. These blots were incubated with antibodies directed against respective proteins as described in Methods.
Molecular weights are indicated.
H. Luss
¨ et al.r CardioÕascular Research 37 (1998) 606–617
613
tion were chosen. The results of quantitative PCR for PLB
and SERCA are given in Table 2. Levels of PLB and
SERCA mRNAs were not changed either in hibernation or
in stunning when compared to controls.
3.6. Quantification of proteins
PLB, SERCA, CSQ, and TnI were easily detected in
porcine cardiac preparations by Western blotting ŽFig. 4..
The linearity of the radioactivity counted within the
immunoreactive bands from the amount of protein loaded
per lane was established for all proteins investigated. This
is shown in Fig. 5 for SERCA as a representative example.
Loading 5 to 40 mg of protein per lane resulted in a linear
increase in the radioactivity of the respective band on the
blot measured in PhosphorImager units. Thus using 25 mg
of protein per lane enabled us to measure both an increase
or a decrease of the SERCA protein level. Because of the
fact that PLB, CSQ as well as TnI exhibited comparable
linearity curves, we were able to quantitate all proteins on
the same blot.
In homogenates of biopsies obtained at 5 and 85 min of
ischaemia as well as at 30 min reperfusion the ratio
Fig. 5. Western blot analysis of porcine heart homogenates for sarcoplasmic reticulum calcium ATPase. Upper panel: autoradiogram. Ventricular homogenates were prepared as described in Methods subjected to
SDS-PAGE and electrophoretically transferred to nitrocellulose membranes. These blots were incubated with a mouse monoclonal antibody
directed against SERCA. Different amounts of protein Žas indicated. were
loaded per lane. Bottom panel: quantification of SERCA. Bands were
quantitated using a PhosphorImager. Abscissa: protein loaded per lane.
Ordinate: phosphorImager units. Values are means"SEM Ž ns8..
nylon membranes ŽSouthern blotting.. Hybridisation using
pig PLB and SERCA cDNAs as probes revealed prominent bands on autoradiographs of the Southern blots ŽFig.
2, B. corresponding to the bands representing the PCR
products on the ethidium bromide stained agarose gels
ŽFig. 2, A.. Thus identity of the PCR products was confirmed.
Using increasing amounts of RNA subjected to different
cycle numbers a series of quantitative PCRs was performed to establish the linear range of amplification. Representative results for SERCA are shown in Fig. 3. Increasing amounts of RNA Ž20, 40, and 60 ng. resulted in
increasing amounts of PCR product measured as incorporated w a- 32 Px in cpm. The amount of amplification product
increased linearly Žin the half logarithmic scale. from cycle
18 to cycle 22 ŽFig. 3.. Therefore in all following quantitative PCRs, SERCA mRNA was determined using 40 ng
RNA applying 22 cycles of amplification. Similar results
were obtained for PLB Ždata not shown. such that the
same conditions Ž40 ng RNA and 22 cycles. for quantifica-
Fig. 6. Bar graph showing the ratio of PLB to SERCA protein
ŽPLBrSERCA upper panel. protein levels of phospholamban ŽPLB middle panel. and sarcoplasmic reticulum calcium ATPase ŽSERCA bottom
panel. quantitated by Western blotting. Homogenates from porcine heart
biopsies after 5 and 85 min ischaemia and after 30 min of reperfusion
from the LAD perfusion bed compared to controls from the LCX
perfusion bed ŽLCX-Ctr.. Crude homogenates were obtained from biopsies at the depicted time points and Western blot analysis was performed
as described in Methods. Values are means"SEM. Numbers in columns
are numbers of animals.
614
H. Luss
¨ et al.r CardioÕascular Research 37 (1998) 606–617
Fig. 7. Bar graph showing protein levels of calsequestrin ŽCSQ upper
panel. and troponin inhibitor ŽTnI bottom panel. quantitated by Western
blotting. Homogenates from porcine heart biopsies after 5 and 85 min
ischaemia and after 30 min of reperfusion from the LAD perfusion bed
compared to controls from the LCX perfusion bed ŽLCX-Ctr.. Crude
homogenates were obtained from biopsies at the depicted time points and
Western blot analysis was performed as described in Methods. Values are
means"SEM. Numbers in columns are numbers of animals.
PLBrSERCA ŽFig. 6, upper panel. was not different from
the control. The absolute amounts of both proteins at 5 and
85 min of ischaemia were not different from the controls
ŽFig. 6, middle and bottom panels.. In homogenates of
biopsies obtained at 5 and 85 min of ischaemia the amounts
of PLB and SERCA proteins were unchanged compared to
controls ŽFig. 6.. Even in biopsies from the reperfused
myocardium taken 30 min after onset of reperfusion no
evidence either for an increase or a decrease in PLB and
SERCA levels could be obtained ŽFig. 6.. Similar results
were obtained for CSQ and TnI ŽFig. 7.. As shown in Fig.
7 TnI levels were not different from control levels. On
inspection of the autoradiographs no evidence for the
presence of TnI degradation products in lower molecular
weight regions was found.
4. Discussion
The cellular mechanisms and expressional changes that
lead to reduced myocardial contractility in short-term hibernation and stunning are not known. Thus the main goal
of the present study was to test the hypothesis that altered
expression of calcium regulatory proteins is an important
cause for reduced contractile function in short-term hibernation andror stunning.
The short-term hibernating porcine myocardium studied
here is characterised by a reduced contractile function
during 90 min of persistent low-flow ischaemia and recovery of an initially impaired metabolism over time. This is
an established and accepted model used in former work of
our group w4,7–9,21x. Using regional injection of microspheres, regional blood flow to the LCX Žcontrol. myocardium could not be determined. However, regional
myocardial function of the LCX-perfusion territory, which
was measured throughout the experiment, was normal,
suggesting also a sufficient perfusion of the LCX myocardium.
During ischaemia an endocardial to epicardial gradient
in regional myocardial blood flow exists, favouring subepicardial perfusion. Collateral blood flow was not measured
in the present study. However, in a previous study, using
the same animal model, collateral blood flow in the bed
perfused by the occluded LAD coronary artery was virtually absent w8x. As the pig does not have an extensive
collateral circulation w8x, the gradient of subendocardial to
subepicardial blood flow is smaller than in species with an
extensive collateral circulation such as the dog w34x. However, in anaesthetised dogs despite a marked perfusion
heterogeneity, the metabolic response to ischaemia is similar in the inner and outer layers of the myocardium. The
decrease in the subepicardial energy rich phosphates during progressive degrees of coronary stenosis is related to
subendocardial ischaemia, even in the absence of subepicardial hypoperfusion relative to baseline w35x. Thus the
consequences of transmural perfusion heterogeneity for
mRNA and protein expression during ischaemia are probably of only minor importance. The mechanical and haemodynamic data in the animals used for this study are comparable to previously published observations.
The time points for the biopsies during ischaemia and
reperfusion were chosen in order to facilitate comparisons
with earlier biochemical studies of our group. There we
characterised the b-adrenoceptor density, ATP and creatine
phosphate content under comparable conditions. At the
same time points which we characterised in earlier work
no alterations in the expression of calcium regulatory
proteins were observed.
Changes of mRNA levels do occur more rapidly than
changes of protein levels. PLB and SERCA are important
proteins for cardiac contractility and were reported to be
decreased in human end-stage heart failure w23–25x. However, there is evidence that protein and mRNA levels of
PLB and SERCA do not always correlate w25,26x. Because
of these findings we studied PLB and SERCA both at
protein and mRNA levels.
One may question whether we might have failed to
detect an increase in PLB and SERCA as a result of
reduced recovery of SR proteins in or after ischaemia.
However, the quotient of PLB versus SERCA, which has
been extensively used to assess SR function w16x, should
not be affected by loss of SR proteins during preparation
H. Luss
¨ et al.r CardioÕascular Research 37 (1998) 606–617
of samples from ischaemic hearts. This quotient remained
unaltered during the whole time course of the experiments.
More directly we also measured another SR protein which
has been used by various groups as an internal standard for
SR recovery w14x, namely, calsequestrin. The level of
calsequestrin was, however, not reduced. Thus preferential
loss of SR proteins is quite unlikely to hamper the interpretation of our data.
The time frame of our model may have been too short
in order to detect any changes in mRNA and protein
levels. However, as early as 2 h after thyroid hormone was
given to hypothyroid rats an increased expression of
SERCA mRNA was noted w36x. In our study samples in
stunning were obtained 120 min after the beginning of
initial ischaemia which is in a comparable time frame.
Moreover, it can be argued that even if transcriptional
activity is not yet altered in hibernation or stunning in our
model proteolytic enzymes which act very rapidly may
have degraded SR proteins. The proteolytic degradation
would be detectable as a rapid and substantial decrease in
SR protein levels measured by Western blots. Indeed
others have shown that 1 h of global ischaemia can
proteolyse TnI w18x which is an important regulator of
calcium responsiveness at the level of the myofilaments.
Gao et al. have speculated that TnI might undergo degradation by calcium activated proteases e.g. calpain in stunning
w19x. However they did not actually measure TnI levels.
We could not detect an increase or a decrease in TnI levels
neither during ischaemia nor during reperfusion. The fact
that there was no detectable decrease in TnI protein levels
argues against massive proteolysis by e.g. calpain in our
model. Thus the systolic contractile dysfunction observed
in hibernation and stunning was not related to changes of
TnI.
The idea that altered expression of calcium regulatory
proteins underlies reduced contractile function was supported by the finding that ablation of the PLB gene
enhanced w17x whereas overexpression depressed contractile function in transgenic mice w15x. However, the present
study clearly indicates that PLB at protein and RNA levels
is not altered in short-term hibernation or stunning, and
thus is not responsible for the reduced contractile function
under these conditions. In addition it was conceivable that
diminished expression of SERCA might underlie contractile failure. For instance, in hypothyroidism w16x and aortic
banding w13x reduced contractility was accompanied by
decreased SERCA levels. Furthermore, a previous study
w37x presented evidence for an increased expression of
mRNAs of PLB and SERCA after short coronary occlusions Žtwo times for 10 min. and reperfusion in open-chest
pig hearts. In apparent contrast, we did not detect an
increase in PLB and SERCA at protein and RNA levels in
stunning.
Clearly the findings by Frass et al. w37x are important
and may well simulate a clinically extremely typical situation namely repetitive stunning. However, our model is
615
critically different. In the present model stunning occurs
after low-flow ischaemia without complete occlusion of
the vessel. Thus, it is reasonable to expect different biochemical findings in a model of total coronary occlusions
inducing stunning compared to stunning after prolonged
reduction of coronary flow. Frass et al. w37x reported
increased mRNA levels for PLB and SERCA in stunned
porcine hearts starting to increase at approximately 90 min
of reperfusion after the second 10 min coronary occlusion.
In the present study, biopsies were taken at 30 min reperfusion following a 90 min moderate ischaemia period.
Thus, changes of PLB and SERCA mRNA levels which
might have occurred later during the protocol were not
detected. However, as myocardial stunning is present from
the beginning of reperfusion, but PLB and SERCA changes
occur no earlier than 90 min after restoration of flow, the
importance of these changes for myocardial stunning appear to be minimal.
What other possibilities are likely to account for the
decrease in contractility in the present model?
Posttranslational modifications of proteins including
glycosylation, disulfide formation might alter the function
of contractile proteins. These parameters might be affected
by generation of free radicals w38x. Furthermore, contractility depends on the phosphorylation state of a variety of
cardiac proteins. This regulation can proceed very rapidly
because the phosphorylation state of proteins can change
within seconds through the action of protein kinases and
phosphatases w39x. Thus increased or decreased phosphorylation of cardiac regulatory proteins by autonomous regulation of kinases or phosphatases could be responsible for
the diminished myocardial contractile function during
short-term hibernation and stunning.
Finally, other target proteins might be altered. In reperfusion after global ischaemia in rat hearts, spectrin, a
protein of poorly defined function, was proteolysed most
probably by calpain w40x. Other still undefined proteins
may also be affected. However, due to the small sample
size of biopsies Žless than 10 mg wet weight. we could not
test these attractive hypotheses experimentally at this time.
In summary, here we demonstrate for the first time that
neither changes of PLB nor SERCA are associated with
the contractile dysfunction observed in short-term hibernation and stunning in the in situ porcine heart. It is suggested that altered expression of as yet undefined target
proteins or posttranslational modifications of SR proteins
are likely to be involved under these experimental conditions.
Acknowledgements
Supported by Deutsche Forschungsgemeinschaft and
Interdisziplinares
Klinisches Forschungszentrum ŽIKF.
¨
Munster,
TP B1, BMBF 01 KS9604. The excellent techni¨
cal assistance of M. Beckhove is gratefully acknowledged.
616
H. Luss
¨ et al.r CardioÕascular Research 37 (1998) 606–617
We thank Dr. C. Martin for the chemical analyses and P.
Gres for their technical support. We thank Dr. Wuytack for
providing us with porcine PLB and SERCA cDNAs. We
are grateful to Drs. L.R. Jones and G.S. Bodor for providing us with antibodies.
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