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Cardiovascular Research 1998 37(3):606-617; doi:10.1016/S0008-6363(97)00238-1
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

Expression of calcium regulatory proteins in short-term hibernation and stunning in the in situ porcine heart1

Hartmut Lüssa,*, Peter Bokniéka, Gerd Heuschb, Frank Ulrich Müllera, Joachim Neumanna, Wilhelm Schmitza and Rainer Schulzb

aInstitut für Pharmakologie und Toxikologie, Westfälische Wilhelms-Universität Münster, Domagkstr. 12, D-48129 Münster, Germany
bAbteilung für Pathophysiologie, Zentrum für Innere Medizin des Universitätsklinikums Essen, D-45122 Essen, Germany

* Corresponding author. Tel. (+49-251) 835 5510; Fax (+49-251) 835 5501.

Received 21 April 1997; accepted 11 September 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
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 (microsonometry) by ~50%. In transmural biopsies obtained during ischaemia and reperfusion creatine phosphate as well as the expression of sarcoplasmic reticulum calcium ATPase (SERCA), phospholamban (PLB), calsequestrin (CSQ), and troponin inhibitor (TnI) were determined. Results: During ischaemia creatine phosphate, after an initial reduction, recovered back to control values, and necrosis was absent (hibernation). After 90 min of ischaemia the myocardium was reperfused for 120 min but regional contractile function continued to be depressed (stunning). 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.

KEYWORDS Calsequestrin; Short-term hibernation; Ischaemia; Phospholamban; Reperfusion; Sarcoplasmic reticulum calcium ATPase; Swine; Troponin inhibitor


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Short-term myocardial hibernation is characterised by perfusion-contraction matching, recovery of metabolic markers, persistence of inotropic reserve and absence of necrosis [1–9]. Stunned myocardium is characterised by prolonged, but reversible postischaemic contractile dysfunction despite restoration of blood flow by reperfusion [10, 11]. Calcium responsiveness is decreased in both short-term hibernating and stunned myocardium [4]. The precise mechanisms that underlie hibernation and stunning remain to be elucidated.

There is evidence that stunning is accompanied by cytosolic calcium overload (for review see [10, 12]). 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 (through 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 (SERCA) [13, 14]and stored by e.g. calseqestrin (CSQ) [14]. Depressed function of SERCA could result from reduced protein levels or enhanced expression of its repressor protein phospholamban (PLB) [13, 15–17]. 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. Indeed degradation of the inhibitory subunit of troponin (TnI), a component of the myofilament, was observed in a model of global ischaemia [18]and this finding led others to speculate that degradation of TnI could be responsible for the decreased contractility in stunning [19]. 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, β-adrenoceptor density was unchanged during short-term hibernation [9]. Moreover, neither adenosine nor ATP-dependent potassium channels were involved in the development of short-term hibernation in anaesthetised swine [20, 21]. In isolated perfused hearts [22]calcium levels were reduced during low-flow 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 (e.g. TnI), or in the SR (PLB, 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 [23–25]. However, there is evidence that protein and mRNA levels of PLB and SERCA do not always correlate [25, 26]. 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 [7–9]. Biopsies were taken at defined time-points during ischaemia and reperfusion and analysed for the expression of calcium regulatory proteins.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
The present study has been approved by the local authorities of the district of Düsseldorf and experimental protocols were in accordance with the guiding principles of the American Physiological Society.

2.1 Experimental model
Seven Göttinger miniswine of either sex (weight: 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 (1 g im) and then anaesthetised with thiopental (500 mg iv). The trachea was intubated through a midline cervical incision and anaesthesia was then maintained by enflurane (1 to 1.5%) with an oxygen/nitrous oxide mixture (40:60%).

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 (see below). Rectal temperature was measured and body temperature was kept above 37°C using a heated surgical table and drapes. Arterial blood gases were monitored periodically throughout the study (Radiometer, Copenhagen, Denmark).

A left lateral thoracotomy was performed in the fourth intercostal space and the pericardium was opened. Through the apex a micromanometer (P7, 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 (System 6, Triton Technologies, San Diego, CA, USA) were implanted in the left ventricular myocardium to measure the thickness of anterior and posterior (control) walls. Swine were anticoagulated with 20 000 IU sodium heparin, additional doses (10 000 IU) were given hourly. The proximal left anterior descending coronary artery (LAD) 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 (Hugo Sachs Elektronik type 215/T, 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 (LVPP).

2.2 Regional myocardial function
End-diastole was defined as the point when left ventricular dP/dt 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 dP/dt. 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 instantaneous left ventricular pressure-wall thickness products over the time of the cardiac cycle with the equation:


Formula

where ed=end-diastole, n=actual cardiac cycle, m=sampling point within cardiac cycle n at a sampling frequency of 200 Hz, LVPn.m=instantaneous left ventricular (LV) pressure within cardiac cycle n and at sampling point m, LVPn.min=minimum LV pressure within cardiac cycle n, and WTh=wall thickness. The maximal work value during systole is reported as the work index (WI) [4].

2.3 Regional myocardial blood flow
To determine regional myocardial blood flow and its distribution throughout the LAD perfusion bed radiolabeled (141Ce, 114In, 103Ru, 95Nb, or 46Sc, NEN/DuPont, Bad Homburg, Germany) microspheres (15 µm diameter) were injected into the coronary perfusion circuit (1–2x105 suspended in 1 ml saline). This procedure for determining blood flow has been validated extensively [8]. 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 (digital 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 (measured by summing the radioactivity in all isotope-containing myocardial samples). Thus, in the present study no reference sample was needed.

2.4 Regional myocardial metabolism
The high energy phosphate (ATP and creatine phosphate, CrP) contents were measured in transmural myocardial biopsies. The analytical procedures (bioluminescence) have been described previously in detail [4, 7]. Biopsy specimens (approximately 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 (using 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 –80°C as described before [7]. 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 (Cavitron/LexO2-Con-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 [7].

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 (pH 7.4) containing 1.0% triphenyl tetrazolium chloride (TTC, Sigma, Deisenhofen, Germany) and 8% dextran (mol wt. 77 800) for 20 min at 37°C to identify infarcted tissue [7].

2.6 Experimental protocol
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 (normally within 30 min after the cannulation procedure). In all swine, a period of controlled hypoperfusion of the LAD for 90 min (ischaemia) 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 (5 min 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 (LCX) which served as controls.


Figure 1
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  		Fig. 1 Protocol of ischaemia (90 min) and reperfusion (120 min). Biopsies were taken at 5 and 85 min ischaemia and after 30 min of reperfusion from the left anterior descending coronary artery (LAD) perfusion bed. Control biopsies were obtained in parallel at the 30 min reperfusion time-point from the left circumflex coronary artery (LCX) perfusion bed.

 
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 [27]was employed. Biopsies were homogenised using a microdismembrator (Braun Melsungen, Melsungen, Germany) in 1 ml TriStar ReagentTM (AGS, 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 µl homogenate 160 µl 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 (0.5 µg) of each RNA preparation were visualised using SYBR-Green stain (Biozym, Hess. Oldendorf, Germany) on 1% denaturing agarose minigels. First strand cDNA was reverse transcribed from 1 µg of total RNA in 10 µl of 50 mM Tris-HCl (pH 8.3), 40 mM KCl, 6.0 mM MgCl2, 1.0 mM each dNTP (Pharmacia, Uppsala, Sweden), 5.0 mM DL-dithiothreitol, 50 µg/ml bovine serum albumin (BSA), 10 units of human placental RNAse inhibitor (AGS, Heidelberg, Germany), and 30 units of TrueScriptTM reverse transcriptase (AGS, Heidelberg, Germany) at 41°C 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 [28]and SERCA [29]cDNA sequences were employed to detect respective mRNAs by RT-PCR. The forward primers for PLB and SERCA were 5'-CCA GCT AAA CAC CGA TAA GAC-3' and 5'-CTG TCC ATG TCA CTC CAC TTC-3' respectively. The reverse primers were 5'-CCC TTC TTC ATG GGA TGA CAG-3' and 5'-GGG TAA GGT TTC AGA ACC TCA-3'. 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 µl containing 20 mM Tris-HCl (pH 8.55 at 25°C), 16 mM (NH4)2SO4, 200 µM each dNTP, 1.5–2.0 mM MgCl2 and 1.5 units Taq DNA polymerase (AGS, Heidelberg, Germany). Each reaction was subjected to 18–24 cycles of denaturation (1 min at 94°C), annealing (2 min at 51°C for PLB and 59°C for SERCA), and extension (2 min at 72°C). All PCR reactions were performed in a thermal cycler (Omnigene, model TR3 CM220, MWG-Biotech, Ebersberg, Germany). Sizes of PCR products were compared to DNA size markers (MBI Fermentas, Vilnius, Latvia). MgCl2 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 µCi [{alpha}-32P]-dCTP (NEN DuPont, Bad Homburg, Germany). PCR products were visualised on 2% agarose gels, cut out and counted in a liquid scintillation counter (Tri-Carb, Canberra Packard, Dreieich, Germany). Background values (in 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. [30]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 (cpm) of incorporated [{alpha}-32P] 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 [31].

In order to confirm the identity of the PCR products Southern blotting was performed. Briefly PCR products were blotted overnight to nylon membranes (Amersham Buchler, Braunschweig, Germany) by capillary transfer in 20xSSC. After prehybridisation of the membranes in a solution containing 50% deionised formamide, 5xDenhardt solution, 0.9 M NaCl, 60 mM NaH2PO4, 6.0 mM EDTA, 0.2 mg/ml tRNA from yeast, and 0.1% sodium dodecyl sulfate (SDS) at pH 7.4, membranes were hybridised overnight at 42°C in the same buffer containing the probes, which were labeled with [{alpha}-32P]-dCTP (NEN DuPont, Bad Homburg, Germany) by random priming (Megaprime-kit, Amersham Buchler, Braunschweig, Germany). Activity of the probes was 1–2x106 dpm/ml. Hybridised membranes were washed at a stringency of 0.2xSSC, 0.1% SDS at 65°C and exposed to PhosphorImager screens and visualised in a PhosphorImager (Molecular Dynamics, Krefeld, Germany). A ~0.8 kb EcoRI fragment of the pig PLB cDNA [28], and a ~3.9 kb EcoRI fragment of pig SERCA class I cDNA [29]were used as probes (kind 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 (Bio-Rad, München, 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 (Schleicher and Schuell, Dassel, Germany) in 50 mM sodium phosphate buffer (pH 7.4) 180 min at 1.5 A at 4°C as reported [32]. Then membranes were blocked in Tris buffered saline containing 2.0% BSA for 30 min and incubated overnight at 4°C with antibodies directed against PLB (monoclonal, A1, 1:2500, Phosphoprotein Research, England), SERCA (monoclonal, AB 465, 1:500), calsequestrin (polyclonal, 1:5000), and TnI (1:3000, 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: Formula -labeled goat-anti-mouse IgG, dilution 1:500, was used to detect PLB, and Formula -labeled protein A (ICN 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 (Pharmacia, Uppsala, Sweden) consisting of rabbit muscle phosphorylase b (94 kDa), bovine serum albumin (67 kDa), egg white ovalbumin (43 kDa), bovine erythrocyte carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20 kDa), and bovine milk lactalbumin (14.4 kDa). The amount of radioactivity was quantitated in a PhosphorImager using the ImageQuant software (Molecular 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 (Gould 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 (approximately 33 consecutive beats over at least two complete respiratory cycles) using CORDAT II software [33]. Haemodynamic parameters reported are heart rate (HR), left ventricular end-diastolic and peak pressure (LVEDP), left ventricular peak pressure (LVPP), the maximum of the first derivative of left ventricular pressure (LV dP/dtmax), and mean coronary arterial pressure (CAP). Regional wall function parameters include systolic anterior wall thickening (AWT) and posterior wall thickening (PWT) as well as the anterior work index (AWI). Metabolic parameters include the myocardial contents of ATP and CrP, the consumption of oxygen, and lactate (positive 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 (Urbana, 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.

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
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
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.


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  		Table 1 Haemodynamics, blood flow, regional myocardial function, and metabolism during control conditions, ischaemia and reperfusion

 
3.1 Haemodynamics and blood flow
The pump blood flow to the LAD perfusion bed during control conditions averaged 31.4±7.2 ml/min, and was decreased to 17.6±4.0 ml/min (p<0.05) with ischaemia. At 30 min reperfusion, pump blood flow was increased above the preischaemic flow (47.5±18.3 ml/min, 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 (CAP) fell significantly from 120±5 mmHg (control) to 45±2 mmHg (5 min ischaemia). Concomitantly transmural (TMF), subepicardial (Epi), and subendocardial blood flows (Endo) within the anterior wall decreased (Table 1). Prolongation of ischaemia to 85 min did not result in any further changes in these parameters (Table 1).

Upon reperfusion CAP, transmural and subendocardial blood flows returned to control values (Table 1).

3.2 Regional myocardial function
After 5 min ischaemia, anterior wall thickening (AWT) was decreased from 30±4% (control) to 16±4% and stayed approximately at that level after 85 min of ischaemia. AWT was still reduced after 30 min of reperfusion (Table 1). The anterior work index (AWI) fell from 260±32 mmHg·mm (control) to 128±22 mmHg·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 (PWT) 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 (MVO2) and CrP content were decreased. Myocardial lactate consumption (MVLac) was reversed to net lactate production (Table 1).

When ischaemia was prolonged to 85 min, MVO2 remained diminished but lactate production tended to be attenuated (Table 1). After 85 min of ischaemia CrP content had recovered to values that were not significantly different from preischaemic values (Table 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. MVO2 and MVLac tended to return to normal although MVLac was still significantly different from control values (Table 1).

3.4 Myocardial viability and biopsies
Myocardial necrosis (TTC-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 (data not shown). The yield of RNA amounted to 2–4 µg 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 (Fig. 2, A). These PCR products were blotted to nylon membranes (Southern blotting). Hybridisation using pig PLB and SERCA cDNAs as probes revealed prominent bands on autoradiographs of the Southern blots (Fig. 2, B) corresponding to the bands representing the PCR products on the ethidium bromide stained agarose gels (Fig. 2, A). Thus identity of the PCR products was confirmed.


Figure 2
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  		Fig. 2 Phospholamban (PLB) and sarcoplasmic reticulum calcium ATPase (SERCA) mRNA in porcine heart. A) RT-PCR analysis of total RNA from normal porcine left ventricle for PLB (lane 1) and SERCA (lane 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 (bp) were detected as compared to DNA size markers (lane M) indicating the presence of PLB and SERCA mRNAs respectively. (B) 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.

 
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 (20, 40, and 60 ng) resulted in increasing amounts of PCR product measured as incorporated [{alpha}-32P] in cpm. The amount of amplification product increased linearly (in the half logarithmic scale) from cycle 18 to cycle 22 (Fig. 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 (data not shown) such that the same conditions (40 ng RNA and 22 cycles) for quantification 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.


Figure 3
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  		Fig. 3 Quantitative RT-PCR for sarcoplasmic reticulum calcium ATPase (SERCA) — amplification product as a function of cycle number and amount of RNA. Semilogarithmic plot of [{alpha}-32P] incorporated into SERCA amplification products by RT-PCR as described in Methods using different amounts of total RNA (inset). After completion of the indicated number of cycles (abscissa) aliquots were removed from the PCR reactions and analysed as described in Methods. The amount of PCR product (ordinate: incorporated [{alpha}-32P] in cpm) increased exponentially up to cycle 22. Values are means±SEM (n=5–8) of 4 separate experiments.

 

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  		Table 2 Levels of PLB and SERCA mRNAs in porcine heart biopsies during ischaemia and reperfusion versus controls

 
3.6 Quantification of proteins
PLB, SERCA, CSQ, and TnI were easily detected in porcine cardiac preparations by Western blotting (Fig. 4).


Figure 4
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  		Fig. 4 Western blot analysis of porcine heart homogenates for phospholamban (PLB) sarcoplasmic reticulum calcium ATPase (SERCA) calsequestrin (CSQ) and troponin inhibitor (TnI). 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.

 
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 µg 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 µg 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.


Figure 5
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  		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 (as 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 (n=8).

 
In homogenates of biopsies obtained at 5 and 85 min of ischaemia as well as at 30 min reperfusion the ratio PLB/SERCA (Fig. 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 (Fig. 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 (Fig. 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 (Fig. 6). Similar results were obtained for CSQ and TnI (Fig. 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.


Figure 6
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  		Fig. 6 Bar graph showing the ratio of PLB to SERCA protein (PLB/SERCA upper panel) protein levels of phospholamban (PLB middle panel) and sarcoplasmic reticulum calcium ATPase (SERCA 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 (LCX-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.

 

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  		Fig. 7 Bar graph showing protein levels of calsequestrin (CSQ upper panel) and troponin inhibitor (TnI 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 (LCX-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.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
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 and/or 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 [4, 7–9, 21]. Using regional injection of microspheres, regional blood flow to the LCX (control) 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 [8]. As the pig does not have an extensive collateral circulation [8], the gradient of subendocardial to subepicardial blood flow is smaller than in species with an extensive collateral circulation such as the dog [34]. 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 [35]. 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 β-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 [23–25]. However, there is evidence that protein and mRNA levels of PLB and SERCA do not always correlate [25, 26]. 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 [16], should not be affected by loss of SR proteins during preparation 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 [14], 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 [36]. 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 [18]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 [19]. 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 [17]whereas overexpression depressed contractile function in transgenic mice [15]. 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 [16]and aortic banding [13]reduced contractility was accompanied by decreased SERCA levels. Furthermore, a previous study [37]presented evidence for an increased expression of mRNAs of PLB and SERCA after short coronary occlusions (two 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. [37]are important and may well simulate a clinically extremely typical situation namely repetitive stunning. However, our model is 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. [37]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 [38]. 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 [39]. 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 [40]. Other still undefined proteins may also be affected. However, due to the small sample size of biopsies (less 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.

Time for primary review 31 days.


   Acknowledgements
 
Supported by Deutsche Forschungsgemeinschaft and Interdisziplinäres Klinisches Forschungszentrum (IKF) Münster, TP B1, BMBF 01 KS9604. The excellent technical assistance of M. Beckhove is gratefully acknowledged. 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.


   Notes
 
1 Dedicated to Professor Dr. Hasso Scholz, Hamburg, on the occasion of his 60th birthday. Back


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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