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Cardiovascular Research 1998 38(1):169-180; doi:10.1016/S0008-6363(97)00283-6
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

Effects of sustained low-flow ischemia on myocardial function and calcium-regulating proteins in adult and senescent rat hearts1

Patrick Assayaga,b,c, Danièle Charlemagnec, Isabelle Martyd, Joël de Leirisa, Anne Marie Lomprée, François Bouchera, Paul-Etienne Valèreb, Sylviane Lortetf, Bernard Swynghedauwc and Sophie Bessea,*

aGroupe de Physiopathologie Cellulaire Cardiaque, ESA CNRS 5077, Université Joseph Fourier, 38000 Grenoble, France
bService de Cardiologie, Hôpital Bichat-Claude Bernard, 75018 Paris, France
cINSERM Unité 127, IFR Circulation, Hôpital Lariboisière, 75010 Paris, France
dLaboratoire de Biophysique Moléculaire et Cellulaire, CEA-DBMS, URA CNRS 520, 38000 Grenoble, France
eURA CNRS 1131, Université Paris-Sud, 91405 Orsay, France
fFaculté de Pharmacie, Université Aix-Marseille II, 13000 Marseille, France

* Corresponding author. Tel. (+33) 4 76 51 46 71; Fax (+33) 4 76 51 26 59.

Received 4 August 1997; accepted 31 October 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Both aging and myocardial ischemia are associated with alterations of calcium-regulating proteins. We investigated the effects of graded levels of low-flow ischemia on myocardial function and on SR Ca2+-ATPase (SERCA2), Na+-Ca2+ exchanger (NCx) and ryanodine receptor (RyR2), at mRNA and protein levels in both adult and senescent myocardium. Methods: Isolated hearts from 4 and 24 month old (mo) rats were retrogradely perfused during 180 min at 100% (100% CF, n=11 and n=11 respectively), 30% (30% CF, n=10 and n=12) or 15% (15% CF, n=13 and n=8) of their initial coronary flow, and active tension and coronary resistance (in % of their baseline value) were recorded. After 180 min of perfusion, NCx, RyR2 and SERCA2 mRNAs (in % of age-matched 100% CF group value) and protein levels were quantitated in the left ventricles by slot blot and Western blot analysis, respectively. Results: In 24 mo hearts, low-flow ischemia induced a greater fall in active tension (–65±7% vs. –40±4% in 4 mo 30% CF, p<0.01 and –82±2% vs. –60±5% in 4 mo 15% CF groups, p<0.05 after 15 min of ischemia) and a greater increase in coronary resistance (+357±44% vs. +196±39% in 4 mo 30% CF, p<0.05 and +807±158% vs. +292±61% in 4 mo 15% CF groups, p<0.001 after 15 min of ischemia). An increased accumulation of SERCA2 (+36%) and NCx (+46%) transcripts, but not RyR2, already occurred in 24 mo 30% CF group while the 3 transcripts accumulated in 24 mo 15% CF group. In 4 mo rats SERCA2 (+26%), NCx (+35%) and RyR2 (+81%) mRNA levels only increased in the 15% CF group. Corresponding calcium-regulating protein levels were unaltered whatever the degree of flow reduction in both 4 mo and 24 mo hearts. Conclusion: Low-flow ischemia does not induce calcium-regulating protein loss in both adult and senescent hearts. The increase in mRNAs coding for calcium-handling proteins and the impairment of myocardial function which occur at a lesser degree of coronary flow reduction in senescent hearts, indicate a higher vulnerability to low-flow ischemia during aging.

KEYWORDS Aging; Ischemia; Contractility; Sarcoplasmic reticulum; Calcium; Rat


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Recent reports have indicated that no-flow ischemia activates protooncogenes and heat shock proteins [1, 2]but also genes coding for anti-oxidant enzymes [3], heme oxygenase [4]and β1-adrenergic receptor [5]. During reperfusion following coronary occlusion in young pigs, an increased transcription of genes encoding calsequestrin and sarcoplasmic reticulum (SR) Ca2+-ATPase has also been described [6, 7]. It has been suggested that this gene activation could be triggered as part of a repair process by the proteins damaged during ischemia [2, 6, 7].

Ischemic stress induces functional alterations of membrane proteins such as decreased activity of Na+-K+ ATPase, SR Ca2+-ATPase, Na+-Ca2+ exchanger and ryanodine receptor [8, 9], and protein damage has been suspected during no-flow ischemia [10, 11]. Several protein alterations are likely to result from degradation by activated proteases [12, 13]. A selective degradation of troponin T and I during ischemia [14]and of cytoskeletal proteins [13, 15]during reperfusion have been reported. However, few data are available [6, 7, 12]concerning changes in gene expression or content of calcium regulating proteins during ischemia.

Until now, alterations in the expression of membrane proteins have been documented mostly during no-flow ischemia with [6, 7]or without [5]reperfusion in isolated heart or coronary occlusion in vivo [6, 7]. The effects of low-flow ischemia on calcium-regulating proteins have not been examined despite the fact that these proteins are the main determinants of calcium overload [16]. The first aim of this study was to investigate the effects of graded reductions in coronary flow on SR Ca2+-ATPase, Na+-Ca2+ exchanger and ryanodine receptor at both mRNA and protein levels.

During senescence, calcium-regulating protein alterations are well documented and very similar to those observed in models of pressure overload [17, 18]. The activity of the Na+-Ca2+ exchanger is decreased [19], and both activity and gene expression of the SR Ca2+-ATPase are reduced [20–22]. In addition, the aged heart has a decreased tolerance to calcium [23], and greater calcium overload and contractile dysfunction have been reported during no-flow ischemia [24, 25]. However, age-related effects of low-flow ischemia on myocardial function remain presently unknown and changes in gene expression or content of calcium-regulating proteins have not yet been investigated in the ischemic senescent myocardium. The second aim of this study was to determine the effects of low-flow ischemia on these proteins in the senescent heart, which is characterized by marked perturbations of calcium homeostasis during ischemia [24].


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Animals and experimental groups
Four (adult) and 24 (senescent) month old male Wistar rats were obtained from IFFA CREDO (Lyon, France). In the Wistar rat population, the spontaneous mortality rate is 50% at 24 months of age. Each age group was subdivided into 3 randomized subgroups: normoxic perfusion at 100% of initial coronary flow (CF) (4 and 24 month old-100% CF groups), ischemic perfusion at either 30% of initial CF (4 and 24 month old-30% CF groups), or 15% of initial CF (4 and 24 month old-15% CF groups). Anesthesia was induced by intraperitoneal injection of thiopental sodium (Nesdonal, Specia, Rhône-Poulenc Rorer, Paris, France) at the dose of 50 mg/kg and 30 mg/kg in 4 and 24 month old rats respectively. The experiments were conducted in accordance with the institutional guidelines and with the guidelines formulated by the European Community for use of experimental animals (L358-86/609/EEC).

2.2 Isolated heart preparation
After anesthesia, the hearts were rapidly excised and perfused according to Langendorff under a hydrostatic pressure of 75 mmHg. Perfusion conditions were identical in adult and senescent animals. The perfusate was a Krebs Henseleit solution containing (mmol.L–1): NaCl 118, NaHCO3 25, KCl 4.8, KH2PO4 1.2, MgCl2 1.2, CaCl2 1.2, glucose 11 (pH=7.4, 37°C) and was constantly bubbled with 95% O2, 5% CO2. The range of partial O2 pressure, measured at the aortic cannula level, was 620–680 mmHg. The hearts were paced at 240 beats min–1 at 200% of threshold with 2 ms stimulation duration (SD9 stimulator, Grass Instrument, Quincy, Mass).

After 15 min of Langendorff perfusion, the resting force was adjusted by hook traction in each heart to produce maximal developed force. Active developed tension (AT; active force per g of Heart Weight, g.g–1 of HW) and resting tension (RT; resting force per g of Heart Weight, g.g–1 of HW) were then recorded during the entire perfusion period, the hook being attached to a force transducer (type 351, Hugo Sachs Electronik, March-Hugstetten, Germany) connected to a Gould recorder (2000 model, Gould Electronic, Cleveland, Ohio). Myocardial contractile force was measured using a hook attached to the apex of the heart rather than an intraventricular balloon to avoid balloon-induced pressure gradient inside the ventricular wall, which could modify the perfusion gradient between epicardial and endocardial layers.

2.3 Perfusion protocol
After 15 min of Langendorff perfusion, baseline values of coronary flow (CF) were measured then the system was switched to flow-controlled perfusion, each heart being constantly perfused at its own initial CF with a peristaltic pump (Minipulse 3, Gilson, Villiers le bel, France). Coronary pressure (CP) was recorded with a pressure transducer (P10EZ model, Gould Electronic, Cleveland, Ohio) throughout the experiment, and the coronary resistance calculated (CR: ratio of CP to CF.g–1 of HW). After a 10 min equilibration in flow-controlled perfusion, baseline values of RT, AT, CP and CR were measured.

The pump flow rate was maintained at 100% of initial CF during 180 min for the 100% CF-normoxic groups. For the 30% CF-ischemic and 15% CF-ischemic groups, the pump flow rate was reduced to 30% or 15% of the initial CF for 180 min to induce a moderate or a severe low-flow ischemia, respectively. An acrylic cover was placed over the perfusion chamber to maintain the temperature at 37°C, and the temperature was monitored throughout the experiments, using a thermoprobe inserted in the right ventricle and connected to an electronic thermometer (model 7002 CH, Jenco Electronic). At the end of perfusion, hearts were immediately dismounted, blotted and weighed quickly. LV with septum were separated from RV, weighed, immediately frozen in liquid nitrogen and stored 1–3 months at –80°C until RNA extraction or protein determination.

2.4 Northern and slot blots analysis
Total RNA according to Chomczinsky and Sacchi [26]was isolated from individual left ventricles rather than from isolated myocytes since during cardiocyte purification different subpopulations of cells may be selected depending on the physiopathological conditions. For Northern blot analysis, 20 µg of total RNA were denatured in 50% formamide, 2.2 M formaldehyde and 1 X MOPS buffer (pH=8.0), size-fractionated on 1% agarose gels under denaturing conditions and then transferred to a nylon Hybond N membrane (Amersham, Les Ulis, France). For slot blot analysis, 1, 2.5, 5, 10 and 15 µg of denatured total RNAs were applied directly to nylon Hybond N membranes. Membranes were submitted to ultra-violet irradiation to covalently link the RNA samples [21, 22].

The rat cardiac ryanodine receptor type 2 (RyR2) cDNA probe (nucleotides 8604 to 9145) was synthesized from RNA by reverse transcription followed by polymerase chain reaction amplification, cloned between the XhoI and BamHI sites of Bluescript II KS and checked by sequencing [27]. The rat cardiac sodium calcium exchanger (NCx) cDNA probe (nucleotides 2360–2850) (gift of K. Boheler) is a KpnI—SacI restriction fragment subcloned in Bluescript II SK [22], the rat cardiac SR Ca2+-ATPase type 2 (SERCA2) cDNA (nucleotides 2618–3124) probe is a XhoI—HindIII restriction fragment subcloned in Bluescript II KS [28], the 18S oligonucleotide probe is a 24 mer oligonucleotide complementary to nucleotides 1046–1070 of the rat 18S RNA [21, 29]and the oligo d(T) probe is a 25–30 mer oligo d(T) (Pharmacia, Les Ulis, France) [21, 29]. The cDNA probes were labelled by random priming with {alpha} (32P) dCTP with a Rediprime Kit (Amersham, Les Ulis, France) and the synthetic oligonucleotides with T4 polynucleotide kinase and {gamma} (32P) ATP.

Northern blots were sequentially hybridized with NCx cDNA, RyR2 cDNA, SERCA2 cDNA and 18S oligonucleotide. Prehybridization and hybridization with NCx, RyR2 and SERCA2 probes were performed in 50% formamide, 5 X Denhardt's solution, 5 X standard saline phosphate ethylene-diamine-tetra-acetic acid (SSPE), 0.1% sodium dodecyl sulfate (SDS), 200 µg/mL herring sperm DNA and 20 µg/mL poly (A+) at 42°C. Washing conditions were as follows: (1) NCx probe: washed in 0.5 X standard saline citrate (SSC)–0.1%SDS at 45°C, (2) RyR2 and SERCA2 probes: washed in 0.5 X SSC–0.1%SDS at 50°C. The hybridizations with the 18S oligonucleotide probe and washes were performed as previously described [21, 29]. The washed Northern blots were exposed to X-ray films (Hyperfilm, Amersham, Les Ulis, France) with Quanta III intensifying screens for 16 h to 8 days at –70°C. Fig. 1 illustrates the specificity of SERCA2, NCx, RyR2 and 18S probes. Since RyR2 transcript is too large (16 kb) to be transferred efficiently from an RNA agarose gel [27, 30], RyR2 cDNA specificity was also checked by RNAse Protection Assay [27]. However, Northern blots do not allow adequate illustration or quantitation of RyR2 mRNAs. Therefore, the RNA slot blot system was used to quantitate all transcripts.


Figure 1
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  		Fig. 1 Autoradiographs of Northern hybridization analysis of 20 µg of total RNA from left ventricle of adult rats. Northern blot was sequentially hybridized with Na+-Ca2+ exchanger (NCx), ryanodine receptor (RyR2), SR Ca2+-ATPase (SERCA2) cDNA and 18S oligonucleotide probes as described in Methods.

 
Slot blots were sequentially hybridized with NCx cDNA, RyR2 cDNA, SERCA2 cDNA, 18S oligonucleotide and oligo d(T) probes. Prehybridization, hybridization and washes were performed as described above for Northern blots. The hybridizations with oligo d(T) and washes were performed as previously described [21, 29]. Slot blots were exposed in intensifying screens (Fuji imaging plate type BAS IIIS, Fuji Co, Tokyo, Japan) and then analysed with a Bioimaging analyser (BAS 1000 Mac BAS, Fuji Co, Tokyo, Japan). The level of each mRNA species, of poly (A+) mRNA and of 18S RNA was determined by scanning densitometry (Mac BAS software 1.01, Fuji Co, Tokyo, Japan) of slot blot membrane autoradiograms. Linearity between densitometric scores and RNA content was checked for the serial dilutions from each RNA sample. The densitometric scores of SERCA2, RyR2 and NCx mRNAs were normalized to poly (A+) mRNA densitometric score obtained by slot blot hybridization with the oligo d(T) probe.

2.5 Western blot analysis
Crude microsomal preparations were extracted from individual left ventricles as previously described [27]and used to quantify RyR2, NCx and SERCA2 proteins by immunoblotting i.e. Western blot technique [31]. For ryanodine receptor quantification, 30 µg of proteins were loaded in a 5–15% polyacrylamide gel [32]. After electrophoretic separation, the proteins were transferred to a nylon Immobilon P membrane (Millipore, Bedford, MA) for 4 h at 1.1 A. The membranes were then incubated overnight at 4°C with 1:5000 diluted anti-ryanodine polyclonal antibodies [32]followed by 2 h incubation at room temperature with a 1:10 000 diluted goat horseradish peroxidase-conjugated antirabbit IgG (Biosource International, Camarillo, CA). For both NCx and SERCA2, 30 µg of proteins were separated on a 8.5% polyacrylamide gel and transferred to an Immobilon P membrane during 2 h at 0.6 A. After an overnight incubation at 4°C either with a 1:5000 diluted anti-NCx R3F1 monoclonal antibody [33]or with a 1:5000 diluted anti-SERCA2 monoclonal antibody (Affinity Bioreagents, Golden, CO) [34], the membranes were incubated for 2 h at room temperature with a 1:7500 goat horseradish peroxidase-conjugated antimouse IgG (Cappel, Organon Teknika Corp, Durham, NC).

After incubation with a chemoluminescent reagent (Specichrom, Speci SA, Sainte Foy Les Lyon, France), the membranes were exposed to X-ray films (Hyperfilm βmax, Amersham, Les Ulis, France). All membranes were stained with Coomassie blue R250 for determination of myosin heavy chain (MHC) [27]. The level of calcium-regulating proteins was determined by scanning densitometry (NIH Image software 1.60), and the densitometric scores of the RyR2, NCx and SERCA2 specific bands on autoradiography were normalized to MHC densitometric score.

2.6 Statistical analysis
The values presented are expressed as mean±SEM. The statistical significance of differences between the various groups was determined by one-way analysis of variance and group-to-group comparisons were made by two-tailed unpaired Student's t-test. A p value <0.05 was considered to be statistically significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Baseline parameters
The switch from Langendorff to flow-controlled perfusion did not significantly modify coronary pressure and active and resting tensions in any of the experimental groups studied (data not shown).

The initial coronary flow, active and resting tensions, coronary pressure and resistance were identical between experimental groups of the same age (Table 1). However, initial coronary flow and active tension were depressed in senescent hearts as compared to the adults (Table 1). Resting tension, adjusted by hook traction in each heart to produce maximal developed tension, was lower in the 24 month old groups, indicating that the senescent hearts were stiffer than the adult hearts (Table 1). Coronary pressure and coronary resistance were not significantly different in 4 and 24 month old groups (Table 1).


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  		Table 1 Baseline values of mechanical parameters before the 180 min of normoxic or ischemic perfusion

 
3.2 Active and resting tensions during ischemia
Normoxic perfusion for 180 min (100% CF groups) induced a mild decrease in active tension, which was more pronounced in 24 month old rats (Fig. 2). The slight increase in resting tension was identical in 4 and 24 month old rats (Fig. 3).


Figure 2
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  		Fig. 2 Effects of low-flow ischemia on active developed tension in both 4 and 24 month old hearts. Active tension was recorded at 1, 5, 15, 30, 60, 120 and 180 min in hearts perfused at 100% (4 and 24 month old-100% CF groups), 30% (4 and 24 month old-30% CF groups) and 15% (4 and 24 month old-15% CF groups) of initial coronary flow. Active tension was expressed in % of baseline value.

*p<0.05 versus 4 month old perfusion-matched group;

{dagger}p<0.05 versus 100% CF age-matched group;

{ddagger}p<0.05 15% CF group versus 30% CF age-matched group.

 

Figure 3
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  		Fig. 3 Effects of low-flow ischemia on resting tension in both 4 and 24 month old hearts. Resting tension was expressed in % of baseline value. See legend of Figure 2 for description of groups and for symbols of statistical significance.

 
Low-flow ischemia induced an early and sustained fall in active tension in all groups (Fig. 2). However, it was much pronounced in senescent animals, especially under the less severe ischemic conditions. While reduction to 30% of CF induced only a moderate decline in 4 month old rats, in 24 month old rats it generated a fall identical to that seen at 15% of CF in adult animals (Fig. 2). Perfusion at 15% of CF severely decreased active tension at both ages, but more dramatically in senescent rats during the first 15 min of ischemia (Fig. 2). Perfusion at 30% of CF resulted in a mild and similar increase in resting tension in 4 and 24 month old hearts (Fig. 3). More severe reduction of coronary flow increased resting tension in both age groups, but this increase was more important in senescent hearts (Fig. 3).

3.3 Coronary resistance during ischemia
During 180 min of normoxic perfusion, coronary resistance was similar in 4 and 24 month old rats (Fig. 4). In 4 month old rats, reduction at 30% of CF induced only a mild and late increase in the coronary resistance, whereas the same reduction of CF induced an earlier and more important increase in 24 month old hearts (Fig. 4). Perfusion at 15% of CF increased the coronary resistance in both age groups but this increase occurred earlier and was more marked in senescent hearts (Fig. 4).


Figure 4
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  		Fig. 4 Effects of low-flow ischemia on coronary resistance in both 4 and 24 month old hearts. Coronary resistance was expressed in % of baseline value. See legend of Figure 2 for description of groups and for symbols of statistical significance.

 
3.4 mRNA quantification
Total RNA yields were similar in all experimental groups (Table 2). The quantity of mRNA measured by oligo d(T) hybridization (poly (A+)/18S ratio) was also similar in all groups, except in the 24 month old-15% CF group in which it was decreased (Table 2). After 180 min of normoxic perfusion, the SERCA2 mRNA level was lower in 24 than in 4 month old rats (Fig. 5Fig. 6) while NCx and RyR2 mRNA levels were not affected (Fig. 6).


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  		Table 2 Extraction yields of total RNAs and proteins, total mRNAs content and Myosin Heavy Chain protein content in adult and senescent rats during low-flow ischemia

 

Figure 5
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  		Fig. 5 Panel A: Northern blot analysis of Na+-Ca2+ exchanger (NCx) and SR Ca2+-ATPase (SERCA2) transcripts after 180 min of normoxic and ischemic perfusions in 4 and 24 month old rats. Total RNA (20 µg) from left ventricle of 4 and 24 month old rats (4 and 24 month old-100% CF, 4 and 24 month old-30% CF, 4 and 24 month old-15% CF groups) are sequentially hybridized with specific probes as described in Methods. Northern blot analysis of ryanodine receptor (RyR2) transcripts, not representative of RyR2 mRNA changes as indicated in Methods, is not shown. Panel B: ethidium bromide staining of the agarose gel.

 

Figure 6
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  		Fig. 6 Na+-Ca2+ exchanger (NCx), ryanodine receptor (RyR2) and SR Ca2+-ATPase (SERCA2) mRNA levels after 180 min of normoxic perfusion (4 month old-100% CF, and 24 month old-100% CF groups), or moderate (4 month old-30% CF, and 24 month old-30% CF groups) or severe (4 month old-15% CF, and 24 month old-15% CF groups) ischemic perfusion. See legend of Figure 2 for symbols of statistical significance.

 
Low-flow ischemia resulted in an increased accumulation of all three mRNA species, which was dependent on both age and degree of CF reduction. NCx, RyR2 and SERCA2 mRNA accumulations were observed only after severe ischemia (15% CF) in 4 month old rats (+35%, +81% and +26% vs. 4 month-100% CF group values, respectively) (Figs. 5 and 6Go). In contrast, a lesser degree of flow reduction (30% CF) was sufficient to increase NCx and SERCA2 mRNA levels in 24 month old rats (+46% and +36% vs. 24 month-100% CF group values, respectively), while RyR2 mRNA level remained unchanged as compared to 24 month-100% CF group (Fig. 6). More severe ischemia (15% CF) doubled the accumulation of the three mRNAs compared to the 100% CF group and these increases were significantly greater than those observed in adult rats (Fig. 6).

3.5 Protein quantification
The microsomal protein yield as well as the MHC protein content were similar in all experimental groups (Table 2). NCx, RyR2 and SERCA2 protein contents were significantly lower in the normoxic senescent myocardium (–36%, –39% and –32% in 24 vs. 4 month-100% CF groups, respectively) (Fig. 7Fig. 8). Low-flow ischemia did not modify NCx, RyR2 and SERCA2 protein contents in any of the groups (Figs. 7 and 8Go). No sign of protein degradation was observed in Western blots for the three calcium-regulating proteins studied, whatever the age of rats or the level of CF reduction (Fig. 7).


Figure 7
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  		Fig. 7 Western blot analysis showing ryanodine receptor (RyR2), Na+-Ca2+ exchanger (NCx) and SR Ca2+-ATPase (SERCA2) proteins after 180 min of normoxic and ischemic perfusions in 4 and 24 month old rats. Crude microsomal preparations (30 µg) from left ventricle of 4 and 24 month old rats (4 and 24 month old-100% CF, 4 and 24 month old-30% CF, 4 and 24 month old-15% CF experimental groups) were incubated with specific antibodies as described in Methods.

 

Figure 8
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  		Fig. 8 Na+-Ca2+ exchanger (NCx), ryanodine receptor (RyR2) and SR Ca2+-ATPase (SERCA2) protein normalized by myosin heavy chain protein levels after 180 min of normoxic perfusion (4 month old-100% CF, and 24 month old-100% CF groups), or moderate (4 month old-30% CF, and 24 month old-30% CF groups) or severe (4 month old-15% CF, and 24 month old-15% CF groups) ischemic perfusion.

*p<0.05 vs. 4 month old coronary perfusion-matched group.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The major findings of this study were the following: (1) low-flow ischemia results in a significant increase in mRNA species encoding SERCA2, NCx and RyR2 without any loss or degradation of the corresponding proteins, (2) in senescent hearts, the myocardial dysfunction and the increase in mRNAs coding for calcium-handling proteins occur at a lesser degree of CF reduction, indicating a higher vulnerability to low-flow ischemia during aging.

4.1 Gene expression during low-flow ischemia
In adult rats, no-flow ischemia activates several ‘immediate early genes’ coding for protooncogenes and for heat shock proteins [1–3]. More recent studies have reported that ischemia and/or reperfusion also activate or repress genes encoding constitutive proteins [5–7, 35, 36]. No-flow ischemia induces β1-adrenergic receptor mRNA accumulation [5], while coronary ligation decreases G{alpha}s and G{alpha}i protein mRNAs associated with a decrease in the corresponding proteins [35]. However, the effect of ischemia on the expression of calcium-regulating proteins is not known. SERCA2 mRNAs have been shown to increase in a model of reperfusion following brief coronary occlusion in young pigs [6], but alterations in NCx or RyR2 mRNAs are presently unknown whatever the model of ischemia. Our model of low-flow ischemia demonstrates that partial reduction of CF increases SERCA2, NCx and RyR2 transcripts in both 4 and 24 month old rats. SR Ca2+-ATPase mRNA accumulation probably results from an increased transcription of the SERCA2 gene [7]. The accumulation of RyR2 and NCx mRNAs could be due to activated transcription and/or changes in mRNA stability.

In ischemia as in other physiopathological states such as heart failure or aging, quantification of mRNA levels is difficult. The glyceraldehyde 3-phospho deshydrogenase (GAPDH) mRNA, often used to normalize the measurements [2, 6]is in fact increased during ischemia [7]. In order to document changes in specific transcripts among the total mRNA population, we normalized our results relative to the poly (A+) mRNAs. SERCA2, RyR2 and NCx mRNA levels were unchanged in the normoxic groups as compared to in vivo values [20–22], indicating the stability of mRNAs coding for calcium-regulating proteins during normoxic perfusion in both adult and aged myocardium. In senescent hearts submitted to severe reduction of coronary flow, total mRNA content decreased indicating a downregulation of total mRNA population and/or decreased stability, in contrast to other ischemic groups where total mRNA content remained unchanged. This specific accumulation of SERCA2, RyR2 and NCx transcripts during ischemia is likely to be related to mRNA upregulation and/or increased stability.

4.2 Triggers for mRNA upregulation?
It has been suggested that increased transcription of the SERCA2 gene could result from SR Ca2+-ATPase degradation, and could in turn act as a repair process [2, 6, 7]. Such a degradation has been documented for contractile and cytoskeletal proteins after total ischemia [13–15]. However in our model, low-flow ischemia does not result in SERCA2, RyR2 and NCx protein degradation, and this finding remains true whatever the age or the degree of CF reduction. The observed mRNA accumulations are not triggered by proteolysis and could not act as a repair process.

Several factors have been suspected to trigger gene activation during ischemia and/or reperfusion. Hormonal regulation [3]can be ruled out in our ex vivo conditions. Reperfusion is not needed since both low-flow ischemia, and no-flow ischemia without reperfusion [5, 35]induce mRNA changes. Ischemia per se must generate a signal responsible for the gene activation. Cytosolic calcium overload [6, 7]could be a transacting factor. Cytosolic calcium overload induces a marked and sustained increase in nuclear calcium concentration [37]which could act as a transcriptional signal. Nevertheless no functional calcium response element has been identified on SERCA2, NCx or RyR2 genes. Oxygen deprivation and/or oxygen free radical production may have a causal role. Indeed, hypoxia is known to activate the myocardial expression of heat shock proteins, heme oxygenase [3]and β1 adreno-receptor [38], and the overexpression of the heme oxygenase during ischemia-reperfusion is prevented by anti-oxidant enzymes [4].

4.3 Vulnerability of senescent myocardium to ischemia
In the senescent heart, a moderate CF reduction induces both important myocardial dysfunction and mRNA upregulation, which resemble the effects of severe ischemia in adult hearts. These results indicate a higher vulnerability of the senescent heart to low-flow ischemia as previously reported for no-flow ischemia [24, 25]. This reduced tolerance has several explanations.

Aging is associated with contractile dysfunction [17, 21, 22, 39]and alterations in coronary perfusion [40–42]. The coronary reserve is reduced [40]as a result of impaired vascular tone, altered endothelial function [41], fibrosis [29]and reduced coronary bed density [42]. Consequently, a moderate reduction of CF in the aged heart is sufficient to induce a marked impairment of coronary resistance and active tension.

Aging is also associated with several alterations in calcium homeostasis. A reduced threshold for calcium overload, which results in mechanical dysfunction and arrhythmias [23], has been observed in senescent hearts. We found that the 3 calcium-regulating proteins are decreased in the normoxic senescent heart in accordance with previous in vivo results [43, 44]. Consequently, both calcium extrusion and uptake are reduced [17–19], leading to a greater calcium overload during ischemia [24], which does not result from modifications in the buffering capacity of the ischemic heart [24, 45]. Moreover, in the senescent heart myocyte loss compensated by hypertrophy of the remaining myocytes, fibrosis and changes in membrane protein densities [17, 18]are associated with increased functional heterogeneity [23]. All these alterations probably account for the profound contractile impairment observed at a lower CF reduction in the senescent myocardium. They may also explain the increased expression of specific gene products.

As previously reported, the capacity of the aged myocardium to alter genetic expression in response to mechanical overload is preserved [21, 46–48]. Our study demonstrates that the capacity of gene response in senescent heart is also preserved during ischemic stress despite the age-related decrease of SERCA2 gene expression [20–22].

4.4 Myocardial ischemia as opposed to mechanical overload
The alterations in gene expression which occur during ischemia are nearly opposite to those observed during cardiac overload. Ischemia is associated with the upregulation of mRNAs coding for membrane proteins such as β1-adrenergic receptor and calcium-regulating proteins, which does not result from protein loss, even if sustained ischemia is associated with prolonged functional abnormalities of the Ca2+-handling system [8, 9, 11]. Whether the overexpression of these calcium-regulating proteins would be beneficial or detrimental in the recovery phase of ischemia or in case of subsequent episodes of ischemia-reperfusion is unknown at the moment. In contrast, cardiac overload is associated with a decrease in both protein densities and mRNA levels of ryanodine receptor and SR Ca2+-ATPase [27, 49, 50]. These modifications participate in a general process of adaptation and are clearly of different origin.

Obviously in our isolated heart model, accumulated mRNAs can not be translated in 3 h of perfusion. It is the reason why, to investigate proteins, some authors have used an in vivo model of ischemia [35]. However, such a model of coronary artery ligation is associated with cardiac overload [51]and does not allow to analyze separately the consequences of ischemia and of cardiac overload, which have opposite effects on gene expression. In certain clinical or experimental settings, ischemia may be associated with cardiac overload and could explain discrepancies frequently reported between clinical molecular biological investigations and experimental models.

In conclusion, low-flow ischemia induces SERCA2, NCx and RyR2 mRNA accumulations without alterations in the corresponding protein contents. The mechanism(s) of this calcium-regulating protein mRNA upregulation, which depends on the degree of coronary flow reduction, remains to be determined. During aging, both marked contractile impairment and mRNA upregulation occur for a lesser degree of CF reduction, indicating that the senescent myocardium is more vulnerable to low-flow ischemia, but has maintained its capacity to respond to ischemic stress.

Time for primary review 30 days.


   Acknowledgements
 
The authors thank Dr. Claude Sebban and Dr. Brigitte Decros for kindly providing senescent rats, Dr. Kenneth Boheler for the gift of the rat sodium-calcium exchanger cDNA, Pascal Trouvé for technical assistance. They wish to thank especially Dr. Alice Barrieux for helpful discussion and Prof. Kenneth D. Philipson for the kind gift of rat sodium-calcium exchanger antibody. This study was supported in part by grants from Fédération Française de Cardiologie, Fondation pour la Recherche Médicale and Société Française d'Hypertension Artérielle.


   Notes
 
1 Presented in part at the XVIIIth Congress of the European Society of Cardiology, Birmingham, UK, 1996 and at the 69th Scientific Sessions of the American Heart Association, New Orleans, USA, 1996. Back


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

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