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Circulation Research. 2004;95:e85-e95
Published online before print November 4, 2004, doi: 10.1161/01.RES.0000149531.02904.09
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(Circulation Research. 2004;95:e85.)
© 2004 American Heart Association, Inc.

UltraRapid Communication

Alterations in Myofilament Function Contribute to Left Ventricular Dysfunction in Pigs Early After Myocardial Infarction

J. van der Velden, D. Merkus, B.R. Klarenbeek, A.T. James, N.M. Boontje, D.H.W. Dekkers, G.J.M. Stienen, J.M.J. Lamers, D.J. Duncker

From the Laboratory for Physiology (J.v.d.V., B.R.K., A.T.J., N.M.B., G.J.M.S.), Institute for Cardiovascular Research, VU University Medical Center, Amsterdam; Experimental Cardiology, Thoraxcenter (D.M., D.J.D.) and Department of Biochemistry (D.H.W.D., J.M.J.L.), Cardiovascular Research School Cardiovasculaire Onderzoekschool Erasmus Universiteit Rotterdam, Erasmus Medical Center, University Medical Center Rotterdam, Rotterdam, the Netherlands.

Correspondence to J. van der Velden, PhD, Laboratory for Physiology, VU University Medical Center, Van der Boechorststraat 7, 1081 BT Amsterdam, the Netherlands. E-mail j.vandervelden{at}vumc.nl


*    Abstract
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 *Abstract
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 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
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Myocardial infarction (MI) initiates cardiac remodeling, depresses pump function, and predisposes to heart failure. This study was designed to identify early alterations in Ca2+ handling and myofilament proteins, which may contribute to contractile dysfunction and reduced ß-adrenergic responsiveness in postinfarct remodeled myocardium. Protein composition and contractile function of skinned cardiomyocytes were studied in remote, noninfarcted left ventricular (LV) subendocardium from pigs 3 weeks after MI caused by permanent left circumflex artery (LCx) ligation and in sham-operated pigs. LCx ligation induced a 19% increase in LV weight, a 69% increase in LV end-diastolic area, and a decrease in ejection fraction from 54±5% to 35±4% (all P<0.05), whereas cardiac responsiveness to exercise-induced increases in circulating noradrenaline levels was blunted. Endogenous protein kinase A (PKA) was significantly reduced in remote myocardium of MI animals, and a negative correlation (R=0.62; P<0.05) was found between cAMP levels and LV weight-to-body weight ratio. Furthermore, SERCA2a expression was 23% lower after MI compared with sham. Maximal isometric force generated by isolated skinned myocytes was significantly lower after MI than in sham (15.4±1.5 versus 19.2±0.9 kN/m2; P<0.05), which might be attributable to a small degree of troponin I (TnI) degradation observed in remodeled postinfarct myocardium. An increase in Ca2+ sensitivity of force (pCa50) was observed after MI compared with sham ({Delta}pCa50=0.17), which was abolished by incubating myocytes with exogenous PKA, indicating that the increased Ca2+ sensitivity resulted from reduced TnI phosphorylation. In conclusion, remodeling of noninfarcted pig myocardium is associated with decreased SERCA2a and myofilament function, which may contribute to depressed LV function. The full text of this article is available online at http://circres.ahajournals.org.


Key Words: infarction • left ventricular remodeling • cardiomyocyte function • Ca2+ handling


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
One of the principle risk factors for the development of heart failure in humans is myocardial infarction (MI). MI results in cardiac remodeling consisting of left ventricular (LV) dilation and hypertrophy. Although this remodeling is aimed to maintain pump function, global contractility of the remodeled heart is depressed.1,2 A number of factors, including changes in cardiac structure (LV dilation), apoptotic cell death, abnormal energy metabolism, and neurohumoral disturbances, have been implicated in the initiation and progression of heart failure.3,4 However, the contribution of depressed contractility and abnormal Ca2+ handling of individual cardiomyocytes in remote noninfarcted myocardium to global LV pump dysfunction early after MI is still incompletely understood.3–7 Because ischemic heart disease has become the most prevalent cause for heart failure, it is important to obtain further insight in the mechanisms that contribute to global LV dysfunction early after infarction.

Contractile function of cardiomyocytes is determined by the expression and phosphorylation status of Ca2+ handling and myofilament proteins. Moreover, activation of the sympathetic nervous system increases cardiomyocyte contractile function via ß-adrenergic receptor-stimulated phosphorylation of several target proteins, such as phospholamban and troponin I.8 Hence, alterations of steps within the ß-adrenergic signaling pathway during infarction-induced myocardial remodeling will also influence contractile performance of the heart. Until now, most studies focused on changes in Ca2+ handling and myofilament proteins involved in reversible contractile dysfunction during short-term ischemia and stunning.9–17 Only few studies addressed the initial protein changes in remote noninfarcted remodeled myocardium.18,19 Yet the functional consequences of protein changes during early myocardial remodeling on an ischemic insult remain to be elucidated.

In light of these considerations, the aims of the present study were to characterize in vivo the early impairment of LV ß-adrenergic responsiveness after MI and to identify underlying causative factors such as modifications in Ca2+ handling and myofilament proteins of remote noninfarcted remodeled myocardium. MI was produced by permanent ligation of the left circumflex coronary artery, which resulted in a 15% to 25% loss of viable LV myocardium. This intervention is associated with mild-to-moderate LV dysfunction.2,20 To reveal the functional consequences of myocardial remodeling in vivo, LV function was determined at rest and during treadmill exercise in pigs 3 weeks after MI or sham procedure, whereas functional properties of the contractile apparatus were studied by measuring maximal isometric force and its Ca2+ sensitivity in mechanically isolated single skinned cardiomyocytes. The latter method allows direct correlation between myofilament protein composition and force characteristics. Furthermore, key components of the ß-adrenergic signaling pathway and Ca2+ handling and myofilament (target) proteins were analyzed by 1D- and 2D-gel electrophoresis and Western immunoblotting in remote noninfarcted LV subendocardial tissue.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Induction of MI
Experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication 86 to 23, revised 1996), and with approval of the Animal Care Committee of the Erasmus Medical Center. Forty-five 2- to 3-month-old Yorkshire–Landrace pigs of either sex entered the study. Pigs were sedated (ketamine, 20 mg/kg intramuscularly [IM], and midazolam, 0.5 mg/kg IM), anesthetized (thiopental, 10 mg/kg intravenous [IV]), intubated, and ventilated with O2 and N2O, to which 0.1% to 1% (vol/vol) isoflurane was added.20,21 Anesthesia was maintained with midazolam (2 mg/kg followed by 1 mg/kg per hour IV) and fentanyl (10 µg/kg per hour IV). Under sterile conditions, the chest was opened via the fourth left intercostal space and a 4-cm incision was made in the pericardium at the site of the origin of the left circumflex coronary artery (LCx). Then, the LCx was dissected out and a suture was placed around it. The LCx was permanently ligated to produce MI (n=23), whereas in the animals from the sham group (n=22) the suture was removed. The chest was closed and the animals were allowed to recover, receiving analgesia (0.3 mg buprenorphine IM) for 2 days and antibiotic prophylaxis (25 mg/kg amoxicillin and 5 mg/kg gentamycin IV) for 5 days.20,21

Exercise Study
In chronically instrumented animals (12 sham and 11 MI), cardiac responsiveness to sympathetic stimulation was studied. LV function and circulating noradrenaline levels were determined in response to treadmill exercise 3 weeks after surgery.2

Surgical Instrumentation
Pigs were sedated (ketamine, 20 mg/kg IM), anesthetized (thiopental, 10 mg/kg IV), intubated, and ventilated with O2 and N2O, to which 0.2% to 1% (vol/vol) isoflurane was added. Anesthesia was maintained with midazolam (2 mg/kg+1 mg/kg per hour IV) and fentanyl (10 µg/kg per hour IV). Under sterile conditions, the chest was opened via the fourth left intercostal space and a fluid-filled polyvinylchloride catheter was inserted into the aortic arch for mean aortic pressure measurement (Combitrans pressure transducers; Braun) and blood sampling for determination of circulating norepinephrine levels. An electromagnetic flow probe (14 to 15 mm; Skalar) was positioned around the ascending aorta for measurement of cardiac output. A microtipped pressure transducer (P4.5; Konigsberg Instruments) was inserted into the LV via the apex, and a polyvinylchloride catheter was also inserted into the LV to calibrate the Konigsberg transducer LV pressure signal. Catheters were tunneled to the back and animals were allowed to recover, receiving analgesia (0.3 mg buprenorphine IM) for 2 days and antibiotic prophylaxis (25 mg/kg amoxicillin and 5 mg/kg gentamycin IV) for 5 days.2,21

Exercise Protocol
After baseline measurements were obtained, a two-stage treadmill exercise protocol was begun (2 and 4 km/h); hemodynamic data and blood samples were collected during the last 30 seconds of each 3 minutes exercise stage.2,21

Echocardiographic Recordings
Three weeks after surgery, 10 sham and 12 MI pigs were sedated with ketamine and midazolam, and 2D echocardiographic recordings of the LV short axis at mid papillary level were obtained and stored for offline analysis. Immediately after echocardiography, pigs were anesthetized with pentobarbital (20 mg/kg IV) and prepared for monitoring of heart rate, cardiac output, mean aortic and pulmonary artery blood pressures, and LV pressure, its first derivative LVdP/dt, and the time constant of exponential LV pressure decay ({tau})20 Subsequently, hearts were arrested and immediately excised. Subendocardial samples were obtained from remote noninfarcted myocardium of the LV anterior free wall, immediately frozen in liquid nitrogen, and stored at –80°C until analysis.

Force Measurements in Single Skinned Cardiomyocytes
Cardiomyocytes were mechanically isolated from subendocardial LV samples (9 sham and 10 MI) and mounted in the experimental set-up as described previously.22,23 Before mechanical isolation, tissue was defrosted in cold relaxing solution (pH 7.0; in mmol/L: free Mg2+, 1; KCl, 145; EGTA, 2; ATP, 4; imidazole, 10). During the isolation, the tissue was kept on ice. Myocytes were permeabilized in relaxing solution containing 1% Triton X-100 (5 minutes) to remove soluble and membrane-bound kinases and phosphatases, which may alter the phosphorylation status of myofibrillar proteins. To remove Triton, cells were washed twice in relaxing solution. Thereafter, a single myocyte was attached between a force transducer and a piezoelectric motor. Isometric force measurements were performed at different [Ca2+], at 15°C, and a sarcomere length, measured in relaxing solution, of 2.2 µm. The diameters of the cardiomyocyte were measured microscopically, in two perpendicular directions. Cross-sectional area was calculated assuming an elliptical cross-section. The composition of relaxing and activating solutions used during force measurements was calculated as described previously.22 The pCa, ie, –log10[Ca2+], of the relaxing and activating solution (pH 7.1) were, respectively, 9 and 4.5. Solutions with intermediate free [Ca2+] were obtained by mixing of the activating and relaxing solutions. After the first control activation at saturating (maximal) [Ca2+] (pCa=4.5), resting sarcomere length was readjusted to 2.2 µm, if necessary. The second control measurement was used to calculate maximal isometric tension (ie, force divided by cross-sectional area). The next force measurements were performed at submaximal [Ca2+], followed by a control measurement. Force values obtained in solutions with submaximal [Ca2+] were normalized to the interpolated control values. Cardiomyocytes were stored on ice to prevent that myofilament-bound kinases and phosphatases alter Ca2+ sensitivity (pCa50) of force during the measurements. Comparing pCa50 values of cardiomyocytes isolated from the same heart, measured at the start and the end of the day, yielded a nonsignificant difference (0.03±0.02 pCa units; n=19). Therefore, we conclude that the residual activity of myofilament-bound kinases/phosphatases does not interfere with the measurements.

After the first force–pCa series, myocytes from sham and MI hearts were incubated in relaxing solution containing the exogenous catalytic subunit of protein kinase A (3 µg/mL [100 U/mL]; Sigma, batch 35H9522) or the exogenous catalytic domain of protein kinase C (PKC) (from rat brain; 0.25 U/mL; Sigma, batch 093K0330) and 6 mmol/L dithiothreitol for 40 minutes at 20°C, after which a second force–pCa series was obtained. The mean parameters of the measurements before protein kinase A (PKA) or PKC treatment were consistent with the observations in the entire sham and MI group studied.

Analysis of ß-adrenergic Pathway Components and Ca2+ Handling Proteins
cAMP
Approximately 20 to 30 mg of frozen anterior endocardial tissue and 0.1 mL frozen 0.1 mol/L HCl was homogenized in liquid nitrogen with a microdismembrator unit (B. Braun Biotech International) at 1700 rpm for 4 minutes in a Teflon vial with a Teflon sphere. Cold 0.1 mol/L HCl was added to 40 tissue volumes and the sample was sonicated for 30 seconds on ice. An aliquot was removed for protein determination using the RCDC protein assay (Bio-Rad Laboratories). The remaining volume was centrifuged for 15 minutes at 9700g at 4°C and the supernatant was stored at –80°C. cAMP concentration was measured using a low pH immunoassay kit (R&D Systems).

PKA
Approximately 20 to 30 mg of frozen tissue was homogenized in a microdismembrator unit as described. Powder was collected in 20 volumes of cold 25 mmol/L Tris/HCl pH 7.4 supplemented with 10 mmol/L 2-mercapto-ethanol, 100 µmol/L IBMX, and protease inhibitor mix Complete (Roche Diagnostics). After sonication for 30 seconds on ice, the sample was centrifuged for 15 minutes at 9700g at 4°C. An aliquot of the supernatant was removed for protein determination. The supernatant was stored at –80°C. Total protein kinase A was measured by means of a nonradioactive protein kinase assay kit (PepTag; Promega) using a saturating level of cAMP in the reaction mixture. The reaction mixture was supplemented with 60 µmol/L dithiothreitol, 1 mmol/L NaF, and 75 µmol/L Cantharidin and was added to 5 µg protein. Because cAMP activates all PKA present in the sample, the signal obtained represents maximal PKA activity, ie, catalytic subunit protein. PKA values were expressed in µg/mg protein using the catalytic subunit of protein kinase A (bovine heart) as a standard.

SERCA2a and Phospholamban
Approximately 40 mg of frozen tissue was homogenized in liquid nitrogen in a microdismembrator unit as described. The frozen powder was resuspended in 20 volumes of cold Laemmli loading buffer. An aliquot was removed, centrifuged briefly, and frozen immediately for later analysis of phospholamban. The remaining volume was heated for 5 minutes at 95°C, cooled on ice, and centrifuged briefly. Aliquots of the supernatants were removed for protein determination. Samples were frozen in liquid nitrogen and stored at –80°C.

Proteins were separated by 1D-SDS polyacrylamide gel electrophoresis (1D-PAGE), using 7.5% to 15% gradient gels.24 Samples for SERCA2a analysis were reheated for 5 minutes at 95°° and 25 µg of protein was applied. Proteins were blotted overnight at 40V onto polyvinylidene difluoride membranes (Immunblot; Bio-Rad). Blots were pre-incubated in Tris-buffered saline with Tween (TTBS)-buffer (10 mmol/L Tris-HCl pH 7.6, 75 mmol/L NaCl, 0.1% Tween) supplemented with 0.5% nonfat milk powder for 1 hour at room temperature and incubated overnight at 4°C with primary antibodies against sarcoplasmic reticulum Ca2+-ATPase 2a (SERCA2a, mouse monoclonal SA-209; BIOMOL Research Laboratories), phospholamban (Plb, mouse monoclonal, Affinity Bioreagents), or serine-phosphorylated phospholamban (Plb-ser-P, rabbit polyclonal; Cyclacel). After washing with TTBS, blots were probed with 125I-labeled secondary antibody in TTBS supplemented with 0.5% milk powder for 3 hours at room temperature [anti-rabbit Ig, F(ab[prime])2 fragment from donkey or anti-mouse Ig, F(ab[prime])2 fragment from sheep; Amersham Pharmacia Biotech]. Blots were washed with TTBS and radioactivity was quantified using a phospho-imaging system (Molecular Imager GS-363; Bio-Rad).

Analysis of Myofilament Proteins
Troponin I (TnI), desmin, and myosin heavy chain were analyzed using 1D-PAGE and Western immunoblotting, as described previously.22,25 The separating gel contained 12% total acrylamide (ratio of acrylamide to bisacrylamide, 200:1; pH 9.3), whereas the stacking gel contained 3.5% total acrylamide (ratio of acrylamide to bisacrylamide, 20:1; pH 6.8). Gels were stained with silver. After electrophoresis, proteins were transferred onto a nitrocellulose membrane. Blots were incubated with primary mouse monoclonal antibodies against desmin (DE-U-10; Sigma) and TnI (1691; Chemicon) and stained using the Vectastain ABC-AmP Immunodetection Kit (Vector Laboratories, Burlingame, Calif).

TnI phosphorylation was analyzed using phospho-specific antibodies; 15% mini 1D-PAGE was performed and proteins were transferred to Hybond-ECL nitrocellulose membranes. Blots were incubated with primary mouse monoclonal antibodies against TnI (8I-7; Spectral Diagnostics Inc), total (phosphorylated and dephosphorylated) TnI (clone 16A11; Research Diagnostics Inc), and phosphorylated TnI (specific for phosphorylated PKA sites serine 22 and 23; clone 5E6; Research Diagnostics Inc), and signals were visualized using a secondary horseradish peroxidase-labeled goat-anti-mouse antibody and enhanced chemiluminescence (ECL plus Western blotting detection; Amersham Biosciences). Basal phosphorylation status of TnI was expressed as the intensity ratio of phosphorylated to total TnI by dividing the signal obtained with antiphosphorylated TnI antibody (5E6) by the signal obtained with total TnI (16A11).

Expression of myosin light chain 1 (MLC-1) and 2 (MLC-2) and troponin T (TnT) was analyzed by 2D-PAGE.25 The phosphorylation status of MLC-2 and TnT could also be determined on these gels. Tissue was treated with trichloroacetic acid to preserve the phosphorylation status of myofilament proteins. Samples (600 µg dry weight) were loaded on immobiline strips with a pH gradient of 4.5 to 5.5 (Amersham Pharmacia Biotech, Uppsala, Sweden). In the second dimension, proteins were separated by 1D-PAGE as described. Gels were stained with Coomassie blue, scanned, and analyzed using Image Quant (Molecular Dynamics). MLC-1 and TnT isoforms were identified using primary mouse monoclonal antibodies in Western immunoblotting: F109:16A12 (Alexis) for MLC-1, and JLT-12 (Sigma) was used for TnT.

Data Analysis
Digital recording and offline analysis of hemodynamic data have been described previously.21 Force–pCa relations were fit to the Hill equation:22

F (Ca2+)/F0=[Ca2+]nH/(Ca50nH+[Ca2+]nH), where F is steady-state force, F0 denotes the steady force at saturating [Ca2+], nH reflects the steepness of the relationship, and Ca50 (or pCa50) represents the midpoint of the relation.

Values are given as means±SEM. The cardiomyocyte force values per heart were averaged to obtain individual values for sham and MI pigs. The mean values for sham and MI pigs were compared with unpaired Student t tests. Paired Student t tests were used when comparing force values of single cardiomyocytes before and after PKA/PKC treatment. Analysis of covariance for repeated measures was used to test for statistically significant differences in relations between hemodynamic variables and noradrenaline (with noradrenaline as covariate) in MI versus sham. Linear regression analysis was performed using Prism (version 3.00). Significance was accepted when P<0.05.


*    Results
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 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
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Global LV Response to Exercise
Myocardial responsiveness to exercise-induced increases in noradrenaline was blunted in MI compared with sham. Moreover, despite elevated exercise-induced increases in circulating levels of noradrenaline, MI pigs were unable to reach the same levels of cardiac output, global LV contractility, and relaxation that were achieved at the highest level of exercise (4 km/h) in sham animals (Figure 1).



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  		Figure 1. Response of hemodynamic variables to endogenous changes in noradrenaline induced by exercise in pigs with (black circles) and without (white circles) MI. Data are shown in each group for resting conditions and during exercise at 2 and 4 km/h. *P<0.05 MI versus sham (by analysis of covariance).

Myocardial Remodeling
After infarction, the surviving LV myocardium hypertrophied, as reflected in the 19% increases in LV weight and LVW/BW ratio (P<0.05; Table 1). MI induced substantial LV dilation, represented by a 69% increase in end-diastolic area (EDA) and a 158% increase in end-systolic area (ESA; both P<0.05). Ejection fraction, calculated as (EDA-ESA)/EDAx100%, was decreased in MI (35±4%) compared with sham (54±5%) animals (P<0.05).


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  		Table 1. Anatomical and Hemodynamical Data

Hemodynamics
Hemodynamic characteristics measured during anesthesia are shown in Table 1. Global LV dysfunction was evidenced by a lower LVdP/dtmax and LVdP/dtmin and a decrease in the less load-dependent LVdP/dt at 40 mm Hg (LVdP/dtP40) in MI compared with sham, whereas the time constant of exponential pressure decay ({tau}) was significantly increased. LV end-diastolic pressure and mean pulmonary artery pressure were higher in MI than in sham.

Force Development in Single Skinned Cardiomyocytes
Force measurements were performed in 31 isolated skinned myocytes from 9 sham hearts and in 32 cells (Figure 2A) from 10 MI hearts. Mean length of the cardiomyocytes between the attachments was 66±4 and 53±2 µm for sham and MI, respectively. The mean width and depth (in µm) amounted to 23±1 and 19±1 in sham and to 24±1 and 21±1 in MI cardiomyocytes. As an example, Figure 2B shows recordings of force development obtained at saturating (pCa 4.5) and at submaximal [Ca2+] (pCa 5.4). Maximal isometric tension (Fmax) was significantly higher in sham (19.2±0.9 kN/m2) compared with MI (15.4±1.5 kN/m2), whereas passive force was similar in both groups (1.9±0.4 and 2.6±0.7 kN/m2 in sham and MI, respectively). Moreover, a highly significant inverse correlation was found between Fmax and LVW/BW (Figure 3A; R=0.78; P<0.001).



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  		Figure 2. A, Single cardiomyocyte from a remodeled heart in relaxing solution glued between a force transducer and a piezoelectric motor. B, Recordings of isometric force development in a cardiomyocyte from a remodeled heart before and after exogenous PKA treatment during maximal (pCa 4.5) and submaximal activation (pCa 5.4). The myocyte was slackened rapidly when force development reached a steady level to determine the baseline level of the force transducer (zero level not visible at sampling rate shown in Figure).



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  		Figure 3. A, Fmax was significantly lower in remodeled (MI) than in sham hearts. A negative correlation was present between Fmax and LVW/BW: Fmax (in kN/m2)=37.3±4.1 to 6.5±1.3 x LVW/BW (in g/kg). B, Force–pCa curves before PKA treatment. Force was divided by cross-sectional area of the cardiomyocytes and expressed in kN/m2. Myocyte values per sham/MI animal were averaged to obtain individual values for sham (n=9) and MI (n=10) hearts. Ca2+ sensitivity was significantly higher in MI compared with sham. C, Normalized force–pCa curves before and after PKA treatment. Force at submaximal [Ca2+] was normalized to the value obtained at maximal [Ca2+], which was set to 1. PKA abolished the difference in Ca2+ sensitivity between sham and remodeled myocardium. D, PKC did not alter Fmax in sham and MI cardiomyocytes.

Ca2+ sensitivity of force was significantly increased in MI (pCa50=5.67±0.04) compared with sham (pCa50=5.50±0.03) animals, whereas the steepness of the force–pCa curves, nH, did not differ between MI (2.55±0.11) and sham (2.95±0.18). pCa50 significantly increased with increasing LVW/BW (R=0.49; P<0.05). The average force–pCa curves shown in Figure 3B indicate that force development in remodeled myocardium is higher than in sham hearts at relatively low [Ca2+] (pCa>5.4), but that force is lower at higher [Ca2+].

Endogenous cAMP and PKA Levels and the Effect of Exogenous PKA on Myofilament Function
The increased Ca2+ sensitivity after MI might be attributable to a reduction of the ß-adrenergically PKA-mediated phosphorylation of troponin I. As shown in Figure 4A, both endogenous cAMP and PKA levels were lower after MI compared with sham (respectively, P=0.07 and P=0.04), and both cAMP (R=0.62; P<0.05) and PKA (R=0.46; P=0.12) inversely correlated with LVW/BW.



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  		Figure 4. A, Both cAMP and total PKA were slightly lower in remodeled (MI) than in sham myocardium (respectively, P=0.07 and P=0.04). B, Western immunoblot analysis of proteins involved in Ca2+ handling revealed a significant decrease in SERCA2a in remodeled (MI) myocardium, whereas expression and phosphorylation of phospholamban was unaltered compared with sham hearts.

To investigate if phosphorylation of TnI by PKA completely or partially reverses the difference in Ca2+ sensitivity between sham and MI, 7 myocytes from 4 sham hearts and 10 myocytes from 5 remodeled hearts were treated with exogenous PKA, which is known to effectively phosphorylate TnI.26 Figure 2B illustrates force registrations obtained in a MI myocyte before and after PKA treatment at maximal and submaximal activation. A minor decrease in maximal force development was observed after PKA, whereas at submaximal [Ca2+] (pCa 5.4) force was markedly reduced. This decrease in force at submaximal [Ca2+] reflects a decreased Ca2+-sensitivity of the contractile apparatus after PKA in myocytes from remodeled myocardium ({Delta}pCa50=0.22±0.03; P<0.05). In cardiomyocytes from sham hearts, PKA treatment caused a smaller shift in pCa50 ({Delta}pCa50=0.12±0.04; P<0.05). Figure 3C illustrates that PKA-mediated phosphorylation of TnI abolished the difference in Ca2+ sensitivity between sham (pCa50=5.40±0.02) and MI (pCa50=5.41±0.01) myocardium. The steepness of the force–pCa curves was not affected in both groups.

Effect of Exogenous PKC on Myofilament Function
The decreased Fmax in remodeled hearts might be attributable to increased protein kinase C activity.27–29 In that case, PKC-mediated phosphorylation would decrease maximal tension in sham myocardium and abolish the difference in Fmax between sham and MI hearts. Exogenous treatment of 6 myocytes from 4 sham hearts with the catalytic domain of PKC did not significantly alter Fmax (respectively, 23.4±3.3 and 24.7±3.9 kN/m2 before and after PKC). In addition, in 3 myocytes from 2 remodeled hearts, Fmax before (15.5±2.9 kN/m2) did not differ from Fmax after PKC treatment (15.9±4.1 kN/m2; Figure 3D). PKC caused a decrease in Ca2+ sensitivity both in sham ({Delta}pCa50=0.12±0.04) and in remodeled ({Delta}pCa50=0.18±0.08) myocytes and reduced the difference in Ca2+-sensitivity between sham and MI hearts.

Ca2+ Handling Proteins
Analysis of the two main proteins involved in Ca2+ uptake by the SR, SERCA2a, and phospholamban (Plb) indicated a 23% decrease in SERCA2a expression in remodeled myocardium compared with sham, whereas no significant difference was found in Plb (Figure 4B). However, the ratio of Plb over SERCA2a did not significantly differ between MI (0.89±0.15) and sham (0.75±0.09) animals. No difference in Plb phosphorylation (Ser,16 phosphorylation site for PKA) was observed (Figure 4B). SERCA2a expression did not correlate with LVW/BW (R=0.22; P=0.54).

Myofilament Protein Composition
Ischemia and ischemia/reperfusion (stunning) are known to induce proteolysis of myofilament proteins (TnI, TnT, MLC-1, and desmin), as well as alterations in phosphorylation status (TnI, TnT).9,14,15,30 To detect degradation of contractile proteins, protein composition was analyzed by 1D-PAGE in 5 sham and 6 MI hearts. Figure 5A illustrates a silver-stained gel of remodeled and sham myocardium. Densitometric analysis did not reveal differences in the composition of contractile proteins between sham and MI pigs. Western immunoblotting confirmed that desmin was not degraded in the remote myocardium of postinfarct hearts (Figure 5B). In addition, no degradation of TnI was found when using antibody clone 1691 (Figure 5B), whereas minute amounts (<4%; as percentage of total TnI) of a TnI degradation product were found in remodeled myocardium when using clone 8I-7 (Figure 5C), directed against the central region of TnI.31 The assay was validated by using a sham sample (sample C, Figure 5C), which was left for 1 hour at 37°C to induce TnI degradation (17% of total TnI).



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  		Figure 5. A, Silver-stained gel of contractile proteins in a remodeled (lane 1) and sham (lane 2) sample, preceded by a molecular weight marker. Protein bands include: ß-MHC, ß-myosin heavy chain; MyBP-C, myosin binding protein C; TnT, troponin T; TM, tropomyosin; TnI, troponin I; MLC-1 and MLC-2, myosin light chain 1 and 2. B, Immunodetection of desmin and TnI in a remodeled (lane 1) and sham (lane 2) sample. C, Western blot demonstrating the presence of a TnI degradation product (arrow) in remodeled myocardium (MI). A sham sample, left for 1 hour at 37°C to induce TnI degradation, served as positive control (C). S, sham samples. D, Cardiomyocytes were mechanically isolated from sham tissue samples left at 20°C for 10, 30, 60, or 120 minutes. Linear regression analysis results in the following equation: Fmax=18.2±1.4 to 1.8±0.3 x TnI breakdown; R=0.97. E, 2D-gel (IEF, isoelectric focusing) to illustrate MLC-1, MLC-2, and TnT composition in myocardium from a MI pig. TnT may be mono- (P) and bisphosphorylated (bisP); MLC-1, unphosphorylated (1) and monophosphorylated (1P); MLC-2 is composed of two isoforms (2 and 2*), which are both partly phosphorylated (2P and 2*P, respectively). F, TnT (spots 1 to 3) isoforms were visualized with specific antibodies in Western immunoblotting. G, Western blot illustrating decreased TnI phosphorylation in remodeled compared with sham myocardium. TnIP, phosphorylated TnI.

To study the effect of TnI degradation on maximal force, sham tissue samples were kept at 20°C for 10, 30, 60, or 120 minutes. Thereafter, one part of each sample was used to measure Fmax in isolated single cardiomyocytes (n=2 per sample), and another part was used to determine TnI degradation by Western immunoblotting. TnI degradation increased with time up to {approx}8% (as percentage of total TnI). Fmax gradually decreased to 20% of Fmax in the untreated sample after 120 minutes (Figure 5D). Ca2+ sensitivity of force increased with time by 0.14 pCa units after 60 minutes. After 120 minute, force development at low [Ca2+] could not be determined accurately because of the low signal-to-noise ratio.

Expression of MLC-1, MLC-2, and TnT was further analyzed in 9 sham and 12 MI samples using Coomassie-stained 2D-gels (Figure 5E). MLC-2 consists of two isoforms (2 and 2*), which are both phosphorylated (2P and 2*P). No differences were present in MLC-2 phosphorylation between sham and MI (Table 2). Recently, Arrell et al32 have shown that MLC-1 may be phosphorylated, as well. In our 2D-gels, two spots were evident at the level of MLC-1, which probably represent the unphosphorylated and phosphorylated forms of MLC-1. The ratio of phosphorylated MLC-1 to total MLC-1 did not differ between sham and remodeled myocardium (Table 2). To assess possible MLC-1 degradation, the ratio of total MLC-1 to total MLC-2 content was analyzed. No significant difference was found in this ratio between sham and MI (Table 2).


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  		Table 2. Densitometric Analysis of 2D-Gels

Immunodetection of TnT indicated three TnT isoforms with different molecular weights (Figure 5F, numbers 1 to 3). Mono- and bisphosphorylation of TnT1 was visible on the 2D-gels (Figure 5E, P and bisP). The amount of mono- and bisphosphorylated TnT1, expressed as a percentage of total TnT1, did not differ between sham and MI pigs (Table 2). The quantities of TnT2 and TnT3 were small in comparison with TnT1 and could not be quantified reliably.

Basal phosphorylation of TnI was determined using phospho-specific antibodies in Western immunoblotting. In Figure 5G, blot signals are shown for 2 sham and 2 MI samples using an antibody against phosphorylated TnI. The ratio of the phospho-TnI to the total (dephospho and phospho) TnI signal was higher, although not significantly, in sham (1.32±0.25; n=4) than in MI (0.84±0.15; n=5; P=0.13) samples, indicating a higher basal TnI phosphorylation status in sham than in remodeled myocardium.


*    Discussion
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Permanent ligation of the LCx induced myocardial remodeling, characterized by LV dilation and hypertrophy. In addition, LCx ligation produced LV dysfunction characterized by a substantial decrease in EF, and decrements in indices of global LV contractility (LVdP/dtmax, LVdP/dtP40) and relaxation (LVdP/dtmin and {tau}) as compared with sham hearts. Our data also demonstrate that remodeling of noninfarcted myocardium is associated with altered myofilament function and a decrease in SERCA2a expression. These changes are consistent with the concept of a reduced cardiomyocyte contractility that contributes to LV dysfunction and impaired global LV contractile reserve during exercise in post-MI remodeled pig hearts.

Altered Signaling
Activation of the sympathetic nervous system enhances pump function via ß-adrenergic receptor-mediated phosphorylation of both Ca2+ handling and contractile proteins. After MI, the ß-adrenergic responsiveness to noradrenaline during treadmill exercise was blunted (Figure 1), consistent with downregulation, uncoupling, and/or downstream signaling defects of the ß-adrenergic receptors. The reduction in ß-adrenergic responsiveness was also evident from decreased levels of endogenous cAMP and PKA in remodeled myocardium compared with sham hearts (Figure 4). The subendocardial LV samples analyzed were obtained from anesthetized pigs, in which noradrenaline levels are comparable to resting values ({approx}50 pg/mL).33 Based on the in vivo measurements of global LV contractility (Figure 1), we presume that the small difference in cAMP level at rest between sham and MI pigs would be enhanced during exercise when noradrenaline levels are elevated.

As a result of ß-adrenergic desensitization, the basal phosphorylation status of Ca2+ handling (eg, phospholamban) and contractile proteins (eg, troponin I) was expected to be reduced, even in the resting state. TnI phosphorylation was lower in remodeled compared with sham myocardium (Figure 5G); however, the phosphorylation level of Plb did not differ. Moreover, exogenous PKA abolished the difference in Ca2+ sensitivity between sham and MI pigs (Figure 3C). This indicates that PKA-mediated phosphorylation of TnI is decreased in remodeled myocardium. The disparity between Plb and TnI phosphorylation may result from compartmentalization of ß-adrenergic signaling involving localized signal transduction.34 Fink et al35 demonstrated that on ß-adrenergic stimulation A-kinase anchoring protein targets PKA to contractile proteins, but not to Plb. Uncoupling of the complex between PKA and A-kinase anchoring protein in remodeled myocardium may explain reduced TnI phosphorylation at a time when Plb phosphorylation is still preserved.

Other signaling pathways may also be involved. Myocardial hypertrophy after MI is thought to result, at least in part, from angiotensin II-induced stimulation of phospholipase C, leading to activation of protein kinase C.20,36 Previously it was shown that angiotensin-converting enzyme inhibition and/or angiotensin II type 1 receptor blockade prevent cardiac remodeling in pigs,20 suggesting the involvement of PKC activation. Increased PKC activity was observed also in rat hearts within 1 to 8 weeks after MI.37 Apart from its role in mediating myocardial hypertrophic growth, PKC may alter cardiomyocyte function via phosphorylation of proteins involved in Ca2+ handling38 and myofilament contraction.39 The direction of the effect of PKC on myofilament Ca2+ sensitivity is still a matter of debate.27 In our study, the catalytic domain of PKC decreased Ca2+ sensitivity in cardiomyocytes from both sham and remodeled hearts. This indicates that increased PKC activity in remodeled hearts would partially counteract the effect on Ca2+ sensitivity of decreased PKA activity.

Apart from TnI, myosin-binding protein C40 and titin41 may be phosphorylated by PKA. We did not analyze the phosphorylation status of these proteins. Hence, we cannot exclude the involvement of myosin-binding protein C and/or titin phosphorylation in the alteration in Ca2+ sensitivity. In addition, the antagonist of kinase activity, phosphatase activity, also could explain a change in myofilament Ca2+ sensitivity via dephosphorylation of contractile proteins. However, basal phosphorylation status of both myosin light chains and TnT did not differ between sham and remodeled hearts. Overall, our data indicate that the increased Ca2+ sensitivity of force in remodeled myocardium is most likely the result of a decreased PKA-mediated phosphorylation of TnI.

Reduced Force-Generating Capacity
Changes in Ca2+ handling proteins alter cardiomyocyte contractile function and may exacerbate pump dysfunction. In the remodeled pig myocardium, a significant decrease was found in the amount of SERCA2a (Figure 4B), which is essential for removal of Ca2+ from the cytosol to facilitate relaxation and accumulation of Ca2+ in the SR for the next contractions. The decreased SERCA2a expression observed is consistent with the observed diastolic (dP/dtmin, {tau}) and systolic (dP/dtmax, dP/dtP40) dysfunction of remodeled myocardium early after infarction. However, future studies that include measurement of the rate of Ca2+ in isolated sarcoplasmic reticulum or myocyte Ca2+ transients are needed to substantiate this contention.

In addition, possible targets within the contractile apparatus should be considered.9,14,15,30,42 The effects of short-term ischemia and stunning have been studied extensively in rodents, in which depressed myocardial function has been ascribed to degradation of myofilament proteins (TnI, TnT, MLC-1, desmin).9,14,15,30 However, in stunned pig myocardium no degradation of TnI was found,16,17 whereas Ca2+ handling was impaired,17 indicating different mechanisms for stunning in rodents and large mammals. In contrast to the studies on stunned pig myocardium16,17 in which acute effects of an ischemic insult were studied, we did find a small amount of TnI proteolysis in remodeled pig myocardium 3 weeks after MI (Figure 5C). Recently, it has been proposed that apart from acute protein loss during an ischemic insult, chronic depletion of contractile proteins, in particular TnI, from viable cells may impair contractility of remodeled myocardium leading to heart failure.18,43 Ricchiuti et al18 reported decreased TnI and TnT expression levels 2 months after MI in remodeled pig myocardium remote from the infarct zone. TnI degradation may have been caused by prolonged activation of calpain-1 because of impaired Ca2+ homeostasis (increased diastolic [Ca2+]) or increased preload.44 Moreover, phosphorylation of TnI has been shown to protect TnI from degradation.31,45 Therefore, the decreased TnI phosphorylation observed in remodeled myocardium (Figure 5G) might render TnI more susceptible to degradation.

Montgomery et al29 found that the decrease in maximal tension in mouse myocardium on endogenous PKC activation was absent in transgenic mouse hearts expressing fast skeletal TnT, indicating a pivotal role for TnT in PKC-mediated depression of maximal force. This observation was confirmed by Sumandea et al, who identified threonine 206 as a functionally critical PKC phosphorylation site within TnT.46 Recently,Pi et al47 reported a decrease in maximum MgATPase activity rate on endogenous PKC activation in transgenic mice, without alterations in maximal force or phosphorylation of TnT. In our experiments, cardiomyocytes were treated with the catalytic domain of PKC. It should be mentioned that the catalytic domain of PKC may differ from PKC isoforms present in the heart with regard to its target specificity. However, in human myocardial tissue this catalytic domain was able to phosphorylate both TnI and TnT, whereas phosphorylation status of MLC-1 and MLC-2 remained unaltered (unpublished data). Hence, the PKC catalytic domain is able to phosphorylate the two main myofilament proteins, which are most likely involved in the reduction of Fmax on PKC activation, as found by Montgomery et al29 and Sumandea et al46 Because treatment of cardiomyocytes with the catalytic domain of PKC did not alter Fmax, whereas TnT phosphorylation status did not differ between sham and remodeled myocardium, we consider it unlikely that PKC-mediated phosphorylation would be the cause of the decreased Fmax of remodeled myocardium.

Recent studies in transgenic mice expressing <20% of the major TnI degradation product (TnI1–193) revealed {approx}50% reduction in maximal force development of isolated trabeculae compared with nontransgenic mice.48,49 This indicates that even a small degree of TnI degradation, as observed in this study, may have a considerable impact on force development. Based on linear regression analysis of the data presented in Figure 5D, 2% TnI degradation would be sufficient to decrease Fmax by 20%, as observed in cardiomyocytes from remodeled pig hearts. Hence, the decrease in Fmax with increasing LV weight (Figure 3A) might be indicative for a loss of myofibrillar proteins from viable cells during ongoing remodeling of the ventricle.

Implications of the Findings
The decrease in Fmax will contribute to reduced myocardial pump function. However, in vivo the normal heart muscle works at submaximal [Ca2+], in which cardiac performance is determined both by Fmax and Ca2+ sensitivity of the myofilaments. In contrast to decreased Fmax, an increase was observed in myofilament Ca2+ sensitivity in MI versus sham animals. The combined effect of decreased Fmax and increased Ca2+ sensitivity, shown in Figure 3B, illustrates that force development by the contractile apparatus is reduced at relatively high [Ca2+], but is increased at low [Ca2+] in remodeled compared with sham hearts. This implies that impaired myofilament function in remote noninfarcted myocardium may contribute to both systolic and diastolic dysfunction of remodeled myocardium, in particular at high levels of exercise (Figure 1).

Previous studies of postinfarct remodeled rat hearts found neither impaired cardiomyocyte function5 nor blunted ß-adrenergic responsiveness6 in remote noninfarcted LV, which is in contrast to our observations in remodeled pig myocardium. The different observations may well be explained by species differences as discussed. The decrease in contractile function within the rat model of MI5,6 was ascribed to changes in LV geometry attributable to apoptosis and alterations in the extracellular matrix. The present study shows that in large mammals alterations at the cellular level in remote noninfarcted myocardium contribute to depressed global LV pump function early after MI.


*    Acknowledgments
 
This study was supported by The Netherlands Heart Foundation grant 99.155 (to G.J.M.S.), grant 2000T038 (to D.J.D.), and grant 2000T042 (to D.M.).


*    Footnotes
 
Original received December 3, 2003; resubmission received September 21, 2004; revised resubmission received October 21, 2004; accepted October 22, 2004.


*    References
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