Cardiovascular Research

Cardiovascular Research 2000 45(4):981-993; doi:10.1016/S0008-6363(99)00374-0
© 2000 by European Society of Cardiology

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Copyright © 2000, European Society of Cardiology

Calpain-I induced alterations in the cytoskeletal structure and impaired mechanical properties of single myocytes of rat heart

Zoltán Papp^a,*, Jolanda van der Velden^b and G.J.M Stienen^b

^aDepartment of Physiology, University Medical School of Debrecen, H-4012 Debrecen, Hungary
^bInstitute for Cardiovascular Research, Laboratory for Physiology, Vrije Universiteit (ICaR-VU), Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands

^* Corresponding author. Tel.: +36-52-416-634; fax: +36-52-432-289 pz{at}phys.dote.hu

Received 27 May 1999; accepted 15 October 1999

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objective: The involvement of Calpain-I mediated proteolysis has been implicated in myofibrillar dysfunction of reperfused myocardium following ischemia (stunning). This study addresses the question whether ultrastructural alterations might be responsible for the depressed contractility. Methods: Mechanical properties and protein composition of isolated myocytes after Calpain-I exposure (1.25 U/ml; 10 min; 15°C; pCa 5.0) and of ischemic rat hearts following reperfusion were characterized. Results: Maximal isometric force (44±5 kN/m²) at pCa 4.5 (pCa=–log[Ca²⁺]) decreased by 42.5% in Triton permeabilized myocytes (n=11) after Calpain-I treatment. Force (and consequent myofilament disarrangement) during Calpain-I treatment was prevented by 40 mM BDM. The contractile force of Calpain-I exposed myocytes was significantly higher at submaximal levels of activation (pCa 5.5, 5.4 and 5.3) before maximal force development (pCa 4.5) than after maximal force development. The pCa₅₀ value (5.40±0.02) determined from these initial test contractures did not differ significantly from that of untreated controls (5.44±0.03). However, after full activation Ca²⁺-sensitivity of force production in Calpain-I treated myocytes was significantly reduced (pCa₅₀ 5.34±0.02). This change in pCa₅₀ was positively correlated with the reduction in maximal isometric force and was accompanied by sarcomere disorder. These findings imply that at least part of the Calpain-I induced mechanical alterations are dependent on force history. Measurements of the rate of force redevelopment after unloaded shortening suggested that Calpain-I did not affect cross-bridge kinetics. SDS gel electrophoresis and Western immunoblotting of Calpain-I treated myocytes revealed desmin degradation. The desmin content of postischemic myocardium was also reduced. Conclusion: Our results indicate that ultrastructural alterations may play an important role in the Calpain-I mediated cardiac dysfunction.

KEYWORDS Contractile apparatus; Contractile function; Myocytes; Stunning

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This article is referred to in the Editorial by Rappaport (pages 810–812) in this issue.

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Recent investigations have made the pathomechanism of reversible postischemic injury, i.e. myocardial stunning increasingly clear. Different experimental approaches converged to the central supposition that calcium overload during late ischemia and upon reperfusion triggers myocardial dysfunction [1–4], which is due to altered myofibrillar properties. The protective role of Ca²⁺-dependent protease inhibitors implicated that Calpain-I (also named µ-Calpain), a ubiquitous intracellular neutral protease, is involved [5–7]. Nevertheless, neither the exact nature of the mechanical dysfunction nor the full extent of the putative intracellular proteolysis is known.

Previous studies on multicellular preparations from rat heart stressed that a major part of the impaired contractility can be explained by the reduction in maximal Ca²⁺-activated force [8,9]. Surprisingly, no changes in this parameter were noticed in earlier studies [10] and when myocyte preparations from stunned porcine hearts were investigated [11,12]. Additional mechanisms, such as altered cross-bridge cycling rates and reduced myofibrillar Ca²⁺-sensitivity were, thus, also implicated [6,11,13].

Gao et al. [14] linked Calpain-I induced selective troponin-I (Tn-I) degradation to the reduction of the Ca²⁺-sensitivity of the myofibrillar system. Experiments with reconstituted troponin regulatory complexes recently confirmed that regulation of the contractile system might indeed be altered in stunned myocardium [15]. However, maximal Ca²⁺-activated force was not affected by this troponin reconstitution and the nature of the altered contractile regulation is controversial as decreased, unchanged or even increased myofibrillar Ca²⁺-sensitivities were all described in postischemic myocardial preparations [6,8–10,12]. Moreover, differences in Tn-I and/or Tn-T phosphorylation [15], binding of cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the M_r 23 000 B-crystalline protein to myofibrils [16], and the formation of covalent Tn complexes [17] were also proposed as possible explanations for altered myofibrillar regulation in stunned myocardium.

Besides proteolysis of regulatory proteins, degradation and/or loss of various cytoskeletal elements such as microtubules [18], -calspectin (spectrin) [19] together with desmin and -actinin [7,9] were also noticed. Moreover, the protective role of 2,3-butanedionemonoxime (BDM) during early reperfusion [20] implied that ultrastructural changes might contribute to the development of postischemic damage.

The present study aimed to distinguish whether regulatory or structural changes are the primary causes of Calpain-I induced myocardial dysfunction. Permeabilized myocytes were used to characterize the Calpain-I dependent changes in maximal isometric force, Ca²⁺-sensitivity of force development and kinetics of force redevelopment after unloaded shortening. The Ca²⁺ concentration required to activate Calpain-I (EC₅₀=2 µM) initiated crossbridge cycling as well. Hence recognition of the pure Calpain-I mediated alterations on contractility was complicated by the concomitant force production. Long lasting contractures have been shown to cause structural alterations [21], moreover, Ca²⁺-dependent proteolysis and force generation might interact [20,21]. BDM, which effectively inhibits force production at Ca²⁺ concentrations of several µM was applied during Calpain-I incubations to distinguish the putative force-dependent and force-independent components of the Calpain-I induced alterations. Using this approach we demonstrate that Calpain-I induces a major reduction in maximal isometric force, and a force history dependent decrease in Ca²⁺-sensitivity without affecting cross-bridge kinetics.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
2.1 Myocyte isolation and experimental set-up
Myocytes were mechanically isolated similarly as described previously [11,22]. Briefly, hearts of Wistar rats (300–600 g) of either sex were rapidly excised following Na-pentorbarbitone (0.1–0.3 ml, i.p.) induced anesthesia and heparin (250 U, intra-caval) injection. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). After excision, the hearts were perfused according to Landgendorff with Tyrode solution (equilibrated with 95% O₂–5% CO₂) containing (in mM): NaCl 128.3, KCl 4.7, CaCl₂ 1.36, MgCl₂ 1.05, NaHCO₃ 20.2, NaH₂PO₄ 0.42, glucose monohydrate 11.1 and 2,3-butanedione monoxime (BDM) 30 (20°C). Then the free right ventricular wall was mechanically disrupted within 5–10 s, using a tissue homogenizer. The resultant suspension of small clumps of myocytes, single myocyte-sized preparations, and cell fragments were skinned by means of 0.3% Triton X-100 (5 min) and kept in relaxing solution at 0°C for 6–24 h. A single myocyte was attached with silicon adhesive to thin stainless steel needles while viewed by an inverted microscope. One needle was attached to a force transducer (SensoNor, Horten, Norway) and the other to a piezoelectric motor (Physike Instrumente, Waldbrunn, Germany), both connected to joystick-controlled micromanipulators. After curing for 50 min, the preparation was transferred from the mounting area to a small temperature controlled well (volume 68 µl), containing relaxing solution, from which the myocyte could be transferred to a similar temperature controlled well containing activating solution. During cell attachment and subsequent force measurements myocytes were viewed at 320x magnification. Images were captured by means of a CCD video camera and stored on a personal computer. Average sarcomere length was determined by means of a spatial Fourier transform as described previously [23] and adjusted to approximately 2.2 µm. The diameters of the preparation were measured microscopically, in two perpendicular directions. Cross-sectional area was calculated assuming an elliptical cross-section.

2.2 Solutions
The composition of the solution used for cell isolation was (in mM): MgCl₂ 1, KCl 100, EGTA 2, Na₂ATP 4, imidazole 10 (pH 7.0, adjusted with KOH). Relaxing and activating solutions for force measurements contained, respectively (in mM): MgCl₂ 6.42 and 6.28, Na₂ATP 5.94 and 6.03, EGTA 5 and 0, CaEGTA 0 and 5.08, moreover, both contained BES 60 and phosphocreatine 14.5 (pH:7.1, adjusted with KOH). The ionic strength of the solutions was adjusted to 200 mM with K-propionate. The pCa, i.e. –log₁₀[Ca²⁺], of the relaxing and activating solution was, respectively, 9 and 4.5. Calculated free Mg²⁺ and MgATP concentrations were 1 and 5 mM, respectively. Solutions with lower free Ca²⁺ concentrations were obtained by appropriate mixing of the activating and relaxing solutions, assuming an apparent stability constant of the Ca–EGTA complex of 10^6.35 [24].

2.3 Force measurements
Isometric force was measured after the preparation was transferred from relaxing to activating solution, by moving the stage of the inverted microscope. When steady force was reached, the length of the myocyte was reduced by 20% within 2 ms using the piezoelectric motor (slack test). As a result of this intervention, force first dropped to zero and then quickly redeveloped to a new steady state level corresponding to the shorter sarcomere-length. After this force redevelopment the myocyte was returned to relaxing solution where the length of the myocyte was reset to the original level allowing the determination of passive force. Maximal active isometric force was calculated from the difference between the zero force level and the peak isometric force following correction for passive force. Experiments were discarded if passive force was higher than 10% of total force. Force redevelopment was fitted with a single exponential to estimate the rate constant of force redevelopment (K_tr) during slack tests. The preparations were not re-stretched prior to K_tr determination to minimize the mechanical stress imposed by the protocol to the minimum. This might influence the determination of the kinetic parameters. Nevertheless, the comparison of rate constants obtained during various phases of the experiments at identical pCa values will reflect changes in overall cross-bridge kinetics and/or in internal shortening [25].

Force and length signals were monitored using an analog pen-recorder and were stored in a personal computer. Sampling rate during experiments was 20 Hz, and 1000 Hz during slack-tests.

2.4 Experimental protocol
Temperature was set to 15°C during force measurements to maintain mechanical stability of the permeabilyzed myocyte preparations. The experimental protocol is schematically illustrated in Fig. 1. During the first phase (phase I) control mechanical properties were determined. The first contracture, during this phase, was induced by saturating calcium concentration (pCa 4.5), then sarcomere length was readjusted to 2.2 µm, if necessary. The second measurement at pCa 4.5 was considered as a measure of maximal force output (P_o) of the preparation. The next three to four measurements were carried out at submaximal Ca²⁺ concentrations, followed by another activation at pCa 4.5. Measurements were continued until a full force–pCa curve was obtained and terminated by a bracketing activation at pCa 4.5. Force production at submaximal levels of activations was normalized to the nearest reference force value obtained at maximal activation to determine myofibrillar Ca²⁺-sensitivity.

View larger version (8K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Experimental protocol: Ca2+-activated force production was determined in permeabilyzed myocytes before Calpain-I treatment (phase I, control) and after Calpain-I treatment (phase II and phase III). Force production during Calpain-I treatment (at pCa 5.0) was inhibited by the simultaneous application of 40 mM BDM. Ca2+ concentration during force measurements was varied between saturating (pCa 4.5, ) and various intermediate Ca2+ levels (x) up to pCa 6.0 during phase I and III. However, during phase II only low force producing intermediate Ca2+ concentrations (between pCa 5.6 and pCa 5.3) were applied. This allowed the characterization of force-independent (phase II) and force-dependent (phase III) components of the Calpain-I mediated effects on myofibrillar mechanics. Symbols at the bottom illustrate the sequence of maximal and submaximal activations.

 
Determination of the control parameters was followed by Calpain-I (Calbiochem) treatment. First, myocytes were bathed in relaxing solution in the presence of Calpain-I (1.25 U/ml) for 20 min to allow diffusion of the enzyme into the intermyofilamental space. Thereafter, Ca²⁺ concentration was raised to pCa 5.0 for 10 min to activate proteolytic activity. During this period force production was prevented by BDM (40 mM). Calpain-I treatment was terminated by reducing the Ca²⁺ concentration back to pCa 9.0. Then, Calpain-I was washed out from the preparation by incubation in relaxing solution for an additional 20 min. This prevented continuation of proteolysis during subsequent Ca²⁺ contractures.

The immediate (force-independent) effect of Calpain-I on force production was tested during phase II at low-force producing submaximal Ca²⁺ concentrations (5.3pCa5.6).

Phase III of the experiment started with a maximal force producing Ca²⁺ concentration (pCa 4.5) followed by others at submaximal Ca²⁺ concentrations and terminated by a final activation again at saturating Ca²⁺ level. To determine myofibrillar Ca²⁺-sensitivity, force production during phase II was normalized to the force obtained at maximal activation at the beginning of phase III. During phase III force production at submaximal levels of activations was normalized to the nearest reference force value obtained at maximal activation. The usual number of force measurements per myocyte amounted to 23, of which at least six were performed at maximal Ca²⁺ concentration. On average, force decline, between the first and final maximal activation before Calpain-I treatment was 5% (see phase I, Fig. 2). Following Calpain-I treatment force decline, between the first and final maximal activation, was somewhat elevated, although, it did not increase above 10% (see phase III, Fig. 2).

View larger version (22K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 (A) Isometric force development in an isolated myocyte before Calpain-I treatment (phase I), after Calpain-I treatment (phase II), and finally, after Calpain-I treatment and full mechanical activation (phase III). Calpain-I treatment is indicated by a vertical arrow. Force values were obtained at saturating Ca2+ concentrations (pCa 4.5; a, c, e, g) during phase I and phase III and at submaximal Ca2+ levels (e.g. pCa 5.4; b, d, f) during phase I, II and III. Zero force and the rate of force redevelopment were determined by reducing the cell length to 80% after reaching peak force in the activating solutions. Initial cell length was restored in relaxing solution allowing determination of passive force. (B) Images (h, i, j) obtained during the experiment in relaxing solution as indicated in (A). Calpain-I decreased isometric force and induced force-dependent deterioration of the cross-striation pattern which was accompanied by subsequent decrease in force at submaximal Ca2+ concentrations (phase II vs. phase III). Peak isometric force: 58.5 kN/m2, passive force: 4.1%.

 
2.5 Protein analysis by SDS-PAGE and Western immunoblot analysis
Analysis of myofibrillar protein composition was performed on myocyte suspensions from the same isolations that were used for the mechanical measurements. Calpain-I treatment of myocytes was carried out at 15°C and inhibited by reducing temperature to 4°C and [Ca²⁺] to pCa 6.4. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described previously [24,26]. The samples were dissolved in buffer containing (in mM): Tris (pH 6.8) 62.5, dithiothreitol 15, phenylmethylsulfonyl fluoride 0.1, leupeptin 0.5 and 1% (w/v) SDS, 0.01% (w/v) bromophenol blue and 15% (v/v) glycerol. Samples without protease inhibitors provided identical results. Comparable samples (5–15 µl1–3 µg dry weight) were loaded in each lane. Silver staining was performed as described by Giulian et al. [26]. Laser scanning densitometry was performed to detect differences in protein composition. Contractile protein bands were identified by their molecular weight and by Western Immunoblotting. Molecular masses of the contractile proteins were determined using molecular mass standards (Bio-Rad, high range: 161-0303 and low range: 161-0304) run under identical conditions. In order to quantify the alterations in protein concentrations following Calpain-I treatment the areas of protein bands were normalized to those of tropomyosin and/or actin. Under our experimental conditions the intensity of these bands showed no Calpain-I dependence. Saturation was tested and subsequently avoided by applying various concentrations of the same test samples on each individual gel. Specific antibodies for identification of protein bands using Western Immunoblotting were obtained from Sigma (anti-desmin Clone DE-U-10, 1:130; anti--actinin Clone EA-53, 1:100), Chemicom (anti Tn-I Clone MAB1691, 1:200), Research Diagnostics (Anti Tn-I Clone C5, 1:300) and Spectral Diagnostic (Anti Tn-I Clone T8-7, 1:200). The procedure used for Western immunoblotting is detailed elsewhere [24].

2.6 Experiments with reperfused Langendorff rat hearts
Excised rat hearts for Langendorff perfusion were initially placed into ice-cold Tyrode solution after which the aortas were quickly cannulated. Coronary perfusion was subsequently maintained at 15 ml/min using 95% O₂–5% CO₂ equilibrated Tyrode solution (in the absence of BDM) at 37°C. Hearts were paced at 320 bpm throughout the experiments, except from the second minute of the non-flow ischemia until the fifth minute of reperfusion. A latex balloon tied to the end of a polyethylene tube was passed into the left ventricle through the mitral valve and connected to a disposable pressure transducer (Ohmeda). The balloon was filled with water to obtain an initial end-diastolic pressure of 2–10 mmHg and kept at the same volume during the rest of the experiment. Representative phases of the left ventricular pressure signal were sampled at 5 kHz. Developed pressure was defined as the difference between peak-systolic pressure and end-diastolic pressure. After 30 min of stabilization, the coronary flow was interrupted by shutting off the perfusion and cross-clamping the perfusion line for either 20 or 40 min. After the period of global ischemia, hearts were reperfused for 20 min during which developed pressure recovered to a new steady state. Another group of hearts was perfused at constant flow (without interruption) for a period of 90 min, i.e. for the total duration of the experiments in which 40 min of ischemia was applied. Protein profiles of these hearts served as non ischemic control. At the end of the experiments, hearts were rapidly disconnected from the experimental apparatus and placed into ice-cold Tyrode solution. Free right ventricular wall was separated from the left ventricle and septum, and stored at –80°C for subsequent SDS-PAGE protein analysis.

2.7 Data analysis
The force–pCa relation was fitted to a modified Hill equation

where P is steady state force. P_o denotes the steady isometric force at saturating Ca²⁺ concentration, the Hill coefficient nHill is a measure of the steepness of the relationship, and Ca₅₀ (or pCa₅₀) represents the mid-point of the relation. Values are given as means±S.E. of n experiments obtained from at least five different hearts. Differences were tested by means of Student's t-test at a 0.05 level of significance (P<0.05).

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
3.1 Effect of Calpain-I in the absence of force production
Force production of a permeabilyzed myocyte before (phase I) and after Calpain-I treatment (phase II and phase III) is illustrated by the recordings shown in Fig. 2A. The maximal force output was tested at saturating Ca²⁺ concentration (pCa 4.5; a, c, e, g), while changes in Ca²⁺-sensitivity were estimated by evoking submaximal activations (e.g. pCa 5.4; b, d, f). The images of the cardiomyocyte recorded in relaxing solution (h, i and j) were used to follow the effect of Calpain-I on myofibrillar integrity. Together with contractures of maximal force production (i.e. at pCa 4.5) they served as a tool to follow the rundown of the preparation.

In the first, control phase (phase I) peak active force level at pCa 5.4 (b) amounted to 71% of that of at pCa 4.5 (a). No irreversible alterations were noticed during this initial phase of the experiments either in terms of mechanical stability (e.g., a and c) or in visual appearance (Fig. 2B, h).

The immediate effect of Calpain-I on force production was tested during phase II of the experiment where only low-force producing submaximal Ca²⁺ concentrations (pCa5.3) were applied. Force-dependent structural changes during this period, therefore, were supposedly still minor. Contractures obtained during this phase already revealed major contractile alterations. At pCa 5.4 isometric force (d) reached only 33% of the value obtained before Calpain-I treatment at the same pCa (b). However, deterioration of the striation pattern was not observed (i).

Phase III of the experiment started with a contracture at saturating Ca²⁺ concentration (pCa 4.5; e) followed by others at submaximal Ca²⁺ concentrations and a final activation at saturating level (pCa 4.5; g). The contractile force during the initial contracture of phase III (pCa 4.5; e) reached only 44% of the control (a,c). Irregularities of the cross-striation pattern became apparent in the preparation following this activation (j). However, average resting sarcomere length was not affected and the final contracture of phase III at pCa 4.5 (g) resulted in a peak force level similar to the first one of phase III (e). This indicates that, despite ultrastructural changes in the resting pattern, the force generating capacity of the preparation during phase III was well preserved.

Normalization of forces at submaximal activation to the maximum values obtained at pCa 4.5 during phase I and phase III resulted in estimates for Ca²⁺-sensitivity for the corresponding phases. It appeared, however, that the choice of reference during phase II is critical. When force at pCa 5.4 (d) during phase II, was normalized to that of pCa 4.5 at the beginning of phase III (e) the relative value (51.6%) was less than that obtained during phase I (71%). Note, that the deviation from phase I (i.e. control) became even higher during phase III. In this case (f) force at pCa 5.4 was only 28% of the peak force at pCa 4.5 (e). This implied that the contractile alterations initiated by Calpain-I were aggravated by maximal force generation and that this resulted in a progressive reduction in the apparent Ca²⁺-sensitivity.

In a series of experiments (n=11) force measurements were extended for a number of different submaximal Ca²⁺ concentrations to construct the relative (Fig. 3A) and normalized (Fig. 3B) force–pCa relations for all of the three different phases of the experimental protocol.

View larger version (9K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Isometric force–pCa relations during control conditions (phase I; ), following Calpain-I treatment (phase II; ) and after Calpain-I treatment and full activation (phase III; ). (A) All force values attained are given as fractions of the maximal force recorded during phase I. (B) Force values of phases II and III are normalized to the maximal force obtained during phase III. Absolute forces during phase II and phase III were significantly lower than those recorded during phase I, isometric forces during phase III at pCa 5.5, 5.4 and 5.3 were significantly lower than those of phase II (indicated by asterisks). The Hill functions fitted to the mean values in (B) illustrate changes in Ca2+-sensitivity during the three phases of the experiments. Means of the fitted parameters obtained during all individual experiments are given in Table 1. Data were obtained from 11 myocytes using the protocol illustrated in Fig. 1. Error bars indicate S.E.

 
As illustrated in Fig. 3A average maximal isometric force at saturating Ca²⁺ concentration was decreased to 57.5%. Contractile force was further reduced during phase III, i.e. after Calpain-I treatment and full activation. The means of absolute force values at intermediate Ca²⁺ concentrations (pCa 5.5, 5.4 and 5.3) were significantly lower after full activation than before (see Table 1 for details).

View this table: [in this window] [in a new window]   Table 1 Parameters of force–pCa relationsa

 
The three normalized sets of data for all individual experiments were fitted with the Hill equation. The mean results of these curve fittings are also summarized in Table 1. Despite the significant alterations in the normalized force values at low Ca²⁺ concentrations statistical comparison revealed no significant difference between the pCa₅₀ values obtained under control conditions (phase I) and following Calpain-I application (phase II). In contrast, the difference between the mean of control pCa₅₀ values and that obtained after Calpain-I treatment and full activation (phase III) was 0.1 and statistically highly significant (P=0.0057). The Hill coefficient (nHill) first increased by 0.88 during phase II, then during phase III it returned back to a level which was not significantly different from the control value.

3.2 Effect of Calpain-I on K_trat maximal and submaximal activations
The rate constant of force redevelopment (K_tr) subsequent to unloaded shortening was determined by fitting the first second (except the first 20 ms) of force redevelopment with a single exponential function at pCa 4.5 and 5.3. For pCa 4.5 this determination was performed during phase I and III and for pCa 5.3 it was performed during phase I, II and III (Fig. 4A). The mean values of K_tr obtained are illustrated in Fig. 4B. Maximal K_tr under control conditions (phase I, pCa 4.5) was 6.65±0.25 l/s (n=11) which is similar to the value obtained previously by others in rat myocardium [27,28]. However, during phase III it significantly dropped to 4.72±0.21 l/s (n=10). In contrast, no significant differences were observed among the means of rate constants determined at pCa 5.3. Control K_tr (phase I) was 5.95±0.50 l/s, while after Calpain-I treatment (phase II) it was 6.06±0.43 l/s and finally after full activation (phase III) it was 6.63±0.72 l/s (n=11, 11 and 10, respectively) at pCa 5.3.

View larger version (17K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 (A) Rate of force redevelopment (Ktr) determined during the first second of force recovery following quick isometric shortenings to 80% of the initial cell length at saturating Ca2+ concentration (pCa 4.5) and at a submaximal Ca2+ concentration (pCa 5.3). Force values are normalized to the same peak to emphasize kinetic differences at pCa 4.5 and to indicate similarities at pCa 5.3. (B) Ktr values: the asterisk indicates significant difference between the means of Ktr obtained during phase I and phase III at pCa 4.5. Error bars indicate S.E.

 
3.3 Relation between the reduction in maximal isometric force and in Ca²⁺-sensitivity of force production
The duration of Calpain-I treatment had a graded effect on the reduction in maximum force. The duration of the Calpain-I treatment was varied between 5 and 20 min. Results of these experiments and those of the previous series shown in Fig. 3 are combined in Fig. 5 (n=32). For each myocyte force–pCa relations were obtained during phase I and phase III. The differences in pCa₅₀ values were expressed as the function of the observed relative isometric force at pCa 4.5 after the Calpain-I treatment (closed circles). In this way force reduction served as a measure of the severity of the Calpain-I treatment. Regression analysis demonstrated that the Calpain-I dependent factors leading to the reduction of maximal isometric force and Ca²⁺-sensitivity were interrelated. Open circles of Fig. 5 illustrate these differences in pCa₅₀ obtained between the control force–pCa relations and those obtained immediately following the Calpain-I treatment (i.e. phase I vs. phase II). These data points fell below the fitted line, indicating smaller changes in Ca²⁺-sensitivity for the same reduction in force.

View larger version (7K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Decrease in Ca2+-sensitivity (pCa50) as a function of relative isometric force at pCa 4.5 after Calpain-I treatment (). The duration of Calpain-I exposure was varied between 5 and 20 min (n=32 myocytes). The change in Ca2+-sensitivity was assessed from the difference between pCa50 values during phase I and phase III. Relative force indicates normalized force obtained during phase III as expressed relative to that of phase I at pCa 4.5. Fitted straight line indicates regression line between the two variables (r=0.76, P<0.0001). The differences between pCa50 values of phase I and phase II (), showed a similar correlation (dashed line, r=0.70, P=0.026) but in general fell below the regression line of the difference between phases I and III.

 
3.4 SDS-PAGE and Western immunoblot analysis of contractile proteins obtained from Calpain-I treated myocytes
Calpain-I dependent alterations in myofibrillar protein composition were evaluated in suspensions of skinned myocytes that were treated with the same concentrations of Calpain-I as those applied during the mechanical measurements at 15°C. To facilitate recognition of protein degradation that might otherwise not be apparent following short Calpain-I exposures, enzyme treatment was extended up to 270 min. This approach revealed that desmin, a Z-disk related protein is removed by Calpain-I from the skinned myocytes with a half time of 20 min. Fig. 6A demonstrates the results of SDS-PAGE. Lane 1 shows typical silver stained myofibrillar protein bands obtained after 270 min incubation in relaxing solution (control). The results obtained after 270 min incubation at pCa 5.0 in the absence of Calpain-I or at pCa 9.0 and in the presence of 1.25 U/ml Calpain-I were identical with the control (not shown). In contrast, marked changes in myofibrillar protein bands were observed when Calpain-I and pCa 5.0 were applied simultaneously indicating that the mechanical and proteolytic changes were indeed the consequence of a Ca²⁺-dependent proteolysis. Protein bands in lane 2 and 3 reflect the results obtained after 90 min of Calpain-I exposure at pCa 5.0 in the absence and presence of 40 mM BDM, respectively. The intensity of the protein band representing desmin (verified by Western Immunoblot, see Fig. 6D) was greatly reduced in both cases. Furthermore, an additional protein band appeared below the level of C-protein at the molecular mass of 146 000, which probably represents a degradation product from another higher-molecular-mass protein. Based on a previous study it might well reflect the degradation product of another Z-disk related protein calspectin (spectrin) [19]. All these changes are clearly visible on the densitometric tracings in panel B. When normalized protein amounts were expressed as fractions of the control, the time-dependent changes in relative protein amounts could be readily evaluated. Soon after the start of the Calpain-I treatment, desmin content was very rapidly reduced and this led to almost complete disappearance after 90 min of enzymatic activity (Fig. 6C). The gradual decline in desmin content during the first 30 min of Calpain-I treatment was verified in separate test incubations. The activity of the Calpain-I batch used in these measurements was less than that used for the myocyte measurements and of those illustrated in Fig. 6C. Therefore, these additional data are not shown. However, these measurements confirmed that the decline in desmin content was well approximated by the dashed line in Fig. 6C between 0 and 30 min, and that there was no significant difference in the effect of Calpain-I in the presence or absence of BDM.

View larger version (20K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Calpain-I dependent alterations in myofibrillar protein profiles of skinned cardiac myocytes. (A) SDS gel from a suspension of skinned myocytes incubated for: 270 min in relaxing solution in the absence of Calpain-I and BDM (lane 1, control), 90 min in the presence of 1.25 U/ml Calpain-I at pCa 5.0 in the absence of 40 mM BDM (lane 2) and 90 min in the presence of 1.25 U/ml Calpain-I at pCa 5.0 in the presence of 40 mM BDM (lane 3). The bands of myosin heavy chain (My), a novel degradation product (D.p.), desmin (De) and actin (Ac) are indicated. (B) Densitometry shows the Calpain-I dependent reduction in desmin and the appearance of a (proteolytic) fragment. (C) the time course of the normalized intensity change of the desmin band based on densitometric area determinations (n=8; mean±S.E.). The means of 30, 60 and 90 min all differed significantly from the control (shown at 0 time). (D) Western immunoblot analysis obtained from control myocytes (lane 1) and from myocytes exposed to Calpain-I for 30 (lane 2) and 270 min (lane 3) both in the presence of BDM using anti-desmin antibody. The reduced intensity at the 55 000 level and the appearance of the new bands below that (at 51 000, 50 000, 48 000, 43 000 and 42 000) confirms desmin degradation. The theoretical location of actin (as reference) is indicated by an asterisk.

 
Western immunoblot analysis using antibodies against desmin, revealed several degradation products at lower molecular masses (Fig. 6D) suggesting that desmin and its breakdown products are proteolyzed.

Careful inspection of all densitometric recordings and Western immunoblots was performed to see if other proteins were also affected. In contrast to the clear time-dependent reduction in desmin content no major alterations could be resolved for Tn-I, Tn-T and for -actinin as might have been expected on the basis of previous studies [7,9,14,17,29]. In case of Tn-I, it was possible to visualize an extra band that could possibly be related to Tn-I degradation using Western immunoblot after 30 min Calpain-I treatment. The impact of this finding, however, is not clear because only one of the three different antibodies gave positive reaction (Spectral Diagnostics, Clone T8-7, not shown), and because of controversial data in the literature [9,14,17,29,30].

3.5 Experiments with reperfused Langendorff hearts
Isolated Langendorff perfused rat hearts were made ischemic by interrupting coronary flow for 20 min at 37°C. Developed pressure quickly dropped to zero, and recovered only partially (to a level between 40 and 60%) after 20 min of reperfusion in four out of six hearts consistent with myocardial stunning. In the two other hearts developed pressure returned to the preischemic level. In a different group of hearts (n=4) mean developed pressure after 40 min of ischemia and 20 min of subsequent reperfusion decreased to 27±22% level of the preischemic value (Fig. 7B). In a third group of hearts (isochronal control of the 40 min ischemia group, n=3) coronary perfusion was maintained continuously for 90 min. Determination of desmin content of perfused hearts using SDS-PAGE was complicated by the presence of a band near desmin. The intensity of this band could be greatly reduced by Triton treatment, suggesting that it originated from a membrane-associated protein. This might explain why desmin degradation was not noticed earlier on SDS gels. Triton treatment (30 min, 0.3 vol%) resulted in a protein distribution of the Langendorff perfused hearts similar to that of cell suspensions (Fig. 6A) and enabled quantification of the desmin band. Fig. 7A demonstrates characteristic protein profiles obtained from the tissue samples of the three experimental groups. Desmin contents of ischemic–reperfused myocardia were expressed as the fractions of the means obtained in the control group (Fig. 7B). Fig. 7B implicates that following 20 min of ischemia and 20 min of reperfusion the amount of desmin was reduced by 24±6% (P<0.05). The reduction in the relative desmin amount after 40 min of ischemia and 20 min of reperfusion was 27±7%. Our analysis did not reveal additional changes in other protein amounts. However, we cannot exclude that they might be found using SDS-gels with different characteristics or Western immunoblotting.

View larger version (15K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 7 Myocardial stunning and changes in myofibrillar desmin levels in reperfused Langendorff hearts. (A) Silver stained SDS gel of Triton treated cardiac tissue obtained from hearts: perfused continuously for 90 min (control, lane 1), exposed to global ischemia for 20 min and subsequently to 20 min of reperfusion (lane 2), exposed to global ischemia for 40 min and subsequently to 20 min of reperfusion (lane 3). The level of desmin (De) and actin (Ac) are indicated on the left. (B) Illustration of the decrease in developed pressure (DP) following 20 and 40 min of global ischemias and 20 min of reperfusion (DP(20') n=6; DP(40') n=4) relative to the preischemic level (100%, DP(C) n=3). B also illustrates the reduction of relative desmin amounts of the 20 and 40 min groups (DE(20'); DE(40')) relative to control (DE(C)). Significant differences are indicated by asterisks. Error bars indicate S.E.

 

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
It is unclear whether myofibrillar dysfunction of stunned myocardium is related to altered thin filament regulation, damaged cytoskeletal structures, or both [9]. Part of the controversy arises from the different results regarding postischemic myocardial mechanics [6,9–11,13]. Furthermore, technical limitations complicate the issue when the subtle changes in myofibrillar protein composition of stunned myocardium are characterized [7,9,16,18,19,30]. Nevertheless, consensus has been reached on the basic concept that the postischemic alterations are Ca²⁺-dependent [31]. Moreover, activation of Calpain-I, a Ca²⁺-dependent neutral protease, has been implicated [6,7,14], and an increasing body of evidence pointed to the involvement of Tn-I degradation in the pathogenesis of myocardial stunning [9,14,17].

This study suggests that desmin degradation plays a significant role in the Calpain-I induced myofibrillar dysfunction.

4.1 Force generation exacerbates the development of myofibrillar dysfunction
Previous experiments with Calpain-I were performed on multicellular cardiac preparations [6,14] at Ca²⁺ concentrations resulting in significant force production. This study is the first in which the proteolytic effect of Calpain-I in the absence of force generation was characterized in isolated myocytes. This approach has the advantage that the alignment between the major myofilaments following the Calpain-I treatment is initially preserved. The use of single myocytes, also enabled us to avoid possible complicating factors due to intercellular connections, extracellular matrix and enzyme diffusion. Other parameters of Calpain-I incubation were chosen to be similar to those described earlier [6,14]. The reduction in maximal isometric force to 57.5%, together with the final decrease in Ca²⁺-sensitivity (pCa₅₀ of 0.1 unit corresponding to 940 nM) were also similar to previous reports on postischemic and Calpain-I treated myocardial preparations [6,8].

One of the new findings of this work is the recognition of the force-dependent enhancement of the Calpain-I induced myofibrillar dysfunction. We demonstrate that contractile activation close to saturating Ca²⁺ concentrations is required to reveal a decrease in myofibrillar Ca²⁺-sensitivity (phase II vs. phase III) and to establish visible changes in cross-striation pattern. These observations extend previous findings [20] suggesting increased fragility of the cytoskeletal system early during reperfusion.

4.2 Proteolysis and altered myofibrillar mechanics in stunned myocardium
In stunned myocardium of guinea pig inhomogeneous and time-dependent disappearance of -actinin, desmin and spectrin have been described using immunohystochemical methods [7]. The rapid disappearance of Z-disk proteins was also found in bovine skeletal muscle fibers treated with Calpain-I and Calpain-II in vitro [32]. We provide evidence that desmin is degraded and removed from Calpain-I treated cardiac myocytes from rat. However, it should be noted that alterations in other structural/regulatory proteins are also probable [7,9,18,19,29]. In addition to the proteolysis of desmin a new protein product with 146 000 molecular mass was also observed. This might be the degradation product of -calspectin (spectrin) because a product with a similar molecular mass (150 000) has been previously identified as such [19] both in ischemic–reperfused and in Calpain-I treated rat myocardial preparations.

We could not reconstruct changes in myofibrillar -actinin and in Tn-I levels for short lasting Calpain-I incubations despite the occurrence of the well-defined mechanical alterations. Technical aspects (e.g. limited sensitivity, contaminating proteins, protein reassociation, etc.) might provide partial explanation for these findings [9,29]. The lack of appreciable changes following long lasting Calpain-I incubations, especially in the case of Tn-I, however, is remarkable as this protein was previously reported as a potential target of Calpain-I mediated proteolysis [14,29]. We would like to point out the resemblance between our preparations and myocardial cryosections (rather than purified Tn-I), and that in cryosections appreciable Tn-I degradation by Calpain-I required about 180 times higher enzyme activity/unit volume [29] even at 37°C. Hence, these previous data indicate that desmin in the myofilament lattice is more sensitive for Calpain-I digestion than Tn-I.

4.3 Alterations in cytoskeletal structure and in myofibrillar mechanics
Here we will try to establish a unifying concept for the diverse alterations in myofibrillar mechanics in Calpain-I treated and postischemic myocardium. It is conceivable that changes in regulatory, kinetic and structural properties develop in parallel, because several components of the myofibrillar system are affected by proteolysis [7,9,14,17–19,29]. Therefore, the observed diversity of mechanical alterations may reflect different, however, not exclusive and certainly not independent facets of the postischemic damage.

The temporal correlation between the Calpain-I induced desmin degradation and the observed alterations in the cross-striation pattern suggests that they are interrelated. The role of desmin in the maintenance of lateral alignment of myofibrils has been recently demonstrated in mice lacking desmin [33]. The reduction in maximal isometric force found in Calpain-I treated myocytes may, therefore, be the consequence of loose Z-line structures.

The maximum rate of force redevelopment (pCa 4.5) during slack-test was slower following Calpain-I treatment than under control conditions. This finding is in agreement with that of McDonald et al. [13], who also reported that the maximum rate of unloaded shortening is reduced in porcine skinned cardiac myocytes after stunning. In contrast, however, we found no difference in these rates at intermediate Ca²⁺ levels (pCa 5.3) during any phase of the experiments. In principle, this might imply that cross-bridge kinetics are affected only at high [Ca²⁺]. However, relatively strong contractures may lead to significant internal shortening in Calpain-I exposed myocytes. This would artefactually lower the rate constants [25,27] even in the absence of changes in cross-bridge kinetics. We favor this latter proposal because K_tr at pCa 4.5 decreased below the level obtained at pCa 5.3 after Calpain-I treatment and, therefore, we do not believe that our data indicate real alterations in cross-bridge kinetics.

Previous studies showed no changes in total Ca²⁺-sensitive ATP-ase activity in ischemic [30] and in reperfused [34] myocardium. The reduction in mechanical efficiency, i.e. contractile function–oxygen consumption ratio, has been also documented [35,36]. Therefore, our results are in line with the proposal that postischemic myofibrillar dysfunction is due to an impaired transduction of ATP-ase activity to mechanical force [36].

The force-dependent development of decreased Ca²⁺-sensitivity can be associated to an enhanced internal shortening and may well explain why different laboratories found either increased or decreased values of this parameter in stunned preparations. This is because the amount of force produced during an individual contracture may influence the integrity of stunned myocardium and, therefore, may affect force generation during the subsequent activations. The implication is that the sequence of applied Ca²⁺ concentrations and the choice of control during normalization become critical when the force–pCa relation of post-ischemic myocardium is characterized. This might explain the controversial results on Ca²⁺-sensitivity observed previously [6,8,9]. One may postulate that the vulnerability of the cytoarchitecture might also complicate the quantification of the Hill-coefficient. In agreement with previous reports [6,8], we found no significant change in this parameter when force–pCa relations during phase I and phase III were compared. However the slope of the force–pCa relations appeared to be decreased in those studies where the Ca²⁺-sensitivity was found to be increased [9].

The time-dependent reduction of desmin content was also noticed in Triton-treated ischemic–reperfused Langendorff preparations. This finding extends earlier observations in guinea pig [7] and rat hearts and seems to confirm the link between compromised myocardial performance and postischemic myocardial desmin content. Nevertheless, it should be stressed that the reduction in postischemic developed pressure following 40 min of ischemia and 20 min of reperfusion was larger than the relative decrease in desmin content (Fig. 7B), suggesting that additional factors might also be involved.

The final decline in Ca²⁺-sensitivity of the Calpain-I treated myocytes, as shown in this study, correlated positively to the decrease in maximal isometric force. This implies that the initial enzymatic damage may determine the extent of the subsequent, force-dependent alterations. Cytoskeletal elements appear crucial in this multifactorial process.

Time for primary review 41 days.

	Acknowledgements

 
This study was supported by the NWO-OTKA (Dutch–Hungarian) Research Fund. The authors would like to thank Ruud Zaremba for the technical assistance and Pieter P. de Tombe for advice.

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