Cardiovascular Research

Cardiovascular Research 1998 38(2):414-423; doi:10.1016/S0008-6363(98)00019-4
© 1998 by European Society of Cardiology

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Copyright © 1998, European Society of Cardiology

Force production in mechanically isolated cardiac myocytes from human ventricular muscle tissue

J van der Velden^a,*, L.J Klein^b, M van der Bijl^a, M.A.J.M Huybregts^c, W Stooker^c, J Witkop^c, L Eijsman^c, C.A Visser^b, F.C Visser^b and G.J.M Stienen^a

^aLaboratory for Physiology, Institute for Cardiovascular Research (ICaR-VU), Free University, van der Boechorststraat 7, 1081 BT Amsterdam, Netherlands
^bDepartment of Cardiology, Institute for Cardiovascular Research (ICaR-VU), Free University, Amsterdam, Netherlands
^cDepartment of Cardiac Surgery, Institute for Cardiovascular Research (ICaR-VU), Free University, Amsterdam, Netherlands

^* Corresponding author. Tel.: +31 20 4448122; fax: +31 20 4448255.

Received 22 July 1997; accepted 12 December 1997

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objective: The expression of contractile isoforms changes during various pathological conditions but little is known about the consequences of these changes for the mechanical properties in human ventricular muscle. We investigated the feasibility of simultaneous determination of protein composition and isometric force development in single cardiac myocytes from human ventricular muscle tissue obtained from small biopsies taken during open heart surgery. Methods: Small biopsies of about 3 mg wet weight were taken during open heart surgery from patients with aortic valve stenosis. These biopsies were divided in two parts. One part (2 mg) was used for mechanical isolation of single myocytes and subsequent force measurement while the remaining part was used, in aliquots of 1 µg dry weight, for protein analysis by polyacrylamide gel electrophoresis. The myocytes were attached with silicon glue to a sensitive force transducer and a piezoelectric motor, mounted on an inverted microscope and permeabilized by means of Triton X-100. Force development was studied at various free calcium concentrations. Results: From all biopsies, myocytes could be obtained and the composition of contractile proteins could be determined. The average isometric force (±s.e.m.) at saturating calcium concentration obtained on 20 myocytes from 5 patients amounted to 51±8 kN/m². Force was half maximal at a calcium concentration of 2.47±0.10 µM. Conclusion: These measurements indicate that it is possible to study the correlation between mechanical properties and protein composition in small biopsies from human ventricular muscle.

KEYWORDS Human; Cardiac myocyte; Muscle contraction; Protein composition; Contractile properties

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
During various pathological conditions, such as cardiac pressure or volume overload [1–3], diabetes mellitus [4, 5], and hyper- or hypothyroidism [6–8]changes occur in the composition of the contractile proteins which may influence cardiac pump function. So far, the effects of alterations in contractile proteins on mechanical and energetic properties of the heart were investigated in animal models. However, species differences in protein composition make it difficult to extrapolate these findings to the human heart. At present, therefore little is known about the consequences of changes in protein composition on contractile properties of the human heart.

It is well established that in human ventricular muscle the slow (β) myosin heavy chain (MHC) is predominantly expressed [9], while in atrial tissue a mixture is found of the fast -MHC and the slow β-MHC [10]. From previous animal studies, e.g. [2, 11], it is known that the myosin heavy chain composition is an important determinant of mechanical and energetic properties such as maximum shortening velocity and ATPase activity. As human ventricular tissue mainly consists of the slow β-MHC the impact of myosin heavy chain isoform changes on contractile properties is likely to be small, although the marked difference in tension cost between fast and slow MHC isoforms observed recently in cardiac tissue from guinea pig indicates that a small change in MHC isoforms may have considerable energetic consequences [12].

In human myocardium changes occur, in particular, in isoform composition and/or phosphorylation level of troponin T (TnT) and troponin I (TnI) subunits of the actin filament [3, 13]and myosin light chains (MLC) [14]. To investigate the influence of alterations in contractile proteins on pump function of the human heart, both force measurements and protein analysis have to be performed in the same sample. This is a severe constraint since only small amounts of ventricular tissue become available during open heart surgery or heart catheterizations. Therefore, as a first step, we investigated the feasibility of simultaneous determination of isometric force development in single cardiac myocytes and of protein composition from small biopsies.

For this purpose, small endocardial left ventricular biopsies (3 mm³) were used, obtained during open heart surgery from patients with aortic valve stenosis. To determine isometric force production, we adopted the technique developed and used recently to study contractile properties of single myocytes from rat, pig and guinea pig [15–17]. Contractile protein composition was analysed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and subsequent silver-staining. This yields quantitative information in the high molecular weight region (myosin heavy chains), as well as in the low molecular weight region, where myosin light chains and components of the actin filament are present.

After isolation, the myocytes were treated with Triton X-100 to remove all membrane barriers, while the contractile apparatus remains intact. This method has the distinct advantage that the correlation between contractile properties and protein composition can be studied under standardized conditions (i.e. composition of the intracellular medium, sarcomere length) without the disturbing factors present in the intact heart (i.e. hormonal factors and variable calcium concentrations).

The main objective of this study is to investigate the feasibility of simultaneous determination of the composition of contractile proteins and contractile properties in small ventricular biopsies. In particular, attention is given to the reproducibility of the measurements of isometric force and its calcium sensitivity in different myocytes from the same biopsy. Furthermore, the focus for protein analysis is placed on using only a minimal amount of tissue for a quantitative analysis by SDS–PAGE. As a consequence, the remaining tissue can be used for further detailed analysis by means of two-dimensional gel electrophoresis, molecular sequencing, ELISA or immunohistochemistry.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
2.1 Biopsies
Small endocardial biopsies from the left ventricle of about 3 mm³ were obtained during open heart surgery, after cardioplegic arrest, from five randomly chosen patients with aortic valve stenosis, undergoing valve replacement (New York Heart Association (NYHA) Class I or II; 2 males, 3 females). The clinical data of the patients are given in Table 1. The left ventricular mass and the pressure gradient across the aortic valve, were obtained from 2-D and Doppler echocardiography. No 2-D information was available in one acute patient (no. 2). Left ventricular mass normalized on body surface of patient no. 3 was almost 3-times the normal value (92 g/m²) [18], indicating substantial hypertrophy of the left ventricle. The study was approved by the Ethical Committee of the Academic Hospital of the Free University. The patients gave informed consent and the investigation conforms with the principles outlined in the Declaration of Helsinki (Cardiovasc Res 1997;35:2–3). The biopsies were quickly immersed in a cold relaxing solution containing (in mmol/l): MgCl₂ 1, KCl 100, Ethylene glycol-bis(amino-ethylether)-N,N,N',N'-tetraacetic acid (EGTA) 2, Na₂ATP 4, imidazole 10 (pH 7.0, adjusted with KOH), kept at about 0°C, and transferred from the operation room to the laboratory in approximately 10 min. Biopsies were divided into two parts. One part (2 mg) was used for mechanical isolation of single myocytes and subsequent force measurement, while the remaining part was frozen in liquid nitrogen, freeze-dried, and stored at –80°C until sufficient samples were collected for protein analysis by SDS–PAGE.

View this table: [in this window] [in a new window]   Table 1 Clinical characteristics of the patients

 
2.2 Force measurements on single myocytes
The myocytes were isolated mechanically as described previously [15, 16]. In short, the samples were mechanically disrupted within a few seconds in relaxing solution using a small glass tissue homogenizer which consisted of a rough glass piston rotating (1000 rpm) rotating inside a small cylindrical beaker of about 0.1 ml in volume. This resulted in a suspension of small groups of myocytes, single cell-sized preparations, and cell fragments, which was kept in relaxing solution at 0°C for 10–24 h. Single myocyte-sized preparations were carefully selected on the basis of size (100–150 µm long by 15–30 µm in diameter) and uniformity of the striation pattern. The preparation was attached with silicon adhesive (Dow Corning, MI, USA) to thin stainless-steel needles (tip diameter about 15 µm) extending from a force transducer (SensoNor, Horten, Norway) and a piezoelectric motor (Physike Instrumente, Waldbrunn, Germany) (Fig. 1). The glue was allowed to cure in 45 min. The force transducer as well as the piezoelectric motor were connected to joystick-controlled micromanipulators. A thin square carbon fibre (length 15 mm, thickness 0.5 mm) was glued to the force transducer element to obtain sufficient sensitivity. The force transducer as well as the extension for the piezomotor were mounted at an angle of 75° with respect to the horizontal plane to reduce the influence of surface tension on the output of the force transducer [19]. The total compliance of force transducer and piezoelectric motor amounted to 0.054 µm/µN. To ensure that the preparations were permeabilized and to improve the quality of the image, the preparation was immersed for 45 s in a relaxing solution supplemented with 0.3% Triton X-100. After this Triton treatment, the preparation was transferred to relaxing solution contained in a small well (volume 80 µl) which had a glass bottom to allow inspection of the preparation during the activation–relaxation cycles. From this well the preparation could be transferred to a similar well containing the activating solution by translation of the stage of the inverted microscope (Fig. 1). During cell attachment and subsequent force measurements, the myocytes were viewed by means of an inverted microscope (Axiovert 40, Zeiss, Oberkochen, Germany) at 320x magnification. Images were recorded by means of a CCD video camera (Philips, Eindhoven, the Netherlands) and stored on a personal computer. Average sarcomere length was determined by means of a spatial Fourier transform [20, 21]and adjusted to 2.2 µm. Fig. 2A shows a human Triton-skinned ventricular myocyte in relaxing solution at an average sarcomere length of 2.15 µm.

View larger version (57K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Schematic diagram of the setup to measure isometric force in single skinned myocytes. In the mounting area (a small microscope slide) a small droplet with the myocyte suspension and a thin line of silicon glue were applied. By lateral movement of the stage of the microscope this area was brought within the field of view. Then the myocyte was attached between the force transducer and the piezoelectric motor, whose positions were controlled by two independent micromanipulators. Prior to the actual force measurement, the myocyte was placed in a droplet of relaxing solution which slightly extended above a temperature controlled well. By translating the stage of the microscope, the myocyte could be transferred to the next temperature-controlled well which contained activating solution. The relaxing and activating solutions were maintained at 15°C by means of a water circulation; the inlet (in) and outlet (out) of this circulation were connected to a temperature controlled water bath.

 

View larger version (79K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 (A) Cardiac myocyte glued between a force transducer and a piezoelectric motor, bathed in relaxing solution viewed in the mounting area. Magnification of the microscope was 320x. The rectangle (80 µm x 27 µm) indicates the area selected for calculation of the average sarcomere length. (B) Power spectrum obtained with the spatial Fourier transform to measure sarcomere length of the myocyte shown in A. The peak in the power spectrum corresponded to a mean sarcomere length of 2.15 µm. Power is normalized to the peak value. The width of this distribution at 50% of the maximal value corresponded to 0.12 µm.

 
For the determination of sarcomere length, a rectangular part of the attached myocyte was selected by means of a cursor-controlled window (see Fig. 2A). The spatial Fourier transform of the intensity distribution along each horizontal pixel line within this window was calculated by means of the fast Fourier algorithm. The amplitude spectra obtained from each line were averaged and the position of the first harmonic in the spatial frequency domain was detected from the midpoint of the integrated power spectrum. This median frequency value was used to calculate the average sarcomere length in the selected window. The width of the power spectrum was used as a measure of sarcomere non-uniformity in the selected area. In Fig. 2B the power spectrum is shown calculated from the selected area of the myocyte shown in Fig. 2A. Sarcomere length was monitored during rest and activation. It should be noted that the quality of the image deteriorated when the myocyte was transferred from the droplet on a microscope slide to the wells with relaxing or activating solution (Fig. 3), because of the long path length in solution (7 mm), reflection of incident light and, occasionally, condensation at the bottom of the temperature controlled well. The diameters of the preparation were measured microscopically, in two perpendicular directions, at a magnification of 320x. The width of a myocyte was measured by means of the calibration image of the CCD camera. Its depth was determined by focusing on the lower and upper surface of the myocyte and measuring the displacement of the objective. Cross-sectional area was calculated assuming an elliptical cross-section.

View larger version (103K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Three successive images taken from a myocyte when viewed in the mounting area (A), in a well containing relaxing solution (pCa=9) (B) and in a well containing activating solution (pCa=4.5) (C). Although the image quality in C was rather limited, the sarcomere pattern in the maximally activated myocyte was still distinguishable and an estimate of the average sarcomere length could be obtained.

 
The compositions of the relaxing and activating solutions used for force measurement were, respectively, as follows (in mmol/l): MgCl₂ 5.42 and 5.26, Na₂ATP 5.45 and 5.53, EGTA 7, imidazole 20, creatine phosphate 14.5 (pH 7.0, adjusted with KOH). The ionic strength of the solutions was adjusted to 180 mmol/l with KCl. The pCa, i.e. –₁₀log[Ca²⁺], of the relaxing solution was 9. The activating solution had a pCa of 4.5. The composition of the solutions was calculated by means of a computer program similar to that described by Fabiato [22]. The calculated free Mg²⁺ and MgATP concentrations were 1 mmol/l and 4 mmol/l, respectively. Solutions with lower free Ca²⁺ concentration 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. The temperature of the solutions during the force measurements was controlled at 15±1°C. The force and, occasionally, the displacement signals were stored after analog-to-digital conversion (at 20 Hz) on a personal computer.

The force measurements started with an activation at saturating calcium concentration (pCa 4.5). Thereafter sarcomere length of the preparation was measured in relaxing solution and readjusted to 2.2 µm if necessary and the measurement was repeated. The baseline of the force transducer signal during activation was determined immediately after transfer of the myocyte to the activating solution, before active force development started. This level corresponded to the passive force level. The second measurement at pCa 4.5 was used to calculate maximal force per cross-sectional area. After three to four activations at submaximal calcium concentrations, another measurement was carried out at pCa 4.5. Force was corrected for myocyte deterioration by linear interpolation between maximal force values. The intermediate results, obtained at higher pCa values, were normalized to the interpolated maximal force values. The measurements were continued until a full force–pCa curve was obtained or until the control force was less than 80% of the first control measurement. To obtain passive force the myocyte was slackened in relaxing solution by means of the piezoelectric motor from the initial sarcomere length of 2.2 µm. The decrease in force during this procedure equals the passive force at 2.2 µm sarcomere length. When the motor was not in use, passive force was obtained from the difference in force found when the myocyte was stretched from a resting sarcomere length of 1.7 µm to 2.2 µm.

2.3 Data analysis
The force–pCa relation was fit by a non-linear fit procedure [23]to a modified Hill equation:

where P is steady-state force. P₀ denotes the steady isometric force at saturating Ca²⁺ concentration, nH represents the steepness of the relationship, and K (or pK) represents the Ca²⁺ concentration (or pCa) at which force=0.5xP₀, i.e. the midpoint of the relation. P₀ was determined from the maximal activation at pCa=4.5.

Single classification analysis of variance (ANOVA), with unequal sample sizes, was performed to determine the inter-patient and inter-myocyte variation [24]. Prior to the single classification ANOVA, the variances of the data from all patients were tested on equality, because homogeneity of variances is an important precondition for the single classification ANOVA. Mean values are given±s.e.m. of n experiments.

2.4 Protein analysis by SDS–PAGE
SDS–PAGE [25]was performed using an acrylamide to bis-acrylamide ratio of 200:1 in the separating gel (12% total acrylamide; pH 9.3) and of 20:1 in the stacking gel (3.5% acrylamide; pH 6.8). A Protean II xi cell was used (Bio-Rad, Hercules, CA, USA). The gel dimensions were width: 16 cm, length 20 cm, and thickness 0.75 mm. The stacking gel was 2.5 cm in length. Lane width was 3.5 mm. The freeze-dried samples were weighed first by means of an electrobalance (Cahn, Cerritos, CA, USA) and subsequently dissolved in a buffer, which contained (in mmol/l): Tris (pH 6.8) 62.5, dithiothreitol (DTT) 15, phenylmethylsulfonyl fluoride (PMSF) 0.1, leupeptin 0.5% and 1% (w/v) SDS, 0.01% (w/v) bromophenol blue and 15% (v/v) glycerol. Comparable samples (5 µl; 1 µg) were loaded in each gel lane. The samples were run at constant current (24 mA) for a total of 5 h (approximately 1800 Volthours). Silver staining was performed as described by Giulian et al. [25]. Contractile protein bands were identified by Western immunoblotting using specific antibodies: -MHC antibody (MAb 249-5A4, 1:50), β-MHC antibody (MAb 169-1D5, 1:100), troponin T antibody (clone JLT-12, Sigma, 1:200), troponin I antibody (MAb 1691, Chemicon, 1:200), actin antibody (clone C4, Boehringer Mannheim, 1:100), the myosin light chain antibody (clone MY-21, Sigma, 1:200) and the tropomyosin antibody (clone TM311, Sigma, 1:400). Molecular weights of the contractile proteins were determined using molecular weight standards (Bio-Rad, high range: 161–0303 and low range: 161–0304) run under identical conditions.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
3.1 Force measurements
The mechanical isolation procedure yielded approximately 1% myocytes suitable for mechanical measurements. Cell attachment was successful in 83% of the attempts, which resulted in the determination of isometric force and its calcium sensitivity in 20 myocytes obtained from five patients. The mean length of the preparations between the attachments was (±s.e.m.) 80±5 µm. The mean diameters, measured in two perpendicular directions, amounted to 29±2 µm and 25±2 µm. Cross-sectional area of the myocytes from patient 3 (Table 2) was larger than the cross-sectional area of myocytes from the other patients, which is in accordance with the enlarged left ventricular mass found in this patient, but the difference was not statistically significant. Sarcomere length for each myocyte was derived from the median frequency value of the integrated power spectrum. The mean value of sarcomere length of all myocytes (n=20) at rest (pCa=9) amounted to 2.16±0.01 µm. A measure of the homogeneity of the sarcomere pattern was obtained from the averaged width of the power spectra at 50% of the peak value. This value corresponded to 0.11±0.01 µm.

View this table: [in this window] [in a new window]   Table 2 General overview of the force measurements

 
The force measurements started with an activation at saturating calcium concentration (pCa 4.5). As seen in Fig. 3, the image quality in the well during maximum activation was rather poor. Nevertheless, sarcomere length could be determined in three myocytes, showing a decrease in sarcomere length of 0.07±0.03 µm, i.e. about 3%. It can be calculated that two-thirds of this internal shortening is due to the total compliance of force transducer and piezoelectric motor. After activation at saturating calcium concentration, three to four contractions were carried out at submaximal calcium concentrations, followed by a control measurement at saturating calcium concentration. The usual number of force measurements performed at maximal and submaximal calcium concentrations per myocyte amounted to nine, of which at least three were performed at maximal activation. Only a small decline in force was present in the preparations. Maximal isometric force at the end of the experiment dropped, on average, to 97% of the maximal isometric force measured at the start of the experiment. An example of a series of five successive measurements is shown in Fig. 4. In this case a maximum force of about 30 µN was attained during maximal activation (pCa=4.5) within about 15 s. Maximum force reached at submaximal calcium concentrations (pCa=5.6, 5.8 and 5.4, respectively) was considerably less. Maximal isometric force measured at the end of this series dropped to 88% of the maximal isometric force measured at the start. An example of force pCa curves obtained from two myocytes from one biopsy (patient 4) are shown in Fig. 5A. It can be seen that little variability exists in the relative forces at corresponding pCa values in the two experimental series. In Fig. 5B, the averaged results are shown obtained from 20 myocytes from five patients. An overview of the average results obtained for each patient and the averaged values derived from the whole group, is shown in Table 2. The average isometric force (±s.e.m.) at saturating calcium concentration obtained on 20 myocytes from five patients amounted to 51±8 kN/m². Passive force at a sarcomere length of 2.2 µm was 3±1 kN/m².

View larger version (15K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Recordings of a series of isometric contractions in a single myocyte at saturating calcium concentration (pCa=4.5), at submaximally activating calcium concentrations (pCa=5.6, 5.8 and 5.4) and again at saturating calcium concentration. The abrupt changes in force mark the transitions of the myocyte through the interface between solution and air. The interrupted lines indicate the passive force level, determined immediately after the myocyte was transferred to the activating solution before active force developed. This level was used as the baseline for the determination of active force development. The difference in the baseline of the force transducer in the relaxing and activating solution was due to surface tension (see text).

 

View larger version (16K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 The Ca2+ sensitivity of isometric force. Isometric force for each myocyte was normalized in the control force found at saturating Ca2+ concentration (pCa=4.5). The force–pCa relations were fitted to a modified Hill equation (A). The force–pCa curves from two myocytes obtained from one biopsy (patient 4). (B). Mean force–pCa curve from 20 myocytes (five patients). The error bars indicate the standard error of the mean. Numbers indicate the number of observations at each pCa value.

 
The variability in force per cross-sectional area between different patients and between different myocytes from each individual patient was appreciable. The relative coefficient of variation (i.e. standard deviation expressed as a percentage of the mean) amounted to 32%. The variance of the average force per cross-sectional area between patients was tested and found to be homogeneously distributed. Single classification ANOVA indicated a significant component of added variance in the average for the whole group of patients. It was found that 59% of the total variation was attributed to variability between patients, while the remaining 41% was due to variation within different myocytes from each patient.

It can be seen from the averaged values from all five patients that force was half maximal at a calcium concentration of 2.47±0.10 µM (pK=5.61±0.02). The steepness of the Hill curve (nH) was 3.55±0.27. These parameters resulted from the averaged pK and nH values of all myocytes. These values indicate that little variation was present in the midpoint and steepness of the force–pCa relationship of myocytes, both within the group of patients and within one patient. No added inter-patient variance was present in these parameters.

The averaged results obtained for patient 3 (with enlarged left ventricular mass) did not differ significantly from the averaged values from the other patients.

3.2 Protein analysis
The dry weight of the samples used for analysis of the protein composition was typically 100 µg. A typical silver-stained 12% polyacrylamide gel of the electrophoretically separated proteins (1 µg dry weight per lane 0.8 µg protein) is shown in Fig. 6, lane E. On this gel only one band was present at the level of myosin heavy chains (200 kDa). In Western Immunoblotting this band was shown to consist mainly of the slow β-myosin heavy chain, while only a minor amount of the fast -myosin heavy chain was detected. For further identification of protein bands, antibodies directed against specific contractile proteins were used in Western immunoblotting. Molecular weights of these bands corresponded to 48, 46, 37, 31, 25 and 20 kDa for actin, troponin T, tropomyosin, troponin I, myosin light chain 1 and myosin light chain 2, respectively.

View larger version (30K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Silver-stained SDS–polyacrylamide gel of myocardial contractile proteins. Lane E, endocardial tissue; lane T, Triton-treated endocardial tissue; lane P, tissue from superficial layer of endocardial biopsy. Protein bands identified: MHC=myosin heavy chain; A=actin; TnT=troponin T; TM=tropomyosin; TnI=troponin I; MLC 1, MLC 2=myosin light chain 1 and 2. The protein band present in untreated tissue (E) at approximately 90 kDa (arrow) disappeared after Triton treatment (cf. lanes E and T). The superficial layer only contained trace amounts of myocardial tissue (lane P).

 
Occasionally, we separated the superficial layer, containing mainly connective tissue, from the endocardial biopsies prior to freeze-drying. One µg dry weight of these samples was also loaded on the gels to see if the connective tissue contained protein bands which interfered with the contractile proteins (Fig. 6, lane P). This, however, proved not to be the case, because only trace amounts of contractile proteins were observed. Moreover, we applied little pieces of ventricular tissue stored overnight in a relaxing solution supplemented with 1% of Triton X-100 (Fig. 6, lane T). This effectively removes the sarcolemmal and sarcoreticular membrane and mitochondria [26]. The densitometric profiles of these Triton-treated samples were very similar to those of the samples that were freeze-dried immediately after arrival in the laboratory, except for a low-intensity band in the upper region of the gel. The molecular weight of this band was approximately 90 kDa, which was determined from the migration distance on the gel, calibrated with the high molecular weight standard (Bio-Rad).

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
The results of this study indicate that it is possible to study the correlation between mechanical contractile parameters and protein composition in small biopsies from human ventricular muscle tissue. Furthermore, data are presented from a group of patients with moderate heart failure, which may be compared with the results from donor or explanted hearts.

Endocardial left ventricular biopsies were obtained from patients undergoing surgery because of aortic valve stenosis. This group of patients was chosen because, as a part of the routine surgery, some tissue became available on a regular basis during open heart surgery. Since from all biopsies myocytes could be isolated, the single myocyte proved to be a useful preparation for measuring cardiac contractile properties.

Single cardiac myocytes have the distinct advantage that contractile properties can be measured without disturbing influences of extracellular matrix components, which are likely to change under pathological conditions. Furthermore, the skinning procedure, during which all membranous structures are removed, allows mechanical measurements under strictly controlled conditions (i.e. intracellular environment). Some shortening of sarcomeres was observed during maximal calcium activation (3%), of which 2/3 can be attributed to the compliance of the pins extending from the force transducer and piezoelectric motor. The remaining part resides in the attachment of the myocyte by the silicone glue. This fairly low compliance in series with the myocyte ensures almost isometric (fixed end) contractions, cf. Moss et al. [15, 16].

4.1 Myocyte isolation
Mechanical isolation yielded approximately 1% suitable myocytes, which is somewhat less than the yield of calcium tolerant cells obtained by enzymatic isolation [27, 28]. However, mechanical isolation has several advantages over enzymatic isolation. Firstly, mechanical isolation can be performed on small biopsies, while relatively large tissue samples are required for enzymatic cell isolation. For instance, Beuckelmann et al. [27], who used large wall segments and perfused their enzyme solution via an artery branch, because of the limited access of enzymes to individual myocytes in a biopsy, report a yield of 5–8%. Harding et al. [28]report a variable yield of rod-shaped cells between 1–10%, and occasionally up to 50–70%. Secondly, the enzymatic isolation procedure needs to be adjusted with respect to the structural properties of the tissue. This is a problem especially when using human tissue, because a priori little is known about structural alterations of the tissue. Thirdly, selection of Ca²⁺-tolerant myocytes may cause a bias when a comparison is made between normal and pathological tissue.

The average cross-sectional area of the myocytes found in this study is 57±7x10^–5 mm². Gerdes et al. [29], who used a Coulter Channelyzer to measure cell volume, obtained smaller values for the cross-sectional area of myocytes. In enzymatically isolated intact cells from nonfailing donor hearts they found a value of 29±5x10^–5 mm², while in diseased hearts (ischemic cardiomyopathy) a value of 25±3x10^–5 mm² was found. The larger values found in our study may reflect cardiac hypertrophy due to pressure overload, because all patients had aortic valve stenosis.

4.2 Force measurements
The maximum force per cross-sectional area (±s.e.m.) corresponded to 51±8 kN/m² (n=20). This value is very similar to the values found in permeabilized cardiac trabeculae from rat [30]and guinea pig [12], and in studies on enzymatically isolated myocytes from rat ventricles [31, 32]. However, considerable variation in force per cross-sectional area was present between patients and also within one patient. On the other hand, little variation was observed in the Hill parameters (pK and nH), which were derived from normalized force values. It is very likely that part of the variation is due to inaccuracy in the determination of cross-sectional area of the myocytes. We used an elliptical approximation of the cross-section of the myocytes which in single muscle fibers proved to provide a precision of 4% [33]. However, an added variation component was found between patients, which cannot be explained solely by the inaccuracy in determination of cross-sectional area. Various factors could be involved in creating the variation between patients. The variation among patients could be an indication that severity of the heart disease affects isometric force, since maximal force development has been shown to decrease with heart failure in rat [21]. It can be noted that left ventricular mass was increased in patient 3, while the force measurements from this patient did not differ from the measurements performed on myocytes from other patients. However, based on this set of data we cannot exclude that a correlation exists between left ventricular mass and isometric force and/or its calcium sensitivity. Other explanations for the observed variability could be differences in contractile protein content of the myocytes and irregularities in cross-sectional shape of the cells.

Wolff et al. [13]recently presented data obtained on mechanically isolated myocytes obtained from large (1–3 g) freeze-dried samples from explanted (failing) and transplanted hearts. The values we obtained for the parameters describing the force–pCa relationship (pK and nH) are comparable to those found by Wolff et al. [13]in myocytes from patients with dilated cardiomyopathy. The average force values they obtained were smaller than those found in our study. This could be due to the severity of the disease state (NYHA class III and IV), a difference in the cause of heart disease (i.e. valvular heart disease versus idiopathic and ischemic dilated cardiomyopathy) and/or a difference in the isolation procedure, since they used myocytes prepared from frozen biopsies. This indicates that further work is needed to determine the maximum force capacity of healthy human myocardium and to establish a correlation between the severity of heart failure and maximal isometric force production.

It should be noted that our values were obtained from patients with moderate heart failure and at 15°C. The experimental temperature of 15°C was chosen to allow a comparison with previous animal studies, from which it also became apparent that sarcomere uniformity during maximum force development was not well preserved at higher temperatures. Although maximum force is not very temperature-sensitive [34], caution should be exerted in extrapolation of the values found in this study to cardiac performance at body temperature.

4.3 Protein analysis
The contractile protein composition of each biopsy could be characterized by SDS–gel electrophoresis and subsequent silver staining. In all samples the β-myosin heavy chain was predominantly expressed, which is in accordance with previous findings on human ventricular tissue [9]. The molecular weights determined for myosin light chains 1 and 2 (25 and 20 kDa, respectively) are very similar to those found by Price et al. [35]. The molecular weight found for tropomyosin equals the molecular weight of the β-subunit of the tropomyosin molecule (i.e. 37 kDa) [36].

Analysis of myofibrillar protein composition by SDS–PAGE from the different biopsies revealed no apparent differences in protein composition. However, protein analysis by SDS–PAGE does not exclude differences between contractile proteins with similar molecular weights (e.g. phosphorylated isoforms, mutations). Further analysis, using two-dimensional protein analysis and molecular sequencing, should be performed to complement this picture.

It was found that the extracellular matrix components present in the tissue from the superficial endocardial layer (Fig. 6, lane P) did not significantly interfere with the contractile proteins. Also, only a slight difference was found between untreated and extensively Triton-treated myocardial tissue, suggesting that membraneous structures do not disturb the contractile protein pattern seen on the gel. The extra 90 kDa band in untreated tissue (Fig. 6) could represent a membrane-bound component of the sarcoplasmic reticulum or mitochondria.

With this analysis it is possible to detect changes in the contractile protein composition which occur under pathological conditions using only small amounts of human myocardial tissue. In fact, we only used a small fraction of the biopsy for protein analysis. Hence, a considerable amount of tissue remains for further detailed analysis of protein composition by means of two-dimensional gel electrophoresis, molecular sequencing, Western immunoblotting, ELISA or immunohistochemistry.

Time for primary review 29 days.

	Acknowledgements

 
We thank Dr. K.T. Strang (Madison, WI, USA) and Dr. P.P. de Tombe (Chicago, IL, USA) for their advice on the myocyte measurements and Dr. J. Graham (Madison, WI, USA) for his expert advice on the SDS–PAGE. We thank Dr. A.F.M. Moorman (Amsterdam, Netherlands) for providing us with the monoclonal antibodies directed against - and β-myosin heavy chains. This study was supported by the Netherlands Heart Foundation (Grant 93.067).

	References

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