AJP - Heart Calcium Transients and Cell-Sarcomere
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Am J Physiol Heart Circ Physiol 274: H1481-H1488, 1998;
0363-6135/98 $5.00
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Vol. 274, Issue 5, H1481-H1488, May 1998

The trabecula culture system: a novel technique to study contractile parameters over a multiday time period

P. M. L. Janssen, S. E. Lehnart, J. Prestle, J. C. Lynker, P. Salfeld, H. Just, and G. Hasenfuss

Medizinische Klinik III, Universität Freiburg, D-79106 Freiburg, Germany

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

In the intact heart, various triggers induce alterations in gene expression that impact on contractile function. Because changes in gene expression reflect altered protein expression patterns after 12-48 h, we developed a system in which intact twitching cardiac trabeculae can be studied for multiday periods. Right ventricular trabeculae from pentobarbital sodium-anesthetized rabbits were mounted in a sterile, closed muscle chamber. Over the first 48 h, developed force (Fdev) did not significantly change: 102.3 and 98.9% of the initial Fdev was observed after 24 and 48 h, respectively (n = 8). Also, neither diastolic force, time from peak to 50% relaxation (RT50), nor protein synthesis measured by a [3H]leucine incorporation assay changed significantly over time. Contractile response after >48 h to an increase in extracellular calcium concentration (1.8 to 2.5 mM; Fdev increased 43.5%, n = 2) or to 1 µM isoproterenol (Fdev increased 138.6% and RT50 decreased 34.9%, n = 2) was similar to those observed in freshly dissected preparations. In conclusion, this system can investigate contractile function of multicellular preparations under well-defined physiological conditions after events that alter gene and consequent protein expression.

protein synthesis; gene transfer; myocardial contraction; rabbit

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

CHANGES IN MYOCARDIAL PROTEIN expression patterns have been observed in various pathophysiological processes (13, 25, 28). These changes can be induced by mechanical (26) and pharmaceutical interventions (30) as well as by a variety of gene transfer techniques. If these techniques are applied under in vivo conditions in intact animals, alteration of protein expression occurs and functional consequences can be studied by conventional physiological techniques under in vitro or in vivo conditions.

Functional changes in many other tissues have been studied by using cell lines. However, efforts to produce a stable cardiomyotypic cell line have not yet been successful (24), and functional effects of altered protein expression in the heart can as yet only be studied in isolated intact myocytes in primary culture. Altered function due to gene expression and consequent altered protein expression may be investigated in these primary cultures through assessment of function over several days while continuous stimulation of these myocytes preserves contractile function in this preparation (3). Thus, through cultivation and observation of these stimulated isolated myocytes, several functional parameters can be investigated. Despite many efforts, only very modest advances have been made to devise an effective technique to measure force development in intact myocytes (22). This limits the number of contractile parameters to those that can be measured by studying unloaded contractions of myocytes with optical techniques (6).

For in vitro studies, the isolated cardiac trabeculae preparation has proven to be a powerful and reliable tool to assess functional changes in contractile parameters in both normal and diseased animal and human myocardium (2, 5, 8, 29). Experimentally induced gene induction/repression by pharmacological and mechanical events or by gene transfer techniques (9) is translated into a consequent change in the protein expression pattern typically after 12-48 h. Therefore, long-term culture of intact cardiac muscle preparations under physiological conditions is a prerequisite to correlate functional differences with alterations in protein expression induced by external stimuli, such as adenovirus-mediated gene transfer.

Accordingly, the goal of the present study was twofold. First, we aimed to develop a system in which an intact twitching muscle preparation could be studied for a 48-h period by continuous assessment of contractile function and protein synthesis. Second, we characterized the time course of several contractile parameters and tested whether, after cultivation for >48 h, several known interventions (e.g., beta -adrenergic response, increase in extracellular calcium) would evoke contractile responses similar to those in freshly excised preparations. We successfully met the goals set and developed a system in which contractile function is sufficiently stable over a multiday time span while physiological and pharmacological responses are maintained after long-term cultivation of the muscle preparation.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Muscle preparation and experimental apparatus. Rabbits (female New Zealand White Star, weighing 2-3 kg) were heparinized and anesthetized via an ear vein by infusion of pentobarbital sodium (Nembutal, 60 mg/kg). The hearts were rapidly excised and retrogradely perfused through the aortic stump in a modified Langendorff perfusion system with a Krebs-Henseleit solution containing (in mM) 120 NaCl, 5.0 KCl, 2.0 MgSO4, 1.2 NaH2PO4, 20 NaHCO3, 10 glucose, and 1.0 CaCl2 in equilibrium with 95% O2-5% CO2. After a thorough washout of all the erythrocytes, the perfusate was replaced by a similar solution that contained a lower calcium concentration (0.25 mM) and to which 20 mM 2,3-butanedione monoxime (BDM) was added to prevent spontaneous beating of the heart during dissection (20). Thin, uniform, nonbranched trabeculae were carefully dissected from the free wall of the right ventricle. Muscle dimensions were measured through the binocular dissection microscope at ×40 magnification using a calibration reticle that was mounted in one of the oculars (~10-µm resolving power). The average dimensions at slack length of the preparations used in our studies were 3.5 ± 0.3 mm in length, 290 ± 34 µm in width, and 199 ± 28 µm in thickness (means ± SE, n = 8). Muscles were carefully mounted in the apparatus in the same Krebs-Henseleit solution used for dissection.

Trabeculae were mounted between the basket-shaped extension (29) of the force transducer (KG4, Scientific Instruments, Heidelberg, Germany) and a hooklike extension of a micromanipulator screw with the aid of a dissection microscope. This attachment and mounting procedure was chosen because it proved to minimize damage to the muscle preparation. In several pilot series other attachment procedures (silk sutures, stainless steel microtweezers) were explored, but these led to a substantial increase in the decline of force over time and a substantially lower initial force development per cross-sectional area, most likely due to a larger amount of damaged-end compliance that is unavoidable in these preparations (14, 15, 29). The experimental apparatus (Fig. 1) consisted of a Teflon muscle bath with a volume of 2 ml. The basket-shaped extension of the force transducer and the hooklike extension of the micromanipulator screw were guided through small holes in the wall of the muscle chamber that, after positioning of the basket and hook in the chamber, were sealed with low- to medium-viscosity silicone gel. The solution in the muscle bath was stirred continuously using a miniature Teflon-coated stirring bar. The muscle chamber was sealed air-tight with a Teflon/Plexiglas top.


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  		Fig. 1.   A: schematic diagram of experimental setup. Muscle length could be adjusted with the microscrew, and solutions were exchanged via the solution inlet through which a syringe could protrude into the solution reservoir, thus preventing muscle from being accidentally touched. O2/CO2, 95% O2-5% CO2 gas mixture. Water in gas reservoirs was vigorously bubbled with this gas mixture and preheated to 37°C before use, whereas during the experiment the humidity of gas mixture was kept saturated. B: detail of muscle chamber. The silicone O-ring was used to seal the top onto the muscle chamber, whereas silicone-based vacuum grease was used to seal holes through which the force transducer (FT) and microscrew (MS) protruded. A miniature Teflon-coated stirring bar was used to continuously stir solution in the chamber.

All solutions were bubbled by a 95% O2-5% CO2 mixture before use. In the muscle chamber this gas mixture was pumped from a reservoir over the solution through in- and outflow tubes in the chamber top (which had a volume of ~10 ml), and the pH of the solution in the muscle chamber could be maintained between 7.45 and 7.55 throughout the experiment. The outflow of the gas mixture from the muscle chamber was guided to a second gas reservoir, and the pump was located between the two reservoirs. This construction provides a volume buffer both before and after the experimental chamber to avoid peristaltic pressure changes. Overall pressure in the system was maintained constant with the use of a pressure valve. This valve prevents pressure buildup that arises from both a small amount of electrolysis (due to stimulation) and temperature shifts in the gas lines and reservoirs. At the starting point of the experiments the humidity of the system was saturated to minimize water evaporation of the solution in the muscle chamber. During the experiment the humidity of the gas mixture was continuously saturated by guiding the gas through sterilized water in the second gas reservoir. The temperature of the muscle chamber was maintained constant at 37.0 ± 0.2°C throughout the experiments.

It is imperative for these long-term experiments to work under sterile conditions in order to prevent bacterial and/or fungal contamination. During dissection working conditions were kept as sterile as possible, and instruments were sterilized before the experiment. Before each experiment all parts of the setup that would come in contact with solution or gas mixture were gas or heat sterilized, and all tubing was renewed before each experiment. During the experiment medium changes were carried out under sterile conditions. In addition, penicillin and streptomycin were added to the culture medium (for concentrations, see Experimental protocol ).

Experimental protocol. The trabeculae were mounted in the BDM-containing Krebs-Henseleit solution. This solution was exchanged for a BDM-free Krebs-Henseleit solution with a higher calcium concentration (0.5 mM). Stimulation of the muscle was started after a second wash with this solution. Solution changes were always executed in multiple steps by replacing one-half of the solution at a time so as to ensure that the muscle remained under the surface level of the solution. The muscles were stimulated via the hooklike extension of the micromanipulator and the basket-shaped extension of the force transducer at ~30% over threshold voltage (typically 2-4 V) through 5-ms asymmetric pulses at a frequency of 0.5 Hz. Over a 30-min time frame calcium concentration was raised stepwise to 2.0 mM. The muscle was carefully stretched to the length at which passive force development was ~2-10% of active developed force, reflecting a sarcomere length between 2.1 and 2.2 µm (29). After the muscle was left to equilibrate and stabilize for at least 30 min under these conditions, the Krebs-Henseleit solution was exchanged for a modified cell culture medium (M199, Sigma) containing the following additions (in mM): 2.0 L-carnitine, 5.0 creatine, 5.0 taurine, 2.0 L-glutamine, and 0.2% albumin, 100 IU/ml penicillin, 0.1 mg/ml streptomycin, and 20 IU/l human insulin (Opti-Pen, Hoechst, Germany). Because this medium contained a CaCl2 concentration of 1.75 mM, the change from the Krebs-Henseleit solution (with 2.0 mM calcium) resulted in a decline of force (5-25%). After force had stabilized (after 3-7 min, typically), data collection was started. Both the medium and the gas mixture were changed regularly at intervals of 7-10 h.

After 48 h from the start of data collection in several experiments, one of three different protocols was employed. 1) To test for the existence of contractile reserve of the muscle after a minimum of 48 h in culture, calcium concentration was increased to obtain a final concentration of 2.5 mM in the muscle chamber. Contractile response was then measured for an additional hour (n = 2). 2) To test for the existence of a beta -adrenergic response, isoproterenol was added to a final concentration of 1 µM, after which contractile response was then measured for an additional hour (n = 2). 3) No interventions were made, and the muscle was simply kept contracting as long as possible (n = 2).

Measurement of protein synthesis. Protein synthesis was measured in intervals of 12 h during a 0- to 72-h time frame in separate incubations. Incorporation of [3H]leucine was measured as an index for protein turnover as described by Berk et al. (4). Briefly, after multiple muscles were mounted to small glass capillaries, they were incubated in a modified culture medium (DMEM; for additions, see Experimental protocol). At time t = 0, 12, 24, 36, 48, or 60 h, leucine-containing medium was exchanged for leucine-free medium that contained 37 kBq [3H]leucine; the last three groups were pooled into the group t > 36 h. After 12 h of incubation time in the [3H]leucine-containing solution, muscles were removed from the system and thoroughly washed with PBS solution. The central part of the muscle preparations were homogenized in lysis buffer (10 mM Tris · HCl, 2.0 mM EDTA, 2.0 mM EGTA, and 1% Triton X-100), and proteins were precipitated with 10% TCA. Protein pellets were washed with 100% ethanol, air dried, and redissolved in 0.4 N NaOH, and protein concentrations were determined using a bicinchoninic acid protein assay. Aliquots were measured in a liquid scintillation counter. All protein synthesis data were expressed as dpm per microgram of protein per 12-h period (dpm · µg protein-1 · 12 h-1). To serve as a negative control, during a 24-h period in a separate group of muscles protein synthesis was blocked by addition of 50 µM cycloheximide (CHX; a specific translation blocker) to the incubation solution. This entire protein synthesis protocol was performed three times in total. In an additional set of experiments protein turnover rate was compared between nonstimulated (n = 6) and stimulated (n = 5) trabeculae over a 24-h period.

Data analysis and statistics. After the muscle had been equilibrated in culture medium in the muscle chamber, data collection was started. Twitches were monitored on-line by a custom-designed data acquisition and analysis program (written in LabView, National Instruments). For every twitch, peak systolic and diastolic force were recorded on disk, and every 30 min an entire twitch was recorded (1-kHz sample frequency). In addition, just before and after the post-48-h protocol, several twitches were recorded on disk for off-line analysis. To monitor the contractile parameters during the experiment, each twitch could be optionally analyzed on-line. The following contractile parameters were analyzed: peak developed force (Fdev, in mN/mm2), time from stimulation to peak force (TTP, in ms), diastolic force (Fdiast, in mN/mm2), maximum absolute and relative rates of force development [dF/dtmax, in mN · mm-2 · s-1, and (dF/dtmax)/F, in s-1, respectively], maximum absolute and relative rates of force decline [dF/dtmin, in mN · mm-2 · s-1, and (dF/dtmin)/F, in s-1, respectively], time from peak force to 50% relaxation (RT50, in ms), and times from stimulation to 50 and 90% relaxation (TT50 and TT90, respectively; in ms). The start of the experiment (t = 0 h) was taken as ~5-30 min after the Krebs-Henseleit solution was replaced by the medium solution. The initial developed force (Finit, in mN/mm2) was calculated as the average developed force during the first hour after t = 0.

Preparations that displayed rapid rundown of Fdev (>40% in the first 3 h) or preparations in which Finit was <5 mN/mm2 were discarded from further study. Data are presented as means ± SE unless stated otherwise. Student's t-test for paired data (where applicable) was used to determine significant changes. A value of P < 0.05 was considered significant.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

In total, 11 cardiac trabeculae were initially included in the study. The success rate (defined as the percentage of experiments in which Fdev did not decline >50% over the first 48 h) was 73% (8 of 11). With the exclusion of one muscle that displayed >40% rundown in the first 3 h, the success rate was 80%. Thus 8 of 10 consecutively studied muscles could successfully be cultured for 48 h or more. Figure 2B shows the time courses of Fdev and Fdiast over the duration of a single experiment. The arrows indicate points at which the medium was changed. After each medium change (Fig. 2A), Fdev increased slightly, followed by a recovery over the next 5-15 min. This fluctuation is most likely due to a slight decrease in temperature after a medium change.


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  		Fig. 2.   B: time course of peak force development (Fdev; top) and diastolic force development (Fdiast; bottom) over first 48 h of a single experiment. Arrows indicate times at which medium was changed. A: recording of detailed time courses of Fdev and Fdiast of medium change at time t = 18.3 h. Large peaks in detail tracing prior to medium change (*) arise from noise induced by opening and closing of solution inlet.

The average time courses of Fdiast and Fdev are shown in Fig. 3. As can be seen, Fdev at t = 48 h did not change significantly compared with Finit (t = 0 vs. 48 h: 19.6 ± 5.1 vs. 18.9 ± 5.0 mN/mm2, P = 0.29), and Fdiast did not change significantly (t = 0 vs. 48 h: 2.0 ± 0.7 vs. 2.7 ± 1.0 mN/mm2, P = 0.2). In the most extreme case, Fdev had not significantly declined after 110 h, when the experiment was terminated. The fluctuations in developed force are largely due to the fact that very small changes in both sarcomere length and calcium concentration in the studied range translate into considerable changes in developed force (16, 29). In Table 1, Finit, Fdev at t = 48 h, and the type and results of post-48-h protocols are given for each experiment.


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  		Fig. 3.   Average time course of Fdev (top) and Fdiast (bottom) over first 48 h of all 8 trabeculae included in data analysis. So that each experiment had the same weight in data analysis, for each individual experiment Fdev and Fdiast were calculated as fractions of initial developed force (Finit); average normalized data were later multiplied by average force per cross-sectional area for display purposes. Data are means ± SE.

                              
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  		Table 1.   Individual force development data and results of post-48-h protocols

Although small fluctuations in Fdev were observed in most of the preparations over time (see Figs. 2 and 3), twitch timing was stable over the duration of the experiment. In Fig. 4, timing parameters TTP, TT50, and TT90 are depicted. These parameters did not change significantly over the first 48 h of the experiment (t = 0 vs. 48 h: TTP, 228 ± 17 vs. 230 ± 10 ms, P = 0.85; TT50, 321 ± 25 vs. 338 ± 13 ms, P = 0.52; and TT90, 486 ± 30 vs. 525 ± 39 ms, P = 0.33). Very small fluctuations observed were linked to changes in absolute force development. In the experiments in which the muscles were kept well beyond 96 h (n = 2), a slowdown of the twitch was observed well after the first 48 h. After 96 h from the start of the experiment, the average values of RT50% between t = 96 and t = 100 had increased from 128 to 156 ms, whereas (dF/dtmin)/F had decreased from -7.54 to -6.25 s-1 from the mean value over the first 48 h.


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  		Fig. 4.   Average time courses of time from stimulation to peak developed force (TTP; open circle ) and times from stimulation to 50% (TT50; bullet ) and 90% (TT90; triangle ) relaxation for all experiments (n = 8). Changes in these parameters were not significant over first 48 h. Small fluctuations observed were mostly linked directly to fluctuations in Fdev. Data are means ± SE.

Small changes in the mean rate of force development and rate of relaxation (dF/dtmax and dF/dtmin, respectively) can be observed over time. However, these fluctuations are linked to changes in Fdev; the relationship of dF/dt divided by F did not show change over the time course of the experiments and is given in Fig. 5. After 48 h, (dF/dtmax)/F and (dF/dtmin)/F had not changed significantly (8.9 ± 1.4 vs. 8.7 ± 0.9 s-1, P = 0.84, and -7.8 ± 1.4 vs. -6.4 ± 0.9 s-1, P = 0.31, for t = 0 and 48 h, respectively).


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  		Fig. 5.   Average time courses of maximum rate of force development [(dF/dtmax)/F; top] and maximum rate of force decline [(dF/dtmin)/F; bottom] for all experiments (n = 8; data not normalized). No significant changes in these 2 parameters were observed over first 48 h. Data are means ± SE.

To test whether the muscle still possessed its normal regulatory systems, several post-48-h protocols were executed after the muscle had been in the setup for >48 h. In Fig. 6A the typical response of a trabecula to the addition of 1 µM isoproterenol (thick lines) is shown; Fdev increased on average by 139%, whereas RT50 decreased by 35% and (dF/dtmin)/F increased by 40% (n = 2). In the alternative protocol (Fig. 6B, thick lines), calcium concentration was raised from 1.75 mM (standard medium concentration) to 2.5 mM. An average increase in Fdev of 44% was observed, with no apparent changes in other contractile parameters (n = 2). For individual results of the post-48-h interventions, see also Table 1. In addition, in one preparation stimulation frequency was changed to 0.25, 0.5, 1, 2, and 4 Hz after t = 48 h. Similar to that in freshly dissected preparations, systolic force rose with an increase in frequency, whereas at 4 Hz diastolic force also increased (n = 1, results not shown).


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  		Fig. 6.   Effect of post-48-h protocols. A: contractile response to 1 µM isoproterenol (thick line) of a muscle preparation that had been contracting for >48 h (experiment 271097) in experimental setup. Inset: same tracings expressed as fractions of their individual peak developed tension to show effect on twitch timing more clearly. Small vibrations in control tracing (thin line) arise from solution vibrations caused by stirring that sometimes occur. B: contractile response to increase in extracellular calcium concentration from 1.75 to 2.5 mM (thick line). Inset: effect on twitch timing of same data set (experiment 131097). Control twitches of both protocols were obtained several minutes before interventions. Intervention twitches were obtained after force had stabilized (5-15 min).

In Fig. 7 the results of the protein synthesis protocol are given (n = 3). As can be seen, no significant changes in protein synthesis were observed over the time period studied (t = 0-12 h vs. t >36 h; 652 ± 65 vs. 750 ± 129 dpm · µg protein-1 · 12 h-1, P = 0.54). As a control, protein synthesis was blocked to a background level by 50 µM CHX, which resulted in complete blockage of protein synthesis during the first 24 h of incubation (t = 0-12 vs. CHX; 652 ± 65 vs. 14 ± 3 dpm · µg protein-1 · 12 h-1, P < 0.0001). Although limited to 24 h, protein turnover rate of stimulated (1 Hz, n = 5) trabeculae was slightly higher than the rate in nonstimulated (n = 6) trabeculae (1,094 ± 182 vs. 973 ± 367 dpm · µg protein-1 · 24 h-1, not significant).


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  		Fig. 7.   Rate of protein synthesis in a separate group of muscle preparations. In intervals of 12 h incorporation of [3H]leucine was measured as an indicator of protein synthesis. In control experiment, 50 µM cycloheximide (CHX) was added to block protein synthesis. In t > 36 h group, protein synthesis was also measured in blocks of 12 h, starting at either 36, 48, or 60 h after preparation was mounted. Data are means ± SE.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

In the present study we developed a muscle culture system and protocol that allows long-term investigation of contractile parameters in isolated cardiac muscle preparations. Over time periods exceeding 48 h no significant changes occurred in Fdev or any other contractile parameter investigated. Moreover, after this extended period contractile reserve and normal response to beta -adrenergic stimulation were completely preserved.

Characteristics of the system. The following aspects were deemed crucial for successful long-term experiments on isolated muscle preparations: 1) a sterile, closed muscle incubation chamber, 2) the use of cell culture medium with the proper additions, 3) absence of continuous solution flow through the chamber to prevent water evaporation, and 4) use of a special muscle preparation, attachment, and equilibration protocol.

Average initial developed force of the muscle preparations (at t = 0) used in this study was 19.6 mN/mm2. Regarding the conditions during the experiment, i.e., a calcium concentration of 1.75 mM (which is submaximal; Ref. 16), temperature of 37°C, stimulation frequency of 0.5 Hz, and muscle length slightly below maximal active force development (which corresponds to a sarcomere length between 2.1 and 2.2 µm), this is comparable to other studies using the same attachment method for the muscle (7, 14, 16, 29). The use of this particular attachment method is imperative because this method results in the most stable preparation by minimizing the damage to the ends of the preparation and yields significantly higher forces than other attachment methods employed (results not shown). In cell culture it has been shown that myocytes are capable of repairing a limited amount of damage inflicted during isolation (19). In our experiments it could be that, because the attachment procedure limits damage to the ends of the muscle preparation, this damage might be (albeit partially) repaired, given that in some experiments we observed a small increase in force over time. For extended lengths of time (mostly exceeding 48 h) Fdev showed remarkably little or no decline. In several pilot experiments we employed other attachment procedures (stainless steel miniature tweezers, silk sutures), but we were not able to establish a preparation that showed almost no rundown of force over the first 24 h. Moreover, Fdev of all of the preparations mounted in the chamber with the use of other methods decreased to <50% of Finit in the first 24 h and could not be stably maintained for extended periods. It is also noteworthy that, although measured under slightly different experimental conditions, a continuous medium perfusion with a constant flow rate gave no satisfying results in terms of preparation stability and longevity of the preparation.

Regular changing of the medium and gas mixture in the reservoirs (every 7-11 h) was able to prevent larger fluctuations in contractile parameters that were observed when this medium change was done less frequently (>14 h, measured in pilot experiment). In the latter case, the pH of the medium increases from 7.45 to ~7.65, inducing a slight increase in developed tension (18). Changing the medium then results in a small decline in tension (due to decrease of pH) rather than the slight increase that usually was observed. This transient increase in Fdev is most likely due to a small decrease in temperature (17). Several minutes after the medium was changed (with ~50% of the total medium in the chamber, see MATERIALS AND METHODS), Fdev gradually returned to prechange levels over the next 5-20 min, when the medium solution temperature returned to 37°C.

To demonstrate that a contractile reserve was still present after this long-term culturing of the muscle, the calcium concentration in the medium was increased to a final concentration of 2.5 mM. This resulted in a significant increase in Fdev, a response similar to that in freshly dissected preparations (16). To investigate whether the beta -adrenergic pathway was intact after a minimum of 48 h in culture, isoproterenol (final concentration of 1 µM) was added to the medium. Fdev increased to ~239% of preisoproterenol values. This observed increase in force and decrease in relaxation time (RT50 was reduced by ~35%) is in agreement with previous studies (7, 12) in freshly excised muscle preparations on the effect of beta -adrenergic stimuli. This implies that the beta -adrenergic signaling pathway is still intact and that effects of positive inotropic agents can reliably be studied after 48 h in this system. Moreover, after 48 h of culture time, the preparation displayed preservation of the positive force-frequency relationship.

The absence of decay of force, the stability of the timing parameters, and the intact responses of pathways to increases in calcium concentration and beta -adrenergic stimulation may suggest that the protein metabolism is intact in these preparations. Nonetheless, we tested protein turnover rate directly using a [3H]leucine incorporation assay. In blocks of 12 h, protein synthesis was not altered over a 48-h period, implying that protein turnover is well maintained in these preparations. Although the preparations in this set of experiments were not stimulated, all other experimental conditions were kept similar to the stimulated trabeculae (extracellular calcium concentration, degree of stretch, composition of medium supplements). Moreover, although only measured over the first 24 h, protein turnover rate was slightly higher (but not significantly) in stimulated versus nonstimulated preparations.

Comparison with other methods. In cardiac tissue it has proven difficult to study functional changes after events that lead to altered gene expression. In other tissues stable cell lines have been employed for this purpose. Unfortunately, no stable cardiomyotypic cell line is available for such purposes. Freshly isolated myocytes can be brought into primary culture (11) and when continuously stimulated, unlike nonstimulated myocytes, will maintain contractile function (3). Thus the single intact myocyte can be a valuable tool in the study of cardiac function (6). However, assessment of contractile parameters in the intact stimulated myocyte is limited to unloaded shortening velocity experiments that might not necessarily reflect the loaded contractions as they occur in vivo, or calcium transients for which the measurement is complicated by shortening per se (15) and calibration of the calcium signals (1, 27). Although single-cell experiments are a valuable tool in cardiophysiological research, given the critical role of the extracellular matrix, cell-to-cell connections, electrical and mechanical conductance, and the presence of noncardiac myocytes (e.g., endothelial cells and fibroblasts) in the contractile function of cardiac tissue, the trabecular preparation clearly has several advantages over single-cell experiments in the study of contractile function of the heart (5).

Because an altered protein expression pattern will not have developed within the first few hours after a gene induction, this system allows the study of functional parameters before and after the established protein expression in the same muscle. This not only might reduce variability between groups but also allows for paired testing and the associated reduction in population sample size.

Limitations of the technique. The current configuration of the experimental setup limits the use of very small muscle preparations. Initially, trabeculae as small as 70 µm in width and 40 µm in thickness were mounted in the system. However, the absolute amount of force generated by these preparations was only a factor of four to five times larger than the noise of the force signal. This was due to the relatively low sensitivity of the force transducer, because its basket-shaped extension has to pass through a layer of silicone grease used to seal the bath. Although these preparations could also be maintained for extended periods (and actually seem to display higher-than-average forces per cross-sectional area, likely due to a relatively reduced content of noncontractile material), the obtained signals with low signal-to-noise ratios were not suitable for accurate analysis of contractile parameters. Preparations that are too large, however, might suffer from diffusion problems and may develop a hypoxic core. In fact, the two preparations that were discarded from analysis (although they met the initial conditions) and went into a rigor state at approximately t = 35 h were the largest and second-from-largest preparations with regard to cross-sectional area. This implies that we could successfully culture all muscles within a certain optimal size range; under the conditions of this series of experiments, the optimal size for the preparations ranges from 200 to 350 µm in width and from 150 to 250 µm in thickness. Length of the preparation is less important, although the relative impact of damaged-end compliance might be less in long preparations due to the increased central segment-to-damaged end ratio.

A second limitation is that the absolute mass of these preparations (236 ± 67 µg, n = 8) might limit analysis of protein content in some cases. This could be overcome by running several parallel experiments. This has not only the advantage of increased muscle mass through pooling but also overcomes the fact that, with a single setup, one experiment (including building up, cleaning, and sterilizing) takes 4-6 days and thus yields only one data set per week.

Possible applications. In this culturing model, contractile parameters are stable over a long period of time, thereby extending the useful life of continuously twitch-contracting cardiac preparations. This system now provides a novel tool in which long-term effects of interest in pharmacological and toxicological studies, as well as biochemical studies of molecules with low turnover rates, can be investigated. This tool provides a possible way to study the aforementioned effects, and the extended life of a preparation might allow more experimental time for investigations on the same preparation.

Both maximal rate of force development and maximal rate of force decline showed no apparent change over the studied period. Moreover, no significant changes were observed in the TTP and in RT50, indicating that not only was force development stable but also twitch timing was not altered by culturing over periods >48 h. The very small fluctuations observed in timing parameters were directly linked to the fluctuations in Fdev. This is due to the fact that an increase in force development is correlated with an increase in twitch duration (14). Thus interventions/events that have been shown to impair relaxation of cardiac muscle (and thus alter twitch timing), such as acute global ischemia (21) and congestive heart failure (10), might be investigated using this developed "trabecula culture system."

Also, this novel tool can be usefully employed in the study of contractile effects of alterations in protein expression induced by physiological or pharmacological interventions or by altered gene expression through introduction of genetic material via viral transfer systems or other methods. The time necessary from a trigger event that initiates altered protein expression to the actual functional incorporation of the protein into the metabolism of the cells exceeds the time in which muscle preparations can be studied with methods available thus far. Thus, by abridging the period needed for cells to express functional differences in this muscle culturing tool, the system might be specifically useful in studies on these trigger events such as mechanical stress (23), gene transfer (9), or hormones (4).

    ACKNOWLEDGEMENTS

We gratefully acknowledge Dr. K. Güth, Scientific Instruments, Heidelberg, Germany, for technical assistance in the design of the setup.

    FOOTNOTES

This project was supported in part by a grant from the Deutsche Forschungsgemeinschaft (HA 1233/3-2) and by a grant from the Zentrum Klinische Forschung, Universitäts Klinik Freiburg, Freiburg, Germany.

Address for reprint requests: P. M. L. Janssen, c/o G. Hasenfuss, Molecular Cardiology, Med. Klinik III, Universität Freiburg, Breisacherstrasse 33, D-79106 Freiburg, Germany.

Received 24 November 1997; accepted in final form 20 January 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Heart Circ Physiol 274(5):H1481-H1488
0363-6135/98 $5.00 Copyright © 1998 the American Physiological Society


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