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Am J Physiol Heart Circ Physiol 294: H532-H540, 2008. First published October 26, 2007; doi:10.1152/ajpheart.00649.2007
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INNOVATIVE METHODOLOGY

A simple device to apply equibiaxial strain to cells cultured on flexible membranes

Obaida R. Rana,1 Carsten Zobel,2 Esra Saygili,2 Klara Brixius,2 Felix Gramley,1 Thomas Schimpf,1 Karl Mischke,1 Dirk Frechen,1 Christian Knackstedt,1 Robert H. G. Schwinger,3 Patrick Schauerte,1 and Erol Saygili1

1Department of Cardiology, University Rheinisch-Westfaelische Technische Hochschule Aachen, Aachen; 2Laboratory of Muscle Research and Molecular Cardiology, Department of Internal Medicine III, University of Cologne, Cologne; and 3Medical Clinic II, Klinikum Weiden, Weiden, Germany

Submitted 5 June 2007 ; accepted in final form 24 October 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSIONS
 REFERENCES
 
The biomechanical environment to which cells are exposed is important to their normal growth, development, interaction, and function. Accordingly, there has been much interest in studying the role of biomechanical forces in cell biology and pathophysiology. This has led to the introduction and even commercialization of many experimental devices. Many of the early devices were limited by the heterogeneity of deformation of cells cultivated in different locations of the culture plate membranes and were also attached with complicated technical/electronic efforts resulting in a restriction of the reproducibility of these devices. The objective of this study was to design and build a simple device to allow the application of dose-dependent homogeneous equibiaxial static stretch to cells cultured on flexible silicone membranes to investigate biological and biomedical questions. In addition, cultured neonatal rat atrial cardiomyocytes were stretched with the proposed device with different strain gradients. For the first time with this study we could demonstrate that stretch up to 21% caused dose-dependent changes in biological markers such as the calcineurin activity, modulatory calcineurin-interacting protein-1, voltage-gated potassium channel isoform 4.2, and voltage-gated K+ channel-interacting proteins-2 gene expression and transient outward potassium current densities but not the protein-to-DNA ratio and atrial natriuretic peptide mRNA. With both markers mentioned last, dose-dependent stretch alterations could only be achieved with stretch up to 13%. The simple and low-cost device presented here might be applied to a wide range of experimental settings in different fields of research.

equibiaxial stretch; hypertrophy; calcineurin

TISSUE ENGINEERING PLAYS more and more an important role in current science. An important domain of tissue engineering is the investigation of cellular responses to different physiological and pathological influences. This allows a better understanding of cellular mechanisms such as intracellular transduction pathways or genetic modulations. One such physiological or pathological stimulus for cells in the organism is mechanical stretch. Cells from tissues such as lung, heart, skeletal, and smooth muscles and also cells within the scope of neoplastic genesis are subjected to biomechanical environments sufficient to produce stretches up to 20% (24, 25, 30, 39, 43, 44). To study the role of biomechanical forces, different experimental devices to apply mechanical strain have been described previously (1, 4, 14, 16, 17, 21, 26, 27, 29, 35). All apparatus described previously are attached with complicated technical and/or electronic efforts so that the ability of reproduction is limited and not accessible for every scientist. The objective of this study was to design and build a simple, practical, and cost-effective device for applying dose-dependent homogeneous equibiaxial strain to cells cultured on deformable silicone membranes. The method of application of static stretch to deformable silicone membranes with the proposed apparatus is basically similar to the novel system described by Lee and coworkers (18) with the distinction that our apparatus is easy to reproduce, cost-effective and uses commercially available six-well culture dishes (for example, Flexercell, Bioflex Collagen I plates) so that different conditions (for example, pharmacological treatment) can be evaluated at the same time. Furthermore, the device described by Lee and coworkers can apply stretches up to 10%. The device presented in this work is able to apply stretches up to 21% or even more. In addition, we isolated neonatal rat atrial cardiomyocytes and cultured them on flexible silicone membranes. After 24 h of cell isolation, sufficient for cardiomyocytes to attach on the silicone membranes, application of static stretch with the proposed apparatus was introduced for 48 h with different strain gradients (3, 7, 13, 18, and 21%) in a serum-free condition. To demonstrate the effectiveness of the device, biological markers such as calcineurin activity, MCIP1, atrial natriuretic peptide (ANP), voltage-gated potassium channel (Kv) isoform 4.2, and KChIP2 gene expression, transient outward potassium current (Ito) densities, and the protein-to-DNA ratio were measured after 48 h of stretch in a dose-dependent manner. These approved methods were chosen to provide evidence about stretch-induced modifications in all important cell compartments, such as cytoplasm, cell nucleus, and the cell membrane. This is the first study to directly examine the calcineurin activity in neonatal rat atrial cardiomyocytes under conditions of mechanical stretch. Recently, with this device, we have demonstrated that stretch of neonatal rat ventricular cardiomyocytes resulted in calcineurin activation that could be abolished in the presence of cyclosporine A (46).

It is well known that cardiomyocytes respond to stretch with an increase in intracellular calcium concentration through activation of several ionic channels and exchangers (28, 33, 38). By promoting an increase in intracellular calcium concentration, stretch stimulates the calcium/calmodulin-activated cytoplasmic serine/threonine phosphatase calcineurin, a hypertrophic signaling pathway (22, 40, 41). Calcineurin is responsible for dephosphorylation of nuclear factor of activated T cells (NFATc3), leading to nuclear translocation and transcriptional activation of numerous hypertrophy-associating genes (9, 23). As reported previously, ANP and MCIP1 gene expression was enhanced after stretch with 13% (46). In this study, we examined whether there is a dose-dependent enhancement of ANP and MCIP1 mRNA after equibiaxial stretch or not. The gene expression of MCIP1, which has been suggested to fulfill a negative feedback loop restraining calcineurin signaling, is a well-established marker to gain additional information about the calcineurin activity (6). In neonatal rat atrial cardiomyocytes, we recently found that the Kv4.2 gene expression, generally encoding the Ito in the rat (7, 12, 13, 31, 45), was significantly reduced after 48 h of 13% stretch (31). In this work we also evaluated if stretch with different strain gradients (3, 7, 13, 18 and 21%) would result in a down regulation of Kv4.2 in a dose dependent manner and if these alterations would correlate with the corresponding current densities (Ito). Moreover, we investigated whether stretch would result in alterations of KChIP2 mRNA or not. Recently, An and coworkers (2) demonstrated the association of KChIP2 with Kv4.2, leading to an increase in current amplitude of Ito and an acceleration of recovery from inactivation. Therefore, a decrease in KChIP2 is predicted to cause a reduction in Ito amplitude.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSIONS
 REFERENCES
 
All animal experiments were approved by the local and state Ethics in Animal Research Committee.

Design of equibiaxial stretch device for cell cultures. See Table 1 for equibiaxial stretch device specifications.


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  		Table 1. Equibiaxial stretch device specifications

 
Procedure of device reproduction. This device was designed to satisfy several performance criteria. First and foremost, homogeneity of deformations over the surface of the elastic membranes should be maintained. The other important consideration was the ability to easily reproduce the device. For the reproduction of the device, readers should act upon the following recommendations.

To prepare the device, four screws should be fixed at the corners of the lower Plexiglas plate as shown in Fig. 1A. The polyvinyl chloride (PVC) discs are placed/fixed on the lower Plexiglas plate according to the culture dishes. For instance, two conditions (stretched and un-stretched) with three wells each have to be examined in a six-well culture plate and then three PVC discs should be positioned under three silicone deformable membranes. The decision under which three membranes the PVC discs should be positioned is left to the operator because the tension on the membrane is not transmitted to neighboring membranes. Before carrying on, all parts of the stretch apparatus should be sterilized by using 70% ethyl alcohol or by autoclaving. Now the culture plates, for example, the Bioflex Collagen Plate I (Flexcell), with the cultivated cells can be positioned on the PVC discs according to Fig. 1, B and E. Next, the upper Plexiglas plate can be located on top of the culture plate as shown in Fig. 1, C and E. Now the flexible membranes of the culture plates can be subjected to strain with one, two, three, four and a maximum of five rotations of the screws (1 rotation = 360° turn of screw). According to the rotations of the screws, a maximum of 21% of strain can be achieved. Original images of the proposed stretch device are shown in Fig. 2.


Figure 1
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  		Fig. 1. Set up of stretch device. A: 4 screws are fixed at all 4 corners of the lower Plexiglas plate. The polyvinyl chloride (PVC) discs are placed/fixed according to the culture dish plate on the surface of the lower Plexiglas plate. B: depiction of how the Bioflex Collagen Plate I with the cultivated cells is placed on the PVC discs. C: the upper Plexiglas plate is located on top of the Bioflex Collagen Plate I. D: diagram of how to initiate the process of stretching by screwing the 4 butterfly nuts. Hereby, the upper Plexiglas plate will move down slowly. Now the user is able to define the percentage of strain by alteration of the screw rotations [a maximum of 21% of strain can be achieved with 5 screw rotations (each rotation = 360°)]. Through this procedure, the deformable membranes of the culture plate are exposed to homogeneous equibiaxial static stretch. E: data in A–D from another perspective for only 1 well.

 

Figure 2
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  		Fig. 2. Original images of the stretch device in 2 different perspectives.

 
Cell culture. Neonatal rat atrial myocytes were isolated and cultured as described previously (31). Briefly, 20–30 hearts were obtained from 1- to 3-day-old Wistar rats and digested with collagenase and trypsin. Myocytes were purified by passage through a Percoll gradient. Cells were plated on silicone membranes coated with collagen type I (Flexcell) at a density of 1.5 x 105 cells/well. Cells were grown in DMEM/Ham's F-12 supplemented with 10% horse serum and 5% FBS. After 24 h, the serum medium was removed, and the cells were washed and maintained in serum-free DMEM/Ham's F-12.

Application of homogeneous equibiaxial static stretch. After cell isolation, atrial cardiomyocytes were cultivated for 24 h in DMEM/Ham's F-12 supplemented with 10% horse serum and 5% FCS. The time period of 24 h was sufficient for the cardiomyocytes to attach on the membranes of the Bioflex Collagen I plates (Flexcell). Now static stretch was introduced to the attached myocytes for 48 h in a complete serum-free medium by applying a fixed tension [3, 7, 13, 18, and 21% according to the rotations of the screws 1, 2, 3, 4, and a maximal 5 (1 rotation = 360° turn of screw)]. The percentage of strain was determined microscopically using the Nikon Eclipse TS100 F (Fig. 3). Lines of 2,000 µm were marked with specially made templates 3,000, 6,000, and 9,000 µm from the center of the membranes under unstretched conditions. Static stretch was now applied by rotation of the screws as described earlier. The expansions of the lines were determined with the use of a micrometer sheet under the microscope. The measurements were repeated 15 times with 1, 2, 3, 4, and 5 rotations of the screws in all three areas of the silicone membrane (3,000, 6,000, and 9,000 µm; Fig. 3).


Figure 3
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  		Fig. 3. Schematic presentation of determining the percentage of strain by applying a variable tension [with 1, 2, 3, 4, and a maximum of 5 screw rotations (1 rotation = 360° turn of screw)] to the silicone membranes. The percentage of strain was determined microscopically using the Nikon Eclipse TS100 F. A: lines of 2,000 µm are marked with specially made templates 3,000, 6,000, and 9,000 µm from the center of the membranes under unstretched conditions. B: after application of static stretch by rotation of the screws, the expansions of the lines are determined with the use of a micrometer sheet under the microscope. The measurements were repeated 15 times with 1, 2, 3, 4, and 5 rotations of the screws in all 3 areas of the silicone membrane (3,000, 6,000, and 9,000 µm from the center of the membranes).

 
Measurement of protein/DNA ratio. Cells were lysed after 48 h stretching in lysis buffer containing 2.5 mg/ml SDS and 5% saline-sodium citrate. The amount of protein in the lysates was measured with the Bradford method using the DC Protein Assay (Bio-Rad) as described previously (31, 46). The concentration of DNA contained in the lysis buffer was determined using Hoechst 33258 dye (bis-benzimide). The fluorescence was determined with the fluorescence detection system of PerkinElmer LS50B.

RNA preparation and first-strand cDNA synthesis. Total RNA was extracted from atrial cardiomyocytes using the Qiagen RNeasy Mini Kit and following the manufacturer's instructions. A total of 1 µg of RNA was reverse transcribed using random hexamers from the Fermentas First-strand cDNA Synthesis Kit (no. K1622).

Quantitative real-time RT-PCR. Real-time PCR was performed in 96-well plates on the ABI Prism 7700 Sequence Detection System (ABI). Two-step RT-PCR was performed using dilutions of first-strand cDNA with a final concentration of 1x Assays-On-Demand and 1x TaqMan Universal PCR Master Mix (ABI P/N 4324018). The final reaction volume was 50 µl. Each sample was analyzed in duplicate. The thermal cycler conditions were hold at 95°C for 10 min, followed by 40 cycles of 15 s at 95°C (denaturation) and 1 min at 60°C (annealing/extension). To analyze the data, the comparative 2Formula method of data analysis was used for the relative quantification (20). Data were collected with instrument spectral compensation by Applied Biosystems SDS 1.7 software.

Primers and probes for quantitative real-time RT-PCR. PCR primers and fluorogenic probes for the target gene and the endogenous control were purchased as Assays-On-Demand (Applied Biosystems, Foster City, CA). The assay numbers for the endogenous control and target genes were as follows: Rn00560865_m1 (β2-microglobulin), Rn00561661_m1 [natriuretic peptide precursor type A (Nppa)], Rn00581941_m1 (Kv4.2), Rn00596606_m1 (MCIP1), and Rn01411451_m1 (KChIP2).

Measurement of calcineurin activity. Calcineurin activity was measured with the Biomol Green Cellular Calcineurin Assay Kit Plus (AK816) following the manufacturer's instructions, as described previously (46).

Ito measurements in neonatal rat atrial myocytes. To perform Ito measurements, the silicone membrane was cut into small pieces that were placed in the recording chamber. Before performing the experiments, it was verified that the collagen-coated silicone pieces did not contaminate the voltage control of cells by comparing the capacitance of these cells with the capacitance of cells cultured on glass coated with laminin (data not presented). Current densities were recorded using the whole cell patch-clamp technique with a Heka EPC10 amplifier. Micropipettes were pulled from a thin-walled borosilicate glass using a Flaming-Brown micropipette puller (model P-87; Sutter Instruments). The pipette tip was heat polished with a heating filament (MF-830). When filled with intracellular solutions, tip resistances were typically 2.5–3 M{Omega}. Leakage compensation was not used. Only seals in the gigaohm range were taken for measurements. All experiments were performed at room temperature. To measure Ito, the extracellular solution contained (in mmol/l): 140 NaCl, 4 KCl, 1 MgCl2, 10 HEPES, 2 CaCl2, 10 glucose and 0.5 CdCl2, 10 tetraethylammonium, and 0.001 dofetilide [both last-mentioned drugs were used to inhibit the ultrarapid delayed-rectifier current (Ikur) and inward-rectifier potassium current (Ik), after it was verified that neither drug affects Ito], adjusted to pH 7.4 with 1 M NaOH. The intracellular solution contained (in mmol/l): 90 potassium aspartate, 20 KCl, 10 HEPES, 1 MgCl2, 5 Na2ATP, and 5 EGTA, adjusted to pH 7.3 with 2 M KOH.

Statistical analysis. All values are expressed as means ± SE. Comparisons of two groups were made by Student's t-test, and multiple groups were made by one-way ANOVA followed by the least-significant difference post hoc test. Values of P < 0.05 were considered statistically significant. For measurements of calcineurin activity, protein/DNA ratio, and target gene expressions, "n" represents the number of myocyte preparations with each condition studied in triplicate.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSIONS
 REFERENCES
 
Achievement of homogeneous equibiaxial stretch. Because it is known that radial deformation of flexible membranes results in homogeneous equibiaxial strain, different subsequent variants of this approach were obtained (15, 18, 34). The goal of this study was to provide detailed information to assemble a simplified device for in vitro stretch investigations practicable for everyone in different fields of research. This device was designed and built for applying a wide range of homogeneous plane equibiaxial static stretch up to 21% or even more to elastic membranes of commercially available six-well culture dishes. The principle of application of homogeneous equibiaxial static stretch to elastic membranes with the proposed device is basically similar to the method described by Lee and coworkers (18). To demonstrate that our device is able to apply homogeneous strain to elastic membranes, microscopically based measurements of membrane extensions were performed. Figure 4 demonstrates the linear relationship of homogeneous strain achieved in percentage after each rotation of the screws in different areas of the silicone membranes (at 3,000, 6,000, and 9,000 µm distance from the center of the membrane). If a higher or lower percentage of strain is necessary, the height of the PVC discs can be varied. The transmission of mechanical strain to the cultured cells is confirmed by the following data.


Figure 4
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  		Fig. 4. Linear relationship of the screw rotations with the corresponding percentage of strain. All measurements were repeated 15 times with each rotation in each area of the silicone membrane (3,000, 6,000, and 9,000 µm from the center of the membranes). Throughout all measurements in all areas of the silicone membrane there were no significant differences (P < 0.05) in strain, indicating a homogeneous strain of the membrane.

 
Stretch induces activation of calcineurin in a dose-dependent manner. In this study, we demonstrate the repeatable effectiveness and the reliability of the proposed stretch device by measuring the calcineurin activity and MCIP1 gene expression after 48 h of static stretch with different strain gradients (3, 7, 13, 18, and 21%; Fig. 5). For the first time with this study we could demonstrate that mechanical stretch resulted in an increase in the calcineurin activity in neonatal rat atrial cardiomyocytes. In addition, we could show that these alterations were associated with a dose-dependent manner of stretch. Figure 5A shows that stretch with 13% or more resulted in a significant increase (P < 0.05) in the calcineurin activity compared with unstretched control cells [control 0.45 ± 0.02 (n = 5), 3% stretch 0.5 ± 0.04 (n = 5), 7% stretch 0.57 ± 0.02 (n = 5), 13% stretch 0.65 ± 0.03 (n = 5), 18% stretch 0.72 ± 0.05 (n = 5), and 21% stretch 0.8 ± 0.05 (n = 5)]. All further specifications to significant differences are to be taken from the Fig. 5A.


Figure 5
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  		Fig. 5. Atrial myocytes were stretched for 48 h with different strain gradients (3, 7, 13, 18, and 21%) as described in MATERIALS AND METHODS. A: dose-dependent stretch-induced increase in calcineurin activity. Stretch with 13, 18, and 21% resulted in a significant increase in calcineurin activity compared with unstretched control cells (control, n = 5 preparations with 3 samples each; stretch 3, 7, 13, 18, and 21%, n = 5 preparations with 3 samples each). Furthermore, stretch with 18 and 21% resulted in a significant increase in calcineurin activity compared with stretch with 3%. In addition, stretch with 21% resulted in a significant increase in calcineurin activity compared with 7%. B: increase in the relative MCIP1 gene expression by mechanical stretch in a dose-dependent manner up to 21%. Stretch with 13, 18, and 21% resulted in a significant increase in the abundance of MCIP1 mRNA compared with unstretched control cells (control, n = 3 preparations with 3 samples each; stretch 3, 7, 13, 18, and 21%, n = 3 preparations with 3 samples each). Accordingly, stretch with 18 and 21% resulted in a significant increase in the abundance of MCIP1 mRNA compared with stretch with 3%, and stretch with 21% resulted in a significant increase in the relative MCIP1 gene expression compared with 7%. P < 0.05 vs. 13 stretch (*), 18% stretch (**), and 21% stretch (***).

 
Calcineurin is a potential central regulator of cardiomyocyte hypertrophy that also responds to altered calcium handling. Once activated by intracellular increases in calcium, calcineurin mediates the hypertrophic response through its downstream transcriptional effector NFATc3, which is directly dephosphorylated by calcineurin, resulting in nuclear translocation. Previous studies have convincingly demonstrated the sufficiency of calcineurin to mediate cardiac hypertrophy and progressive heart failure (22, 41, 46).

Furthermore, we could show that MCIP1 gene expression, which has been suggested to fulfill a negative feedback loop restraining calcineurin signaling, was upregulated after 48 h of stretch (Fig. 5B). In addition, the stretch-induced increase of MCIP1 mRNA was in a dose-dependent manner, and stretch with 13% or more resulted in a statistically significant upregulation (P < 0.05) of MCIP1 mRNA compared with unstretched cells [control 1 ± 0 (n = 3), 3% stretch 1.19 ± 0.11 (n = 3), 7% stretch 1.48 ± 0.14 (n = 3), 13% stretch 1.82 ± 0.24 (n = 3), 18% stretch 2.01 ± 0.17 (n = 3), and 21% stretch 2.15 ± 0.22 (n = 3)]. These results demonstrate that stretch with the proposed apparatus is associated with changes in biological markers in the cytoplasm and the cell nucleus.

Stretch induces atrial cardiomyocyte hypertrophy through calcineurin activation. In addition, we examined whether stretch would result in an increase in protein/DNA ratio and ANP mRNA expression in a dose-dependent manner, indicating myocyte hypertrophy. Figure 6A demonstrates the stretch-induced dose-dependent upregulation of ANP mRNA with stretch up to 13%. Stretch with 18 and 21% could not further increase the ANP mRNA expression compared with stretch with 13% [control 1 ± 0 (n = 3), 3% stretch 1.12 ± 0.13 (n = 3), 7% stretch 1.24 ± 0.17 (n = 3), 13% stretch 1.43 ± 0.15 (n = 3), 18% stretch 1.47 ± 0.17 (n = 6), and 21% stretch 1.49 ± 0.20 (n = 6)]. Statistically, stretch with 13% and more showed a significant increase (P < 0.05) in the abundance of ANP mRNA compared with unstretched control cells. The results for the measurements of protein/DNA ratio are comparable to the results of ANP mRNA. Similarly, Fig. 6B demonstrates the stretch induced dose-dependent upregulation of the protein/DNA ratio with stretches up to 13%. Accordingly, we could not raise further enhancement of the protein/DNA ratio with 18 and 21% of stretch compared with stretch with 13% [control 1 ± 0 (n = 15), 3% stretch 1.03 ± 0.08 (n = 15), 7% stretch 1.08 ± 0.06 (n = 15), 13% stretch 1.15 ± 0.05 (n = 15), 18% stretch 1.16 ± 0.09 (n = 15), and 21% stretch 1.17 ± 0.12 (n = 15)]. A statistically significant increase (P < 0.05) of the protein/DNA was achieved with stretch of 13% or more compared with control cells.


Figure 6
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  		Fig. 6. Atrial myocytes were stretched for 48 h with different strain gradients (3, 7, 13, 18, and 21%) as described in MATERIALS AND METHODS. Even though A demonstrates the increase in the relative ANP gene expression by mechanical stretch up to 13% in a dose-dependent manner, stretch with 18 and 21% could not further increase the ANP gene expression compared with stretch with 13%. In addition, stretch with 13, 18, and 21% resulted in a significant increase in the abundance of ANP mRNA compared with unstretched control cells (control, n = 3 preparations with 3 samples each; stretch 3, 7, and 13%, n = 3 preparations with 3 samples each; stretch 18 and 21%, n = 6 with 3 samples each). B: stretch-induced increase in the protein-to-DNA ratio in a dose-dependent manner up to 13%. Similarly, stretch with 18 and 21% could not further increase the protein/DNA ratio compared with stretch with 13%, and, accordingly, stretch with 13, 18, and 21% resulted in a significant increase in the protein/DNA ratio compared with unstretched control cells (control, n = 15 preparations with 3 samples each; stretch 3, 7, 13, 18, and 21%, n = 15 preparations with 3 samples each). P < 0.05 vs. 13% stretch (*), 18% stretch (**), and 21% stretch (***).

 
Homogeneous equibiaxial static stretch of atrial cardiomyocytes and electrical remodeling. Moreover, static stretch with the proposed device was not only able to induce changes in the cytoplasm and cell nucleus but also in the cell membrane with alterations in ionic currents (Ito; GoFig. 8). The main gene encoding for the rat atrial Ito under physiological conditions is Kv4.2 (3). Figure 7A shows the stretch-induced dose-dependent downregulation of Kv4.2 gene expression after 48 h of equibiaxial static stretch. Stretch with 13% or more resulted in a significant reduction (P < 0.05) of the abundance of Kv4.2 mRNA compared with unstretched control cells. Furthermore, except the significant difference (P < 0.05) of Kv4.2 mRNA with 3% of stretch compared with 18 and 21%, no further significant differences between the other groups are to be raised [control 1 ± 0 (n = 3), 3% stretch 0.9 ± 0.05 (n = 3), 7% stretch 0.78 ± 0.07 (n = 3), 13% stretch 0.6 ± 0.11 (n = 3), 18% stretch 0.49 ± 0.13 (n = 3), and 21% stretch 0.43 ± 0.15 (n = 3)]. In addition, and for the first time, we could demonstrate that the stretch-induced dose-dependent downregulation of Kv4.2 mRNA correlated with a decrease of the corresponding ionic currents (Ito) in a dose-dependent manner. Current-voltage relationships and typical recordings of Ito with the stated stretch conditions from isolated neonatal rat atrial myocytes are depicted in Fig. 8.


Figure 7
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  		Fig. 7. Atrial myocytes were stretched for 48 h with different strain gradients (3, 7, 13, 18, and 21%) as described in MATERIALS AND METHODS. A: stretch resulted in a dose-dependent decrease in voltage-gated potassium channel (Kv) isoform 4.2 gene expression up to 21% compared with unstretched control cells (control, n = 3 preparations with 3 samples each; stretched 3, 7, 13, 18, and 21%, n = 3 preparations with 3 samples each). Statistically, stretch with 13, 18, and 21% resulted in a significant decrease in the Kv4.2 gene expression compared with control cells. Furthermore, stretch with 18 and 21% resulted in a significant decrease in the abundance of Kv4.2 mRNA compared with stretch with 3%. B: decrease in the relative KChIP2 gene expression by mechanical stretch up to 21% in a dose-dependent manner. Stretch with 13, 18, and 21% resulted in a significant decrease of the KChIP2 mRNA compared with control cells. In addition, stretch with 18 and 21% resulted in a significant decrease of the abundance of the KChIP2 gene expression compared with cells stretched with 3%. Finally, stretch with 21% also resulted in a significant downregulation of the relative KChIP2 gene expression compared with stretch with 13%. P < 0.05 vs. control (*), 3% stretch (**), and 13% stretch (***).

 

Figure 8
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  		Fig. 8. A: typical recordings of transient outward potassium current (Ito) under conditions of mechanical stretch in isolated neonatal rat atrial cardiomyocytes in the presence of tetraethylammonium and dofetilide to inhibit ultrarapid delayed-rectifier current (Ikur) and inward-rectifier potassium current (Ik), after it was verified that neither drug affects Ito (53). The recordings are digital filtered with the Fitmaster Software (Heka, Germany). B: current-voltage (I-V) relation in control cells (13 cells from 9 preparations), cells stretched with 3% (7 cells from 5 preparations), cells stretched with 7% (6 cells from 4 preparations), cells stretched with 13% (9 cells from 5 preparations), cells stretched with 18% (6 cells from 4 preparations), and cells stretched with 21% (5 cells from 4 preparations). All specifications to significant differences are demonstrated. C: cell capacitance of all 46 cells measured for the Ito currents. P < 0.05 vs. control (*) and vs. stretch with 3% (#).

 
The cell capacitance, as an indicator of cell size, also showed a stretch-induced increase in a dose-dependent manner [control 15.67 ± 1.94 pF (n = 13), 3% stretch 15.78 ± 3.93 pF (n = 7), 7% stretch 16.94 ± 3.13 pF (n = 6), 13% stretch 19.57 ± 2.75 pF (n = 9), 18% stretch 20.35 ± 4.83 pF (n = 6), and 21% stretch 21.86 ± 2.18 pF (n = 5)]. Only stretch with 21% showed a significant increase of cell capacitance compared with control cells [control 15.67 ± 1.94 pF (n = 13) vs. 21% stretch 21.86 ± 2.18 pF (n = 5), P < 0.05; Fig. 8C].

Recently, we could show that static stretch of atrial cardiomyocytes with the proposed device resulted also in shortening of action potential duration by downregulation of the Ito, an upregulation of Ikur, and an upregulation of Ik1, correlating with the gene expression data (31). With this work, we complemented our recently presented data for Ito raised with the proposed device in a dose-dependent manner of stretch up to 21%. Moreover, Ito was measured in the presence of 10 mM tetraethylammonium and 0.001 mM dofetilide to suppress Ikur and Ik (19), because in this work our focus was only to measure Ito under conditions of mechanical stretch without involvement of Ikur, which mainly represents the end-pulse steady-state current.

An and coworkers (2) have recently demonstrated the association of KChIP2 with Kv4.2 leading to an increase in current amplitude of Ito. Therefore, a decrease in KChIP2 is predicted to cause a reduction in Ito amplitude. To confirm these findings with a new point of view, in this study we have also investigated the impact of mechanical stretch on KChIP2 mRNA. Stretch resulted in a dose-dependent downregulation of the abundance of KChIP2 mRNA, confirming the findings of An and coworkers (Fig. 7B). Stretch with 13, 18, and 21% resulted in a significant decrease (P < 0.05) in the abundance of KChIP2 mRNA compared with control cells. In addition, stretch with 18 and 21% resulted in a significant decrease (P < 0.05) of KChIP2 mRNA compared with stretch with 3%, and finally stretch with 21% resulted in a significant dose-dependent decrease (P < 0.05) of the amount of KChIP2 mRNA compared with stretch with 13% [control 1 ± 0 (n = 3), 3% stretch 0.85 ± 0.08 (n = 3), 7% stretch 0.64 ± 0.23 (n = 3), 13% stretch 0.44 ± 0.13 (n = 3), 18% stretch 0.31 ± 0.14 (n = 3), and 21% stretch 0.14 ± 0.08 (n = 3)].


   DISCUSSIONS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSIONS
REFERENCES
 
There is presently significant interest in cellular responses to physical forces. This has led to the introduction and even commercialization of many experimental devices. Many studies demonstrate that mechanical stretch can have significant effects on cellular function within physiological and pathological processes (29, 30, 40). Since over 20 years, methods with cyclic and static stretch were often used to apply mechanical load to cells cultured on silicone membranes (1, 21, 26, 27, 35). As with all in vitro systems, each technique has its limitations, and these have been reviewed extensively by Brown (5). However, many devices have been developed to apply stretch to cultured cells, but many of the early devices were limited by the heterogeneity of deformation of cultivated cells in different locations of the elastic membranes. Furthermore, most of the described devices were attached with complicated electronic/technical efforts so that the ability of reproduction of these devices was limited. The objective of this study was to design and build a simple, practical, and cost-effective strain device that imposes homogeneous equibiaxial static stretch to cells cultured on deformable silicone membranes for different fields of research.

The method of application of static stretch to deformable silicone membranes with the proposed device is basically similar to the method described by Lee and coworkers (18) with the distinction that our apparatus is easy to reproduce, cost-effective, and uses commercial available six-well culture dishes (for example, Flexercell, Bioflex Collagen I plates). Furthermore, our device is able to apply stretches up to 21% or even more in contrast to the device described by Lee et al. (10%). The present apparatus has been designed to provide the following features: easy to use, simple to assemble, cost effective, reproducible strain selections up to 21%, simultaneous stretching of multiple samples, with easy installation and switching of individual samples without disturbing the stretching of other samples. The device can be operated in an incubator and sterilized by 70% ethyl alcohol or by autoclaving.

There are several advantages of the present equibiaxial strain system over reported biaxial cell stretch devices (32). Systems involving compressive loading or vacuum suction of an elastic substrate (11, 42) produce strains that are not equibiaxial, which means that stretch along each axis is different and shears may be present. According to the data of Lee et al. (18) and our results, we can conclude that the device described here is sufficient to establish reproducible homogeneous equibiaxial strain up to 21% over a wide range of the elastic membrane. In addition, Lee et al. convincingly demonstrated that the mean equibiaxial strain measured inside the cells cultured on a collagen-coated silicone membrane is not significantly different from the mean strain in the stretched-silicone elastic membrane (18).

In addition, we isolated neonatal rat atrial cardiomyocytes and cultured them on flexible silicone membranes. After 24 h, sufficient for cardiomyocytes to attach on the silicone membranes, application of static stretch with the proposed device was introduced for 48 h with different strain gradients (3, 7, 13, 18, and 21%). To demonstrate the effectiveness of the device, biological markers such as calcineurin activity, MCIP1, ANP, Kv4.2, and KChIP2 gene expression, Ito densities, and protein/DNA ratio were measured after 48 h of stretch in a dose-dependent manner. These approved methods were chosen to provide evidence about stretch-induced modifications in all important cell compartments, such as cytoplasm, cell nucleus, and the cell membrane.

Calcineurin, an intracellular phosphatase, is responsible for dephosphorylation of NFAT, leading to nuclear translocation and transcriptional activation of numerous hypertrophy-associating genes (9, 23). In this study and for the first time, we directly examined the calcineurin activity in neonatal rat atrial cardiomyocytes under conditions of mechanical stretch. We could demonstrate that stretch caused a dose-dependent upregulation of calcineurin. Recently, with this device, we have demonstrated that stretch of neonatal rat ventricular cardiomyocytes resulted in calcineurin activation that could be abolished in the presence of cyclosporine A (46).

As reported previously, ANP and MCIP1 gene expression were enhanced after stretch with 13% (46). In this study, we examined whether there is a dose-dependent enhancement of ANP and MCIP1 mRNA after equibiaxial stretch or not. For the first time we could demonstrate that stretch resulted in a dose-dependent upregulation of the ANP (only up to 13% of stretch) and MCIP1 (up to 21% of stretch) gene expression in neonatal rat atrial cardiomyocytes. The gene expression of MCIP1, which has been suggested to fulfill a negative feedback loop restraining calcineurin signaling, is a well-established marker to gain additional information about the calcineurin activity (6).

In neonatal rat atrial cardiomyocytes, we recently found that the Kv4.2 gene expression, generally encoding the Ito in the rat (7, 12, 13, 31, 45), was significantly reduced after 48 h of stretch with 13% (31). In this work, we also evaluated if stretch with different strain gradients (3, 7, 13, 18, and 21%) would result in a downregulation of Kv4.2 in a dose-dependent manner and if these alterations would correlate with the corresponding current densities (Ito). In a new approach, we could demonstrate that stretch caused a downregulation of Kv4.2 gene expression in a dose-dependent manner. Furthermore, the dose-dependent downregulation of the Kv4.2 mRNA correlated with a downregulation of the Ito density.

Recently, An and coworkers (2) demonstrated the association of KChIP2 with Ito current amplitude, after which one would have to expect a reduction of Ito amplitude by a downregulation of KChIP2 gene expression. This association could be confirmed with the present results. For the first time we could demonstrate that stretch resulted in a downregulation of KChIP2 mRNA in a dose-dependent manner.

Strain with the proposed device resulted in a significant increase in the protein/DNA ratio with 13% of stretch or more. Dose-dependent stretch-induced enhancement of the protein/DNA ratio could only be achieved with stretches up to 13%. Stretch with 18 and 21% could not further increase the protein/DNA ratio compared with stretch with 13%. One explanation for this behavior of cardiomyocytes could be the fact that hypertrophy can only be achieved within a limit and when the limit is passed antagonistic stimuli gain the upper hand so that a further protein synthesis is interrupted.

In summary, we have presented the design and device specifications for the construction of a novel equibiaxial strain apparatus. We characterized the procedure of device production and used element analysis to evaluate the homogeneity of deformation of the elastic membranes exposed to stretch with this device, and measured the strain field experimentally. Finally, we applied static homogeneous equibiaxial strain to cultured neonatal rat atrial cardiomyocytes and investigated the impact of strain to biological markers in a dose-dependent manner.

Environmental changes involved in normal or pathological tissues are supposed to take part at many levels, in organization with morphogenetic movements, or in direct relation with the physical environment in developing or completely differentiated tissues (8, 10, 36, 37). The results obtained from the use of this new device will be valuable for the clarification of the mechanisms of cellular regulation and adaptation to mechanical forces in vivo.


   ACKNOWLEDGMENTS
 
We gratefully acknowledge Prof. M. Kelm (University RWTH Aachen, Germany) and Dr. U. Schotten (University Maastricht, Netherlands) for the realization and complementation of this work. Furthermore, we thank Murat Saygili and Ayaz Hameed Khan for helpful assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: O. R. Rana, Univ. Hospital RWTH Aachen, Dept. I of Internal Medicine, Division of Cardiology, Pulmonary and Vascular Diseases, Pauwelsstr. 30, D-52074 Aachen, Germany (e-mail: orana{at}ukaachen.de)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSIONS
 REFERENCES
 

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