Organ Explant Culture of Neonatal Rat Ventricles: A New Model to Study Gene and Cell Therapy

Testing cardiac gene and cell therapies in vitro requires a tissue substrate that survives for several days in culture while maintaining its physiological properties. The purpose of this study was to test whether culture of intact cardiac tissue of neonatal rat ventricles (organ explant culture) may be used as a model to study gene and cell therapy. We compared (immuno) histology and electrophysiology of organ explant cultures to both freshly isolated neonatal rat ventricular tissue and monolayers. (Immuno) histologic studies showed that organ explant cultures retained their fiber orientation, and that expression patterns of α-actinin, connexin-43, and α-smooth muscle actin did not change during culture. Intracellular voltage recordings showed that spontaneous beating was rare in organ explant cultures (20%) and freshly isolated tissue (17%), but common (82%) in monolayers. Accordingly, resting membrane potential was -83.9±4.4 mV in organ explant cultures, −80.5±3.5 mV in freshly isolated tissue, and −60.9±4.3 mV in monolayers. Conduction velocity, measured by optical mapping, was 18.2±1.0 cm/s in organ explant cultures, 18.0±1.2 cm/s in freshly isolated tissue, and 24.3±0.7 cm/s in monolayers. We found no differences in action potential duration (APD) between organ explant cultures and freshly isolated tissue, while APD of monolayers was prolonged (APD at 70% repolarization 88.8±7.8, 79.1±2.9, and 134.0±4.5 ms, respectively). Organ explant cultures and freshly isolated tissue could be paced up to frequencies within the normal range for neonatal rat (CL 150 ms), while monolayers could not. Successful lentiviral (LV) transduction was shown via Egfp gene transfer. Co-culture of organ explant cultures with spontaneously beating cardiomyocytes increased the occurrence of spontaneous beating activity of organ explant cultures to 86%. We conclude that organ explant cultures of neonatal rat ventricle are structurally and electrophysiologically similar to freshly isolated tissue and a suitable new model to study the effects of gene and cell therapy.


Introduction
New therapies for various cardiovascular diseases are currently being developed with a focus on gene and cell therapy. For gene therapy, lentiviruses are attractive vectors because they are capable of transducing non-replicating cells [1] (e.g., cardiomyocytes), stably integrating the gene of interest into the host genome [2], and carrying relatively large genes [3]. The expression of lentivirally-transduced genes requires several days. This time is needed to allow the viral mRNA to be reverse transcribed into DNA, which is required for subsequent integration and expression. Evaluating the effects of cell therapy may also require several days [4]. Experimental models to test gene and cell therapy for cardiovascular diseases should therefore replicate the in vivo situation over a period of at least days. Although this may be achieved via animal studies, these studies have important drawbacks, including their high cost and labor intensity. Therefore, suitable in vitro models are important, especially as an initial strategy screening tool. While current in vitro models, such as cultured neonatal rat ventricular cardiomyocytes, provide the opportunity of testing cells over 1-2 weeks, important properties of the in vivo situation are lost in this system. For instance, neonatal rat ventricular cardiomyocytes cultured in a syncytium (monolayer) show a flat, star-shaped morphology instead of the normal rodlike shape, along with a reduction in peak transient outward current, sodium current, and calcium current [5]. Furthermore, these cells display a depolarized resting membrane potential (RMP), a reduced action potential upstroke velocity, and spontaneous beating [6,7]. These properties are correlated to the loss of I K1 [8] and the number of myofibroblasts present in these cultures [9]. Miragoli et al. described a fast phenotypic switch from fibroblasts to myofibroblasts when in culture [10]. These myofibroblasts expressed both connexin-43 and connexin-45 at contact sites between each other and with cardiomyocytes. Since myofibroblast have a more depolarized membrane potential, coupling between myocyte and myofibroblast is believed to induce depolarization in the cardiomyocyte, thereby causing depolarization-induced automaticity [11].
Several studies describe methods to limit the changes due to cell culturing, including anisotropic growth and the use of pharmacologic agents and cell separation to reduce the number of (myo) fibroblasts [10,12]. However, a 3-dimensional system would still be preferable over a 2-dimensional cell culture for various reasons, including differences in gene expression, regulation of cell growth and fibroblast morphology [13]. Furthermore, the 3-dimensional structure more closely resembles the in vivo situation and may be particularly relevant when studying electrical phenomena such as current-to-load mismatch [14].
Clearly, the development of a 3-dimensional cardiac in vitro model that remains viable for several days, yet maintains its electrophysiologic properties, is crucial to reliably study various gene-and cell-based therapies. Previous studies have reported on Figure 1. Organ explant cultures are (immuno) histologically similar to freshly isolated tissue. From left to right: organ explant cultures, freshly isolated tissue, and monolayers. A, Hematoxylin-eosin staining. B, Immunohistochemistry for a-actinin (green). C, Immunohistochemistry for connexin-43 (green). D, Immunohistochemistry for a-smooth muscle actin (green). Nuclei are stained with sytox orange (red). doi:10.1371/journal.pone.0059290.g001 short-term culturing of cardiac tissue slices. However, extensive electrophysiologic characterization after several days is lacking from these reports [15][16][17]. Therefore, in the present study, we investigated the (immuno) histologic and electrophysiologic properties of 6-8 days old cultured intact cardiac tissue (organ explant culture). We compared outcomes to both freshly isolated cardiac tissue and to monolayers to address the following questions. (1) Are organ explant cultures viable for 6-8 days? (2) How do the (immuno) histologic and electrophysiologic properties differ among these in vitro systems? (3) Can organ explant cultures be transduced by lentiviral vectors? (4) Can organ explant cultures functionally couple to spontaneously beating neonatal rat ventricular cardiomyocytes?

Preparation of organ explant cultures and monolayers
All animal experiments were approved by the local Animal Experiments Committee (Academic Medical Center, University of Amsterdam; DCA102499) and carried out in accordance with national and institutional guidelines.

Histology
After embedding freshly isolated tissue and organ explant cultures in Tissue-Tek O.C.T (Sakura, # 4583), 10 mm thick sections were cut by microtome and stored at 220uC. For fixation of monolayers, glass coverslips with cells attached were washed with PBS twice and then covered in methanol (220uC) for 2 minutes. Subsequently, the coverslips were washed with PBS 3 times. Fixed monolayers were kept at 4uC.
HE staining. For hematoxylin-eosin (HE) staining, tissue sections were thawed for 2 hrs and fixed in 4% PFA for 10 minutes. Afterwards, both the fixed sections and the previously fixed monolayers were washed with distilled H 2 O, stained with hematoxylin (Sigma) for 5 minutes, washed with H 2 O, and stained with eosin (Sigma) for 30 seconds. After washing with H 2 O, the preparations were shortly rinsed with 70% ethanol, 96% ethanol and 100% ethanol, after which they were left in xylol (Bufa) for 5 minutes and mounted with pertex (Histolab).

Intracellular recordings
To assess RMP, intracellular microelectrode recordings were made of tissue and cell cultures at 37uC in Tyrode's solution, which contained (mM) NaCl 140, KCl 5.4 mM, CaCl 2 1.8, MgCl 2 1.0, glucose 5.5, HEPES 5.0 (pH 7.4 with NaOH). Microelectrodes were filled with 3 M KCl, and tip resistance was 15-35 MV. A reference electrode was placed in the tissue bath. Action potentials were recorded at 16 kHz using a data acquisition system (Biosemi Actiview), and analyzed using custom-made software based on Matlab (Mathworks). We characterized tissue by (1) the presence of spontaneous beating activity, (2) the presence of phase 4 depolarization, defined as a diastolic depolarization rate of at least 5 mV/s measured over the 50 ms interval starting at the maximal diastolic potential (MDP), and (3) MDP or RMP (MDP in case of preparations showing phase 4 depolarization; RMP in case of quiescent tissue). Action potential amplitude, action potential duration (APD), and upstroke velocity were not measured due to difficulty keeping the microelectrode intracellular during contraction, especially in organ cultures.

Optical mapping
To assess conduction velocity (CV) and action potential properties, optical mapping of tissue and cell cultures was done. After exposure to 15 mM di-4-ANEPPS (Invitrogen, #D1199) in EBSS (Gibco, #24010) for 7.5 minutes, explants were placed in an optical mapping set-up in Tyrode's solution (37.060.5uC) containing (mM) NaCl 140, KCl 5.4 mM, CaCl 2 1.8, MgCl 2 1.0, glucose 5.5, HEPES 5.0 (pH 7.4 with NaOH) and blebbistatin 10 mmol/L (Tocris Bioscience, #1760). Six power light emitting diodes (5 W per diode, band-pass filtered 510620 nm) delivered excitation light. Emission fluorescence (filtered .610 nm) was transmitted through a tandem lens system on a Complementary Metal Oxide Semiconductor chip (Micam Ultima) with a spatial resolution of 1006100 pixels. Data acquisition was carried out with a sample frequency of 1 kHz for freshly isolated tissue and organ explant cultures, and 0.5 kHz for monolayers (because of lower fluorescence intensity in monolayers). Data were analyzed using custom made software based on Matlab (Mathworks). We characterized tissue by (1) the presence of spontaneous beating activity, (2) CV, (3) anisotropy ratio, (4) maximal upstroke velocity, (5) APD at 20%, 50%, 70%, and 90% repolarization (APD20, APD50, APD70, APD90), and (6) the ability to stimulate tissue at different cycle lengths (CLs) varying from 500 ms to 100 ms. CV and maximal upstroke velocity were measured at a pacing CL of 500 ms. CV was calculated from activation times -based on time of maximal upstroke velocity -recorded at 2 sites along a line perpendicular to the isochronal lines, when stimulated at 2 times stimulation threshold at a cycle length of 500 ms. The anisotropy ratio was defined as maximal CV divided by CV measured perpendicular to maximal CV. Maximal upstroke velocity was determined in normalized action potentials -baseline was set to 0 and maximal amplitude to 100 -and defined as the maximal rise in percentage of action potential amplitude per ms (d(%APA)/ dt max ). Because of the lower sample frequency, this could not be measured in monolayers.

Gene and cell therapy
A lentiviral Egfp expression vector (pRRL-cPPT-CMV-X2-IRES-Egfp-PRE-SIN lentiviral transfer vector, from here on called LV-Egfp) was made as described previously [20]. Virus was produced via cotransfection of HEK293T cells, and titrated by detection of EGFP expression 3 days after transduction of HeLa cells.
To test the feasibility of gene transfer we injected virus LV-Egfp (1610 6 IU) into organ explant cultures at day 0, before attachment of the tissue to the gel, in the presence of dextran (10 Mg/mL;

Statistical analysis
Data are presented as mean 6 SEM. Statistical tests used include Student's t-test and one-way ANOVA for normally distributed continuous data and Pearson's chi-squared test or Fisher's exact test for categorical data. P,0.05 was considered statistically significant. SPSS 18 was used for statistical analysis.

Histology
Hematoxylin-eosin (HE) stained organ explant cultures had a more loose tissue structure than freshly isolated tissue, but maintained a longitudinal fiber orientation. In contrast, monolayers did not show a longitudinal fiber orientation ( Figure 1A). Immunohistochemistry for the cytoskeletal protein a-actinin, which is localized to the Z-discs, showed a striated pattern in all groups ( Figure 1B). Also, immunostaining for the gap-junction protein connexin-43 showed a similar pattern in all 3 groups, i.e., localization not only to the intercalated discs, but along all cell borders ( Figure 1C). Expression of a-smooth muscle actin, a marker for myofibroblasts and smooth muscle cells, was abundantly present throughout the whole preparation in monolayers, while this was not the case in both freshly isolated tissue and organ explants cultures. In the example of freshly isolated tissue shown in Figure 1D, a-smooth muscle actin was present in vascular smooth muscle cells. Table 1 shows results from microelectrode measurements; Figure 2 depicts typical examples of stimulated action potentials when stimulated at 500 ms CL. Twenty percent of organ explant cultures (n = 10; 4 left ventricle, 3 right ventricle, 3 septum) showed spontaneous beating activity. This proportion was similar to freshly isolated tissue (17% [n = 6; 2 left ventricle, 2 right ventricle, 2 septum], p = NS). In contrast, 82% of monolayers (n = 17) beat spontaneously (p,0.05 vs. both other groups). Accordingly, we observed phase 4 depolarization in 20% of measurements in organ explant cultures, never in freshly isolated tissue, and in 67% of the monolayers (p,0.05 vs. both other groups).

Intracellular recordings
RMP of organ explant cultures was similar to freshly isolated tissue (283.964.4 mV and 280.563.5 mV, respectively, p = NS), while being more depolarized in monolayers (260.964.3 mV; p,0.05). Maximal CV of organ explant cultures was similar to that of freshly isolated tissue (18.261.0 cm/s and 18.661.0 cm/s, respectively, p = NS), but lower than in monolayers (24.360.7 cm/s, p,0.05 vs. both other groups). Electrical conduction remained anisotropic in organ explant cultures (anisotropy ratio 2.560.1), similar to freshly isolated tissue (2.160.2, p = NS), while conduction in monolayers was isotropic (1.260.04, p,0.05 vs. both other groups). Figure 3 shows typical examples We did not observe a difference in d(%APA)/dt max between organ explant cultures and freshly isolated tissue (46.862.4 and 50.461.9 %APA/ms, p = NS).
We further established that the composition of the culture medium was not of influence on monolayer characteristics ( Figure 5). Monolayers grown in organ explant medium (n = 10) showed electrophysiologic characteristics similar to monolayers grown in monolayer medium (n = 9). Both groups showed a high occurrence of spontaneous beating activity (89% vs 90%), similar CL of spontaneous beating activity (399637 vs 479686), and no differences in CV, APD, and capture when stimulated at different CLs (all p = NS).

Gene and cell therapy
After establishing that electrophysiologic and histologic properties of organ explant cultures were highly comparable with freshly isolated tissue, we next tested how well organ explant cultures were suitable for lentiviral gene transfer. Injection of LV-Egfp into the center of organ explant cultures (n = 4) resulted in expression of EGFP at the site of injection ( Figure 6A).
Finally, we investigated whether organ explant cultures may be useful in evaluating the effects of cell therapy, by testing their capability to couple to other cardiomyocytes. Here we found the addition of 0.3610 6 spontaneously active neonatal rat ventricular cardiomyocytes to increase the percentage of spontaneously beating organ explant cultures to 86% (CL 605.8670.8 ms; Figure 6B). In comparison, above reported unmodified organ explant cultures exhibited spontaneous beating only in 28% (n = 32) of the samples (p,0.05). Figure 6C shows a typical example of an isochronal activation map, with spontaneous beating originating from the monolayer of isolated neonatal rat ventricular cardiomyocytes, with capture of the organ explant culture.

Discussion
Interpretation of the results from in vitro studies on gene therapies in culture models of neonatal rat ventricular cardiomyocytes is often confounded by the occurrence of a partial loss of the native physiologic properties. In the present study, we investigated whether a new in vitro model of long-term organ explant culture retained its in vivo properties, and whether this model is suitable for studying gene-and cell-based therapies. Using (immuno) histochemical and electrophysiologic analysis, we demonstrated that long-term heart organ explant cultures were viable up to 8 days in culture, while still retaining multiple critical properties of freshly isolated cardiac tissue. Cardiomyocytes in organ explant culture retained their longitudinal fiber orientation, while expression patterns of the structural proteins a-actinin, connexin-43, and asmooth muscle actin were similar to freshly isolated cardiac tissue. Also, the electrophysiologic properties were largely preserved in organ explant cultures. As in freshly isolated cardiac tissue, most of the cultures did not show spontaneous beating activity and had stable RMP of approximately 285 mV. In addition, CV, anisotropy ratio, maximal upstroke velocity, and APD were unaltered.
In comparison, alterations in (electro) physiologic properties in monolayers were more pronounced. Immunoreactivity for asmooth muscle actin was abundantly present throughout monolayers, indicative for the presence of myofibroblasts. Moreover, monolayers often beat spontaneously, and showed a depolarized RMP, an increased presence of slow diastolic depolarization, a loss of anisotropic conduction, and a prolonged APD as compared to freshly isolated tissue. Also, while organ explant cultures could be paced at frequencies similar to freshly isolated tissue, monolayers were more frequently not captured at short CLs.
Finally, we demonstrated that organ explant cultures can be transduced with LV-Egfp (inferred from the expression of EGFP), and are capable of functional coupling to spontaneously beating neonatal rat ventricular cardiomyocytes.

Origin of a-smooth muscle actin expression in monolayers
The expression of a-smooth muscle actin in organ explant cultures and freshly isolated tissue is related to the presence of smooth muscles in the vasculature [21]. In monolayers, the origin of the a-smooth muscle actin is less clear. While recent reports suggest that this is due to the presence of myofibroblasts [11,12], it could also be the result of dedifferentiation of cardiomyocytes, since a-smooth muscle actin is also temporarily expressed in fetal rat cardiomyocytes [22]. Zhang et al. have shown that cultured cardiomyocytes can dedifferentiate and express early transcription factors, but they did not investigate the expression of a-smooth muscle actin in these cells [23]. In vivo, the expression of a-smooth muscle actin in both adult cardiomyocytes and myofibroblasts has been described in cardiac diseases such as atrial fibrillation and right ventricular pressure overload [24,25].

Myofibroblasts, MDP, and spontaneous activity
Several studies investigated the effects of different amounts of (myo) fibroblasts on MDP and spontaneous beating activity of monolayers [10,26]. In these studies a preplating step was used to separate fibroblasts from cardiomyocytes, and bromodeoxyuridine was added to influence the growth of (myo) fibroblasts present in culture. Miragoli et al. described a decrease in MDP from approximately 280 mV to 250 mV when the amount of myofibroblasts increased. In that study, experiments were performed in 324 days old preparations. Since we want to use our model to further study the effect of lentiviral gene transfer after 6 days, we tried to decrease the number of myofibroblasts this way. However, in our experience, decreasing the amount of (myo) fibroblasts via preplating and bromodeoxyuridine was only effective for a limited amount of time. Alternatively, pharmacologic interventions such as cytochalasin D or latrunculin B have been shown to disturb actin function, restore membrane potential and stop spontaneous beating in cell pairs of myofibroblasts and neonatal rat ventricular cardiomyocytes [12]. Yet, we did not attempt to use this type of interventions as they do not prevent abundant presence of myofibroblasts and membrane potentials remain relatively depolarized at approximately 274 mV.
Another factor that can contribute to spontaneous beating in cultured cells is a decreased inward rectifier current (I K1 ), which has been shown in both adult rat and rabbit ventricular myocytes [8,27]. This progressive decrease in I K1 was correlated to a depolarization of the RMP, and could thus also influence the occurrence of spontaneous beating.
Even in the absence of myofibroblast and with a RMP of approximately 284 mV, some of the organ explant cultures and freshly isolated tissues showed spontaneous beating activity. Spontaneous beating was observed in some preparations where we did not detect phase 4 depolarization. This likely occurred in settings where the impalement site was not at the origin of spontaneous membrane depolarization. We showed that the beating activity in organ explant cultures and freshly isolated tissue did not originate from the dissection border of the tissue, excluding tissue damage as a causative factor in our preparations. Since part of the conduction system is still present in freshly isolated tissue and organ explant cultures, it is possible that spontaneous beating originated from these cells.

Conduction velocity
CV of freshly isolated tissue was relatively low (18.261.0 cm/s) compared to reported values for adult rat ventricle (6668 cm/s) [28]. Only few papers have reported CV in neonatal rat heart [29][30][31], and showed values ranging from 21 cm/s in 2-day old rats to 31 cm/s in 10-day old rats. This increase in CV with increasing age was found in several species [32,33].
We found that CV in monolayers was higher than in organ explant cultures and freshly isolated tissue. This was somewhat surprising, given the fact that organ explant cultures had electrophysiologic properties and tissue architecture that were more favorable (i.e., conducive to high CV) than monolayers. CV has multiple determinants, whose net contributions are difficult to predict. For instance, fiber direction, a determinant of CV [34], appears more favorable for organ explant cultures than monolayers, as longitudinal cell orientation (associated with large CV in the longitudinal direction) was preserved in organ explant cultures, but not in monolayers. Paradoxically, tissue geometry may also provide a potential cause for the lower CV in organ explant cultures. Since a monolayer is a 2-dimensional network, the activation wavefront must travel in a horizontal plane, i.e., the plane that is measured by optical mapping. However, in organ explant cultures, the loose 3-dimensional tissue structure may give rise to activation wavefronts that travel outside the planes of optical measurement. In that case, the CV will be underestimated.
Another factor that can influence CV is the magnitude of the upstroke velocity [35]. However, since the sample time was 2 ms in optical mapping of monolayers, we could not measure upstroke velocity and compare this to organ explant cultures and freshly isolated tissue, as these were mapped with a sample time of 1 ms.
A possible contributor to higher CV in monolayers may be larger cell size. While we did not quantify cell surface area, in both HE staining and immunohistochemistry, cell surface area in monolayers appeared to be larger, which may partly explain larger CV [36,37]. A final determinant of CV to consider is cell-to-cell coupling [36]. However, this determinant is not likely to explain differences in CV, as connexin-43 expression was similar in all groups; being distributed along all cell borders, instead of being localized to the intercalated discs as in adult tissue [38]. We did not investigate Cx45 expression and therefore cannot rule out potential differences among the individual groups. Although the presence and role of connexin-45 during embryonic development have been well established [39], its role in neonatal and adult tissue seems limited, as it has consistently been found in very low quantities in both neonatal and adult heart [40][41][42]. Furthermore, in cell pairs of neonatal rat ventricular cardiomyocytes there is a linear correlation between gap junction conductance and connexin-43 immunosignal [43]. We therefore like to suggest that potential differences in connexin-45 are unlikely to explain the differences in CV in our study. Nevertheless, if there is a difference in expression of connexin-45 in monolayers as compared to freshly isolated tissue and organ explants cultures, this could influence CV, as mixed channels containing both connexin-43 and -45 have a lower channel conductance [44][45][46]. This lower gap junction conductance is expected to decrease CV [47].
While CV is higher in monolayers than organ explant cultures, freshly isolated tissue has a similar CV to organ explant cultures, which leads us to believe that the organ explant cultures in this study showed a CV within the normal range for neonatal rat ventricular tissue.

Action potential duration
The APDs of freshly isolated neonatal rat ventricle and monolayers reported vary between different studies, and the values as reported in this paper are consistent with previously published studies [48][49][50]. The prolonged APD seen in monolayers as compared to freshly isolated tissue can be explained by their depolarized MDP. Due to this depolarization, both the transient outward current (I to ) and the rapid component of the delayed rectifier current (I Kr ) are inactivated more strongly [51][52][53]. The resulting decrease in repolarizing currents is expected to prolong APD. Bursac and colleagues indeed showed that blocking I to with 4-aminopyridine in freshly isolated tissue strongly increased the APD, while blocking I to had no effect on the APD in monolayers [49].

Stimulation at different cycle lengths
Often properties of monolayers are studied at a 2 Hz pacing rate. Since neonatal rat hearts beat at around 350 beats per minute (CL,170 ms; [54,55]), we also investigated capture at shorter CLs. While both organ explant cultures and freshly isolated tissue could still be paced with a CL of 150 ms, capture was frequently lost in monolayers at CLs of around 200 ms. This finding is in accordance with the longer APD found in monolayers.

Conclusion
In conclusion, we developed a method of organ explant culture that provides an in vitro model of cardiac tissue that is structurally and electrophysiologically similar to freshly isolated tissue. In accordance with its tissue architecture, (near) absence of spontaneous beating and myofibroblasts render this model superior to the well established model of neonatal rat ventricular cardiomyocytes cultured in monolayer. Because the cultured organ explant model also appeared readily modified by methods of gene and cell therapy, this model may offer a powerful screening tool for the evaluation of biological approaches aimed at treatment of cardiovascular disease.