INTRODUCTION

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ONE OF THE MOST EXCITING questions of cell biology is the regulation of cell-specific and organ-specific gene expression. To investigate differential gene expression, usually RNA is extracted from different organs and analyzed by RT-PCR or by Northern blot hybridization. However, every organ contains several cell types. In the mammalian heart, for example, only 30% of the cells are cardiac muscle cells (5), with the remainder consisting of endothelial cells, vascular smooth muscle cells, pericytes and fibroblasts, and nerve cells. It would be highly desirable to get more information on the cell-specific expression profile of the different cell types.

Single-cell RT-PCR has proved useful, especially in the field of neuronal ion channels and receptors, and it is still a considerable challenge to try and correlate the mRNA signals found in individual neurons with the ion channels and receptors in these cells (3). Correlation of the properties of heterologously expressed channels and receptors with the properties of channels present in native cells is difficult, because the properties of channels and receptors may change depending on the expression system used. Unfortunately, single cell RT-PCR is not feasible in all cell types. In endothelial cells, for example, the amount of cytoplasm that can be harvested with a patch pipette is extremely small, because the thickness of the cell outside the nuclear region is <1 µm. Moreover, gigaseal formation is impeded by the basement membrane surrounding the endothelial cell layer. Therefore, we have devised an alternative method to get information on the gene expression of coronary endothelial cells. The method is based on separation of different cell types with collagenase and visual recognition and selection of cells that can be identified by their morphology. The cells are taken up by a simple "cell picker," transferred to a reaction tube, and then subjected to RT-PCR. As a test of the reliability and efficiency of the method, we compared the expression of endothelin-1 (ET-1) and troponin T in capillary endothelial cells and cardiomyocytes isolated from guinea pig heart.

	METHODS

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Cell isolation. Isolated cardiomyocytes and capillaries were obtained from guinea pig hearts by enzymatic dispersion. Guinea pigs weighing 250-450 g were decapitated and their hearts rapidly excised. A cannula was attached to the aorta (Langendorff preparation), and the coronary arteries were perfused at a constant flow rate of 10 ml/min using a peristaltic pump. The heart was submerged in a small organ bath warmed to 37°C. The perfusing solution contained (in mM) 130 NaCl, 15 KCl, 2 CaCl₂, 0.8 MgCl₂, 1 NaH₂PO₄, 2 Na-pyruvate, 10 glucose, and 10 HEPES. The pH was 7.4 (adjusted with NaOH); the temperature was 37°C. The elevated K⁺ concentration of the perfusate caused cardiac arrest. Within 15-20 min, coronary perfusion pressure increased to a steady level between 60 and 100 mmHg, indicating recovery of energy metabolism.

Selection of cardiomyocytes. Two milliliters of the mixed cell suspension were layered on storage solution containing 4% bovine serum albumin (BSA), and the heart cells were allowed to settle by gravity sedimentation for 15-20 min (4). This procedure was then repeated, and the final pellet was resuspended in 3-5 ml of storage solution. For cell picking, several drops of the enriched cardiomyocyte suspension were transferred to a 35-mm petri dish filled with storage solution to which 1% BSA had been added to prevent attachment of the cells. The petri dish with the diluted cell suspension was mounted on an inverted microscope (Zeiss IM 35). Up to 1,000 cardiomyocytes were collected under visual control with the cell picker.

Selection of capillary endothelial cells. Single isolated endothelial cells round up and cannot be easily discriminated from fibroblasts, damaged vascular smooth muscle cells, or pericytes. Therefore, capillary fragments were used that can be easily recognized morphologically as a regularly spaced string of nuclei connected by a tube of 3-5 µm in diameter (2). The mixed cell suspension obtained as described above was sieved through a nylon filter (Falcon, mesh size 70 µm) that retained the capillary fragments. The cells were washed with physiological salt solution (PSS) containing (in mM) 135 NaCl, 5 KCl, 1 CaCl₂, 1 MgCl₂, 0.33 NaH₂PO₄, 10 glucose, 2 Na-pyruvate, and 10 HEPES (pH adjusted to 7.4 with NaOH). The capillary fragments were then removed from the sieve by rinsing with PSS. Single drops of the enriched endothelial cell suspension were added to a 35-mm petri dish containing PSS with 1% BSA. Roughly 150 capillary fragments consisting of 6-15 endothelial cells were collected under visual control with the cell picker. Care was taken to exclude capillary fragments to which cells were attached (putative pericytes) or that were contaminated with cell debris.

RNA extraction, RT, and PCR. For the isolation of the total RNA, a commercial kit was used (RNeasy Mini Kit, Qiagen, Chatsworth, CA). Usually, 800-1,000 picked cardiomyocytes or roughly 150 capillary fragments were transferred to 800 µl of lysis solution (RNeasy Mini), and the total RNA was extracted according to the instructions of the manufacturer. One-third of the RNA eluate was reverse transcribed using 200 units of Superscript II reverse transcriptase (GIBCO BRL) and a random hexanucleotide mixture (total volume 25 µl). The PCR was performed with gene-specific, intron-spanning primers of 24-28 nucleotides in length. The total PCR volume was 50 µl, including 2 µl of RT reaction, 50 pmol of each primer, and 2.5 units of AmpliTaq Gold (Applied Biosystems). Thus one RT reaction gave ~10 PCR runs. The tubes were placed in the thermal cycler (model 2400, Applied Biosystems), and the PCR was run with a hot start for 5 min at 94°C (initial melt); then for 40 cycles of 0.5 min at 94°C, 0.5 min at 55°C, and 1 min at 72°C; and, at last, for 5 min at 72°C (final extension). Fifteen microliters of the PCR probe were size fractionated by agarose gel electrophoresis. To verify the identity of the PCR products, DNA fragments of the expected length were isolated and directly sequenced using an ABI prism 310 DNA sequencer (Applied Biosystems).

Cell-specific markers. To control the purity of the cell fractions, we designed intron-spanning primers for cell-specific markers. Troponin T and ET-1 transcripts were used as markers for cardiomyocytes and endothelial cells, respectively. Primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a constitutively and ubiquitously expressed gene, were used to check RT-PCR conditions. Specific primers for guinea pig troponin T were required to obtain distinct PCR signals. The sequences of the guinea pig-specific markers were submitted to GenBank. In the case of GAPDH, primers derived from the human sequence worked well.

	RESULTS

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Cell picking. To study cell-specific gene expression in the different cardiac cells, we have devised a method for the selective isolation of myocytes and capillary fragments. The principle of the design of the cell picker is illustrated in Fig. 1. It consisted of a polyethylene pipette with a tip diameter of ~150 µm, an electric two-way microvalve, and a piece of silicon tubing serving as a siphon. The degree of suction applied could be adjusted by varying the level of the outlet of the tubing, which was usually 1-2 cm below the level of the petri dish mounted on the inverted microscope. The electric valve was a normally closed two-way valve, the opening of which was controlled by a pulse generator (+5 V). Voltage pulses of 20-200 ms in duration (controlled by a potentiometer) could be applied at regular intervals by pressing a knob. The standard interval chosen was 1 s, which was well above the reaction time of the experimenter. Thus the number of suction pulses could be adjusted to the minimum required to transfer the selected cell into the pipette.

View larger version (37K): [in this window] [in a new window]   Fig. 1.   A: principles of cell-picking and RT-PCR procedures. B: schematic drawing of cell picker (for details, see text). Cells were viewed on an inverted microscope (Zeiss IM 35) with phase-contrast optics at ×320 magnification. Suction pulses were controlled by a miniature solenoid valve from Lee (model LFAA 120601H; Westbrook, CT). Interval between pulses was kept constant at 1 s. Duration of pulses and height of water column could be adjusted by experimenter. Usually, duration of suction pulse was ~100 ms, and height of water column was ~2 cm. Amount of fluid taken up into pipette per suction pulse was 1-2 µl.

View larger version (112K): [in this window] [in a new window]   Fig. 2.   Photomicrographs of cell-selection procedure taken at low magnification for better overview. A: diluted suspension of cardiomyocytes enriched by gravity sedimentation as described in METHODS. Arrow indicates a residual large capillary fragment. B: diluted suspension of capillary fragments prepared by sieving as described in METHODS. C and D: cell picking. A clean cardiomyocyte was placed near pipette tip (C; arrow). After a single suction pulse, cell disappeared in pipette (D; arrow); other cells were unaffected and remained in place. Calibration bar applies for A-D.

View larger version (55K): [in this window] [in a new window]   Fig. 3.   Detection of troponin T transcripts in cardiomyocytes and endothelin-1 (ET-1) transcripts in capillary endothelial cells by multicell RT-PCR. Representative 2% agarose gels were loaded with one-third of each PCR probe and stained with ethidium bromide. A: multicell RT-PCR with cardiomyocytes. Lanes contain molecular weight markers and probes with glyceraldehyde-3-phosphate dehydrogenase (GAPDH), troponin T (TropT), and ET-1, respectively. B: multicell RT-PCR with capillary endothelial cells. Lanes contain molecular weight markers and probes with GAPDH, troponin T, and ET-1, respectively.

	DISCUSSION

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We have shown that it is possible to isolate pure heart cell fractions with an easy and highly efficient method. The purity of the collected cell fractions was controlled by RT-PCR with gene-specific, intron-spanning primers. The cell picker described here requires only an inverted microscope, a micromanipulator, and, in addition, some plastic materials and an electric microvalve. Recently, other methods to select single cells or groups of cells have been tried, for example, laser-assisted microdissection (1). This approach is well suited for stained tissue sections, especially under pathophysiological conditions. However, for studying cell-specific gene expression, the method described here is probably simpler, more sensitive, and less expensive.

ET-1, a peptide that was originally isolated from aortic endothelium (6, 8), is one of the endothelium-derived contracting factors. We have detected expression of the ET-1 gene only in freshly isolated coronary endothelial cells and not in cardiomyocytes of guinea pig heart (Fig. 3). In contrast, Suzuki et al. (7) recently reported that ET-1 is expressed in cultured neonatal rat cardiac myocytes. This discrepancy may be attributable to the difference in species or age. However, it cannot be excluded that expression of ET-1 in neonatal rat cardiomyocytes was induced by the cell culture conditions (7). It is well known that the morphology and the pattern of gene expression changes considerably during cell culture. The difference between the results of Suzuki et al. (7) and our results underscores the importance of using freshly isolated cells for studying cell-specific gene expression.

The results presented here suggest that, at least in the adult guinea pig, ET-1 is not constitutively expressed in cardiomyocytes. ET-1 was found to be a specific marker for freshly isolated coronary capillary endothelial cells from guinea pig heart. Troponin T was highly expressed in cardiomyocytes and could not be detected in any of the endothelial cell preparations (Fig. 3). Thus troponin T appears to be a specific marker for cardiac muscle cells.

We have shown that multicell RT-PCR is a suitable method for the detection of gene transcripts in different cell types of the heart. mRNA transcribed at a very low rate can be detected, and, on the other hand, the expression of certain genes in specific cell types can be reliably excluded. Furthermore, the expression of a number of different genes can be analyzed in one cell preparation. With the total RNA from ~1,000 cardiomyocytes or 150 capillary fragments (each containing 6-15 endothelial cells), >30 PCR runs can be performed. It is likely that with minor modifications this method can also be applied to cell-specific RT-PCR analysis in other organs.