Selective gene expression analysis of muscular and vascular components in hearts using laser microdissection method

Abstract

Background. The heart consists of various kinds of cell components. However, it has not been feasible to separately analyze the gene expression of individual components. The laser microdissection (LMD) method, a new technology to collect target cells from the microscopic regions, has been used for malignancies. We sought to establish a method to selectively collect the muscular and vascular regions from the heart sections and to compare the marker gene expressions with this method. Methods and Results. Frozen left ventricle sections were obtained from Wistar-Kyoto rats (WKY) and stroke-prone spontaneously hypertensive rats (SHR-SP) at 24 weeks of age. Using the LMD method, the muscular and vascular regions were selectively collected under microscopic guidance. Real-time RT-PCR analysis showed that brain-type natriuretic peptide (BNP), a marker of cardiac myocytes, was expressed in the muscular samples, but not in the vascular samples, whereas α-smooth muscle actin, a marker of smooth muscle cells, was detected only in the vascular samples. Moreover, SHR-SP had significantly greater BNP upregulation than WKY (P < 0.05) in the muscular samples. Conclusions. The LMD method enabled us to separately collect the muscular and vascular samples from myocardial sections and to selectively evaluate mRNA expressions of the individual tissue component.

Figure 1

(A) Representative microphotographs demonstrating the sampling of vascular area of the myocardial section obtained from SHR-SP using LMD method. (a) Laser cut lines (red lines) were placed on the outside border of the medial smooth muscle layer. (b) Myocardial section after vascular samples were cut off by laser dissection shown. (c) After laser dissection, isolated vascular fragments (red arrows) fell down into the caps of Eppendorf tubes containing TRIzol reagent. The vascular fragments were being lysed in the TRIzol reagent. (B) For sampling myocardial area, laser cut lines were placed not to include microscopically visible vasculatures and infiltrating cells in the area of interest.

Figure 2

(a) Representative RNA electrophoregrams of the muscular samples of WKY (top) and SHR-SP (bottom). (b) Representative amplification plots of BNP mRNA in the muscular and vascular samples of WKY.

Figure 3

BNP and α-SMA mRNA expression in the muscular (closed column) and vascular (open column) samples obtained from the heart of WKY (n = 2). Expression level of the target gene was normalized by β-actin level in each sample.

Figure 4

BNP mRNA expression in the muscular and vascular samples obtained from the heart of WKY (open column) and SHR-SP (closed column). BNP expression levels were expressed as fold change from the muscular sample of WKY. Unpaired t-test was used for comparison of initial data before expression as fold changes. Bar = 1 × SD (n = 3). *P < 0.05 versus WKY.