Matrix metalloproteinase-2, caveolins, focal adhesion kinase and c-Kit in cells of the mouse myocardium

Abstract

Matrix metalloproteinase-2 (MMP-2) may play roles at intracellular and extracellular sites of the heart in ischaemia/reperfusion injury. Caveolins (Cav-1, -2 and -3) are lipid raft proteins which play roles in cell sig-nalling. This study examined, using immunohistochemistry and two photon confocal microscopy, if MMP-2 and caveolins co-localize at the plasma membrane of cardiac cells: cardiomyocytes (CM), fibroblasts (FB) and capillary endothelial cells (CEC) in the left ventricle (LV) of the Cav-1(+/+) and Cav-1(-/-) mouse heart. In Cav-1(+/+) mouse LV MMP-2 and Cav-1 co-localized at CM plasma membranes, and at multiple locations in FB and CEC. MMP-2 co-localized with Cav-2 only at CEC. MMP-2 co-localized with Cav-3 at CM plasma membranes and Z-lines, and partially at FB and CEC. In Cav-1(-/-) LV Cav-1 and MMP-2 were absent or reduced everywhere. Cav-2 appeared at CEC despite the absence of Cav-1. Cav-3 appeared at CM plasma membranes and Z-lines, FB and CEC. Also, FAK in FB and c-Kit in interstitial Cajal-like cells (ICLC) were completely absent. By transmission electron microscopy in Cav-1(+/+), regular size caveolae (Cav) were at CEC, irregular size Cav were at CM and a few were at FB. In Cav-1(-/-) there were few Cav at CM and FB and some at CEC. To conclude, MMP-2 is closely associated with caveolins at FB and CEC as well as at CM. Also, MMP-2 is closely associated with FAK at FB and c-Kit at ICLC. Thus, Cav-1 expression is not necessary for Cav-2 expression. Cav-3 or Cav-3 with Cav-2 has the capability to make Cav.

1

Co-localization of MMP-2 and Caveolins in Cav-1^+/+ and Cav-1^−/− mouse LVM. In Cav-1^+/+ MMP-2 (red) appeared to have good co-localization with Cav-1 (green) at CM plasma membranes (large open arrows), CEC (closed arrows) and FB (small open arrows), and partial co-localization occurs at Z-lines (open arrowheads) (A–C). MMP-2 (red), however, appeared to have good co-localization with Cav-2 (green) only at CEC (closed arrows). FB with MMP-2 appear (open arrows) (E–G). MMP-2 (red) appeared to have good co-localization with Cav-3 (green) at CM plasma membranes (open arrows) and Z-lines (arrowhead), and partial co-localization at FB (closed arrows) (I–K). In Cav-1^−/− Cav-1 is absent and MMP-2 is markedly reduced at CM plasma membrane (arrow), CEC and FB (D). IS refers to interstitial space. Interestingly, Cav-2 (green) appears regardless of Cav-1 only at CEC (H) and Cav-3 (green) appears at CM plasma membranes (large arrows), Z-lines (arrowhead), and FB (small arrows) like in Cav-1^+/+(I). CM is cardiomyocyte and IS is interstitial space. Scale bar is 10 mm for A–H and I, and 20 mm for I–K.

2

Co-localization of MMP-2 and FAK / DDR-2 / vWF / c-Kit in Cav-1^+/+ and Cav-1^−/− mouse LVM. In Cav-1^+/+ co-localization (arrows) of MMP-2 (green) and FAK (red) (A–C), MMP-2 (green) and DDR-2 (red) (E–G), and MMP-2 (red) and vWF (green) (I–K) shows that MMP-2 localizes at FB (nucleus:large asterisks) as well as at CEC (nucleus:small asterisks). MMP-2 (green) also co-localizes (arrows) with c-Kit (red) at ICLC near by CM (M-N). In Cav-1^−/−, surprisingly, FAK (red) and c-Kit (red) are completely absent at FB and ICLC, respectively, except FAK in nuclei of all cells (Dand P).vWF, however, is still present at CEC (I). CM is car-diomyocyte and IS is interstitial space. Scale bar is 10 mm for all images.

3

Co-localization between caveolins in Cav-1^+/+ and Cav-1^−/− mouse LVM. In Cav-1^+/+ 3D reconstructed images (-45° rotation of y-axis) shows Cav-1 (red) localizes at both FB (nucleus: large asterisks) and CEC (nucleus: small asterisks), but Cav-2 (green) localizes only at CEC. Partial co-localization of Cav-1 and Cav-2 occurs at CEC (open arrows), but sometimes Cav-1 without Cav-2 occurs at CEC membrane (closed arrows) (A–C). Cav-1 (red) co-localizes well with Cav-3 (green) at CM plasma membrane (large open arrow), FB (nucleus: large asterisks) interconnecting with tails (small open arrow) and CEC (nucleus: small asterisks). Also, Cav-1 immunoreactivity occurs near CM Z-lines and partial co-localization with Cav-3 (E–G). Cav-2 (green) co-localizes partially with Cav-3 (red) at CEC (arrows). CEC make frequently anastomotic structure (asterisk) (I–K). In Cav-1^−/− Cav-2 (green) immunore-activity occurs regardless of Cav-1 at CEC (d). Cav-3 (green), immunoreactivity occurs also at CM plasma membranes, Z-lines and FB (asterisks) (H). Co-localization (open arrows) of Cav-2 (green) and Cav-3 (red) at CEC is similar to that in Cav-1^+/+(I). CM is cardiomyocyte and IS is interstitial space. Scale bar is 10 mm for all images.

4

Co-localization of Cav-1 and FAK / DDR-2 / vWF / c-Kit in Cav-1^+/+ and Cav-1^−/− mouse LVM. In Cav-1^+/+ Cav-1 (green) co-localized well with FAK (red) at FB with long tails (nucleus: asterisks) (A–C). Cav-1 (red), also, co-localized (arrows) well with vWF (green) at CEC (nucleus: asterisks) (E–G). Cav-1 (red) co-localized (arrows) with DDR-2 (green) at a few FB around venule but didn't co-localize at some FB (asterisks) (I–K). Also, Cav-1 (green) co-localized well with c-Kit (red) (M–O). In Cav-1^−/− FAK and c-Kit disappear completely with Cav-1 at FB and ICLC, respectively (D and P) . However, vWF (green) and DDR-2 (green) appear at CEC and FB, respectively (H and I). CM is cardiomyocyte and IS is interstitial space. Scale bar is 10 mm for all images.

5

Co-localization of Cav-2 and FAK / c-Kit in Cav-1^+/+ and Cav-1^−/− mouse LVM. In Cav-1^+/+ Cav-2 (green) (closed arrow) appears only at CEC (nucleus: small asterisks) and FAK (red) (open arrow) only at FB (nucleus: large asterisks). An anastomotic structure appears (open arrowhead) (A–C). Cav-2 (green) doesn't co-localize with c-Kit (red). Arrow indicates for FB, large asterisk for FB nucleus and small asterisk for CEC nucleus (E–G). In Cav-1^−/− FAK and c-Kit are missing with Cav-1 (D and H). CM is cardiomyocyte and IS is interstitial space. Scale bar is 10 mm for all images.

6

Co-localization of Cav-3 and FAK / DDR-2 / vWF / c-Kit in Cav-1^+/+ and Cav-1^−/− mouse LVM. In Cav-1^+/+ mouse some FB are Cav-3- (green) and FAK-positive (red) and the two proteins are partially co-localized (yellow) (large arrows), besides some FB are Cav-3-positive but FAK-negative (small arrows) (A–C). Some Cav-3-positive FB (red) are DDR-2-positive (green) and the two proteins are partially co-localized in FB tails (arrows) (E–G). vWF-positive CEC (arrows) do not have Cav-3 (I–K). ICLC are both Cav-3- (green) and c-Kit-positive (red) and the two proteins are partially co-localized (yellow) (M–O). In Cav-1^−/− mouse Cav-3 appeared at CM plasma membranes, Z-lines and FB (arrows), but FAK and c-Kit are completely absent (D and P). DDR-2 and vWF shows at FB and CEC, respectively (H and I). Scale bar is 10 μm for a–d and i–p, and 7 μm for e–g.

7

Localization of FAK and vWF and co-localization of DDR-2 and c-Kit in Cav-1^+/+ and Cav-1^−/− mouse LVM. In Cav-1^+/+ mouse FAK (red, open arrows) appears at FB and vWF (green, closed arrows) does at CEC, but these are not co-localized (A–C). Interestingly some DDR-2-positive FB (green, small open arrow) are c-Kit-positive (red, small closed arrows) and these are partially co-localized (yellow, large open arrows) (E–G). In Cav-1^−/− mouse FAK is completely absent but vWF (green, arrows) appears at CEC (D). c-Kit, also, is completely absent but DDR-2 (green, arrows) appears at FB (H). CM is cardiomyocyte and IS is interstitial space. Scale bar is 10 μm for all images.

8

Electron micrographs of CM, FB and CEC in Cav-1^+/+ and Cav-1^−/−mouse LVM. In Cav-1^+/+ mouse A–C shows that CM, FB and CEC are closely located. Some caveolae (Cav, arrows) reveal at CM sar-colemma and a number of (Cav, arrows) reveal at CEC membrane. Well-developed sarcoplasmic retic-ulums (SR, closed thin arrows) are continuously arranged between myofilaments in CM and a few endoplasmic reticulums (ER, open thin arrows) reveal near by Cav at CEC membrane. Sacs of T-tubules (arrowheads) reveal adjacent to Cav and SR of CM and large mito-chondria (m) locate adjacent to sar-colemma. FB does not have Cav but have well-developed rough ER. In Cav-1^−/− mouse D–F shows that CM, FB and CEC are closely located as Cav-1^+/+ mouse. A few Cav (arrow) reveal at the sarcolemma and some flat-shaped SR (closed thin arrows) along the sarcolemma are discontinuously located in CM. Sacs of T-tubules (arrowheads) reveal adjacent to SR. CEC have a few Cav. FB does not have Cav, but a few have it. FB-p, process of fibroblast.