Preservation of Functional Microvascular Bed Is Vital for Long-Term Survival of Cardiac Myocytes Within Large Transmural Post-Myocardial Infarction Scar

Abstract

This study was aimed to understand the mechanism of persistent cardiac myocyte (CM) survival in myocardial infarction (MI) scars. A transmural MI was induced in 12-month-old Sprague-Dawley rats by permanent coronary artery ligation. The hearts were collected 3 days, 1, 2, 4, 8, and 12 weeks after MI and evaluated with histology, immunohistochemistry, and quantitative morphometry. Vasculature patency was assessed in 4-, 8-, and 12-week-old scars by infusion of 15-micron microspheres into the left ventricle before euthanasia. The infarcted/scarred area has a small continually retained population of surviving CMs in subendocardial and subepicardial regions. Surprisingly, whereas the transverse area of subepicardial CMs remained relatively preserved or even enlarged over 12 post-MI weeks, subendocardial CMs underwent progressive atrophy. Nevertheless, the fractional volume of viable CMs remained comparable in mature scars 4, 8, and 12 weeks after MI (3.6 ± 0.4%, 3.4 ± 0.5%, and 2.5 ± 0.3%, respectively). Despite the opposite dynamics of changes in size, CMs of both regions displayed sarcomeres and gap junctions. Most importantly, surviving CMs were always accompanied by patent microvessels linked to a venous network composed of Thebesian veins, intramural sinusoids, and subepicardial veins. Our findings reveal that long-term survival of CMs in transmural post-MI scars is sustained by a local microcirculatory bed.

Keywords: Thebesian vessels; cardiac myocyte survival; microvascular beds; middle-aged rats; myocardial infarction; scar formation; subepicardial veins; venous sinusoids.

Figure 1.

Distribution of surviving cardiac myocytes in a representative 1-week-old transmural post-MI scar visualized with Masson’s trichrome stain (dark red color in A) and double immunofluorescence staining (B–D) with antibodies against cardiac MHC β-isoform (red color) and laminin (green color). In C and D, nuclei are counterstained with DAPI (blue color). (A) Transverse section through the midventricular level of the post-MI heart demonstrating the presence of viable cardiac myocytes in subendocardial (arrows) and subepicardial (arrowheads) regions of the scar. Note that muscle-specific staining (dark red color) seen within the middle region of the scar is associated entirely with the remnants of dead mummified myocytes. (B) Immunofluorescence micrograph of the area outlined by a black box in A showing the viable cardiac myocytes in subendocardial (arrows) and subepicardial (arrowheads) regions of the same scar obtained on an adjacent serial section. Note that remnants of dead mummified myocytes located in the middle region of the scar remain unstained with antibody against cardiac MHC β-isoform (red color), although most of them continue to be outlined by laminin-positive basement membranes. (C) High-power view of the subendocardial region outlined by a white box in B demonstrating the arrangement of viable cardiac myocytes in the discontinuous layer just beneath the endocardium (arrows) as well as in the isolated cluster around a small, thin-walled venous-like vascular channel, which resembles a Thebesian vein or vessel (asterisk). (D) High-power view of the subepicardial region outlined by a white box in B showing the groups of viable cardiac myocytes beneath the epicardium (white dotted line) in close proximity to a large subepicardial vein (V). Scale bars are 600 µm (A, B) and 50 µm (C, D). Abbreviations: MI, myocardial infarction; MHC, myosin heavy chain; DAPI, 4′,6-diamidino-2-phenylindole.

Figure 2.

Distribution of surviving cardiac myocytes in the representative 2-week-old (A, B) and 4-week-old (C, D) transmural post-MI scars visualized by double immunofluorescence staining with antibodies against desmin (red color) and laminin (green color). In all micrographs, nuclei are counterstained with DAPI (blue color). (A and C) Low-power views of the transmural scars demonstrating the discontinuous layers of viable cardiac myocytes beneath the endocardium (arrows) and epicardium (white dotted line). In A, a few isolated clusters of surviving cardiac myocytes are seen intramurally, surrounding the thin-walled sinusoidal vessels (asterisks). It is important to note that in comparison with subepicardial veins (V), the walls of large coronary arteries (A) are composed of desmin-positive vascular smooth muscle cells. Furthermore, a patchy pattern of red immunofluorescence staining detected in the middle portion of the scar extending along the compact laminin-positive bundles presumably belongs to the desmin-expressing myofibroblasts. (B and D) High-power views of the subepicardial regions outlined by the corresponding white boxes in A and C, respectively, demonstrating the large groups of surviving cardiac myocytes just beneath the epicardium (white dotted line) and near the large subepicardial veins (V). Scale bars are 500 µm (A), 100 µm (C), and 50 µm (B, D). Abbreviations: MI, myocardial infarction; DAPI, 4′,6-diamidino-2-phenylindole; MHC, myosin heavy chain.

Figure 3.

Distribution of surviving cardiac myocytes in a representative 12-week-old transmural post-MI scar visualized by double immunofluorescence staining with antibodies against cardiac MHC β-isoform (red color) and laminin (green color). In all micrographs, nuclei are counterstained with DAPI (blue color). (A) Low-power view of the scar demonstrating the discontinuous layers of viable cardiac myocytes beneath the endocardium (arrows) and epicardium (white dotted line). Note that viable cardiac myocytes are present around a large vein (V), whereas they are completely absent in the vicinity of coronary arteries (A). (B) High-power view of the subepicardial region outlined by the white box in A showing the viable cardiac myocytes beneath the epicardium (white dotted line) and around the large subepicardial vein (V). (C) High-power view of the subendocardial region outlined by a white box in A demonstrating the viable cardiac myocytes beneath the endocardium. Scale bars are 100 µm (A) and 50 µm (B, C). Abbreviations: MI, myocardial infarction; MHC, myosin heavy chain; DAPI, 4′,6-diamidino-2-phenylindole.

Figure 4.

Time course of changes in CSA of the cardiac myocytes surviving in subepicardial and subendocardial regions of the transmural scar during 12 post-MI weeks. Data are Mean ± SD; *p<0.05, **p<0.01, and ***p<0.001 versus cardiac myocytes in corresponding groups of the control rats (C); ^††p<0.01 and ^†††p<0.001 versus cardiac myocytes in subendocardial region of the same rats. Abbreviations: CSA, cross-sectional area; MI, myocardial infarction.

Figure 5.

Immunofluorescence micrographs demonstrating the organized sarcomeric striations in cardiac myocytes surviving within 4-week-old (A–D) and 8-week-old (E, F) transmural post-MI scars. The viable cardiac myocytes (red color) are visualized using double immunostaining with antibodies against either cardiac actin (A, B, C, and D) or desmin (C, D) and laminin (green color). In all micrographs, nuclei are counterstained with DAPI (blue color). (A, C, E) Low-power views of the scars demonstrating the discontinuous layers of viable cardiac myocytes beneath the endocardium (arrows) and epicardium (white dotted line in A and C). Note that, in all scars, the clusters of surviving cardiac myocytes are present in close proximity to the large venous vessels (V). It is important to notice that desmin-positive immunostaining in the walls of large coronary arteries (A) is associated with the presence of vascular smooth muscle cells. (B, D, F) High-power views of the regions outlined by the corresponding white boxes in A, C, and E, respectively, exposing the sarcomeric striations in viable cardiac myocytes (arrowheads) surviving beneath the endocardium (arrows in D) and near the large venous vessels (V) located in the subepicardial area. Scale bars are 50 µm. Abbreviations: MI, myocardial infarction; DAPI, 4′,6-diamidino-2-phenylindole.

Figure 6.

Immunofluorescence micrographs displaying the gap junctions between cardiac myocytes surviving within 2-week-old (A) and 4-week-old (B) transmural post-MI scars. The viable cardiac myocytes are outlined using an antibody against laminin (green color), whereas the locations of gap junctions are visualized with an anti-connexin 43 antibody (red color). In both micrographs, nuclei are counterstained with DAPI (blue color). (A) High-power view showing surviving cardiac myocytes beneath the endocardium (arrows). (B) High-power view demonstrating surviving cardiac myocytes in close proximity to a large venous vessel (V) beneath the epicardium (white dotted line). Note that in A and B, the connexin 43 is accumulated on the lateral sides of viable cardiac myocytes displaying the typical gap junction arrangement along the interfacing surfaces of adjacent myocytes (arrowheads). Scale bars are 50 µm. Abbreviations: MI, myocardial infarction; DAPI, 4′,6-diamidino-2-phenylindole.

Figure 7.

Immunofluorescence micrographs demonstrating the presence of functional capillaries among the viable cardiac myocytes in 1-week-old (A, B), 2-week-old (C, D), 8-week-old (E, F), and 12-week-old (G, H) transmural post-MI scars. The cardiac myocytes surviving within subendocardial (A, C, E, G) and subepicardial (B, D, F, H) regions of the corresponding scars are visualized using immunostaining with an antibody against cardiac MHC β-isoform (red color), whereas capillaries (arrowheads) and other vascular structures, including veins (V) and arteries (A), are revealed with the GS-IB₄ lectin staining (green color). Note that in the wall of a former coronary artery, the GS-IB₄ lectin-positive staining is associated with both endothelial and smooth muscle cells. In all micrographs, nuclei are counterstained with DAPI (blue color). In A, C, E, and G, arrows point to the endocardium, whereas in F and H, the white dotted lines mark the epicardial border. Furthermore, in A and B, a white solid line separates the groups of surviving cardiac myocytes from the highly vascularized granulation tissue. It is important to emphasize that all clusters of long-term surviving cardiac myocytes seen beneath the endocardium (A, C, E, G) and epicardium (B, D, F, H), including those surrounding the large veins (A–D and F), expose the presence of well-organized capillary networks. Moreover, the capillary beds accompanying the viable cardiac myocyte for the duration of 12 post-MI weeks remain generally preserved even during maturation of the granulation tissue—a process associated with the disappearance of a majority of microvessels from the scar. Scale bars are 20 µm (A–D, G, and H) and 50 µm (E, F). Abbreviations: MI, myocardial infarction; MHC, myosin heavy chain; GS-IB₄, Griffonia Simplicifolia isolectin IB₄; DAPI, 4′,6-diamidino-2-phenylindole.

Figure 8.

Immunofluorescence micrographs demonstrating the presence of resistance microvessels accompanying the viable cardiac myocytes in 1-week-old (A, B), 2-week-old (C, D), 4-week-old (E, F), and 8-week-old (G, H) transmural post-MI scars. The cardiac myocytes surviving within subendocardial (A, C, E, G) and subepicardial (B, D, F, H) regions of the corresponding scars are visualized using immunostaining with an antibody against cardiac MHC β-isoform (green color), whereas the resistance microvessels are identified with an antibody against α-SM actin (red color in A–G and bright green color in H). Note that in addition to small resistance microvessels (arrowheads and asterisks), the α-SM actin-positive immunostaining is present in the walls of a former coronary artery (A in micrograph C) and large subepicardial veins (V) as well as within the layer of subendocardial myofibroblasts in E and G. In all micrographs, nuclei are counterstained with DAPI (blue color). Furthermore, in A, C, E, and G, arrows point to the endocardium, whereas in B, D, F, and H, the white dotted lines mark the epicardial border. It is important to highlight that although the existence of α-SM actin expression in these microvessels suggests their belonging to resistance vasculature, primarily to small- and medium-sized arterioles, phenotypically, most of them resemble the arterialized thin-walled venous vessels, such as postcapillary venules (arrowheads) and small veins (asterisks). The latter assumption is also corroborated by the fact that according to the pattern of α-SM actin-positive immunoreactivity, such “resistance-like” microvessels often display a discontinuous and relatively thin muscular wall in comparison with their luminal diameter. For comparison, the double arrow in micrograph A points to a relatively typical arteriolar vessel seen inside the developing granulation tissue. Scale bars are 20 µm (A–H). Abbreviations: MI, myocardial infarction; MHC, myosin heavy chain; SM, smooth muscle; DAPI, 4′,6-diamidino-2-phenylindole.

Figure 9.

Immunofluorescence and Masson’s trichrome stained micrographs displaying the viable cardiac myocytes in the vicinity of intramural sinusoidal vessels within 1-week-old (A) and 4-week-old (B, C) transmural post-MI scars. (A) High-power view of an immunofluorescence micrograph from the intramural region of a 1-week-old scar showing the viable cardiac actin-positive myocytes (red color) around the thin-walled sinusoidal vessels (S), which are outlined with the laminin-positive basement membranes (green color). Nuclei are counterstained with DAPI (blue color). Note that one of two depicted sinusoidal vessel (arrowheads) extends for a relatively long distance within the scar. (B) Low-power view of a representative transversal section through a 4-week-old scar stained with Masson’s trichrome stain demonstrating the typical sites of cardiac myocytes survival (dark red color), including the areas beneath the endocardium (arrows) and epicardium, particularly around the large subepicardial veins (V), and, most importantly, along the intramural venous sinusoids (S). Notice a complete absence of viable cardiac myocytes around the arterial vessels (A). (C) High-power view of the area outlined by a black box in B showing the presence of viable cardiac myocytes intramurally, along the thin-walled sinusoidal vessels (S). It is important to emphasize that long-term survival of mature cardiac myocytes around such vascular structures suggests their functional potency for persistent blood perfusion. Scale bars are 50 µm (A), 500 µm (B), and 100 µm (C). Abbreviations: MI, myocardial infarction; DAPI, 4′,6-diamidino-2-phenylindole.

Figure 10.

High-power micrographs demonstrating the existence of the vascular orifices (arrows), probably from Thebesian veins or venous channels, on the endocardial surface of 3-day-old (A, B), 1-week-old (C, D), and 2-week-old (E, F) transmural post-MI scars that connect the subendocardial vascular network associated with surviving cardiac myocytes to the LV cavity. The representative micrographs are stained either with hematoxylin and eosin (A) or immunofluorescence (B–F). In all micrographs, asterisks denote the profiles of the vascular structures, including venous microvessels and sinusoids, whereas arrowheads expose the vascular channels linking endocardial orifices to subendocardial vessels. In immunofluorescence micrographs B, C, D, and F, the viable cardiac myocytes (red color) are visualized using double immunostaining with antibodies against either cardiac actin (B, D, and F) or cardiac MHC β-isoform (C) and laminin (green color). In immunofluorescence micrograph E, the viable cardiac myocytes (green color) are visualized using immunostaining with an anticardiac MHC β-isoform antibody, whereas vascular structures (red color) are revealed with the GS-IB₄ lectin staining. In all immunofluorescence micrographs, nuclei are counterstained with DAPI (blue color). Note, in micrographs A, B, and C, besides the subendocardial region, the surviving cardiac myocytes are also seen intramurally, extending deeper along the venous vascular structures, most likely Thebesian veins and venous sinusoids, which are directly linked to LV cavity. Scale bars are 200 µm (A), 50 µm (C), and 20 µm (B, D, E, and F). Abbreviations: MI, myocardial infarction; MHC, myosin heavy chain; LV, left ventricular; GS-IB₄, Griffonia Simplicifolia isolectin IB₄; DAPI, 4′,6-diamidino-2-phenylindole.

Figure 11.

Hematoxylin and eosin–stained micrographs demonstrating the presence of a microsphere within a venular-like microvessel associated with the group of viable cardiac myocytes in representative 8-week-old transmural post-MI scar. (A) Low-power view of the subepicardial region revealing a microsphere (arrows) surrounded by surviving cardiac myocyte. (B) High-power view of an area outlined by a black box in A displaying the same microsphere (arrows) in a microvessel surrounded by three viable cardiac myocytes (asterisks). (C) High-power view of the same region shown in B (obtained from an adjacent serial section) demonstrating a profile of the microvessel in which a microsphere (arrows) has been detected. In all micrographs, arrows point to the location of a microsphere, whereas in B and C, the asterisks indicate the same cardiac myocytes located in the vicinity of the microsphere-containing microvessel. It is important to emphasize that the microsphere-containing microvessel has an apparent phenotypic resemblance of a venule. A and V indicate an artery and a vein, respectively. Scale bars are 50 µm (A) and 30 µm (B, C). Abbreviation: MI, myocardial infarction.

Figure 12.

Hematoxylin and eosin–stained micrographs demonstrating the presence of microspheres (asterisks) in the lumen of functional microvessels accompanying the viable cardiac myocytes in 4-week-old (A, B), 8-week-old (C, D), and 12-week-old (E, F) transmural post-MI scars. The clusters of cardiac myocytes surviving in subepicardial (A, C, E) and subendocardial (B, D, F) regions of the corresponding scars are outlined by the black dotted lines. In micrographs B, D, and F, arrows point to the endocardium, whereas in micrographs C and E, veins are indicated by V and an artery by A. It is important to emphasize that the presence of microspheres in microcirculatory beds associated with the viable cardiac myocyte suggest their functional potency. Moreover, structural appearance of a majority of thin-walled microsphere-containing microvessels points at their venous origin. Scale bar is 30 µm (A–F). Abbreviation: MI, myocardial infarction.