Cardiac regeneration from activated epicardium

Abstract

In contrast to lower vertebrates, the mammalian heart has a very limited regenerative capacity. Cardiomyocytes, lost after ischemia, are replaced by fibroblasts. Although the human heart is able to form new cardiomyocytes throughout its lifespan, the efficiency of this phenomenon is not enough to substitute sufficient myocardial mass after an infarction. In contrast, zebrafish hearts regenerate through epicardial activation and initiation of myocardial proliferation. With this study we obtain insights into the activation and cellular contribution of the mammalian epicardium in response to ischemia. In a mouse myocardial infarction model we analyzed the spatio-temporal changes in expression of embryonic epicardial, EMT, and stem cell markers and the contribution of cells of the Wt1-lineage to the infarcted area. Though the integrity of the epicardial layer overlaying the infarct is lost immediately after the induction of the ischemia, it was found to be regenerated at three days post infarction. In this regenerated epicardium, the embryonic gene program is transiently re-expressed as well as proliferation. Concomitant with this activation, Wt1-lineage positive subepicardial mesenchyme is formed until two weeks post-infarction. These mesenchymal cells replace the cardiomyocytes lost due to the ischemia and contribute to the fibroblast population, myofibroblasts and coronary endothelium in the infarct, and later also to the cardiomyocyte population. We show that in mice, as in lower vertebrates, an endogenous, epicardium-dependent regenerative response to injury is induced. Although this regenerative response leads to the formation of new cardiomyocytes, their number is insufficient in mice but sufficient in lower vertebrates to replace lost cardiomyocytes. These molecular and cellular analyses provide basic knowledge essential for investigations on the regeneration of the mammalian heart aiming at epicardium-derived cells.

Figure 1. Epicardial activation in response to myocardial ischemia.

Panels A–J show the pattern of expression of cTnI (A,F), Raldh1 (B,G), Raldh2 (C,H), Tbx18 (D,I) and Wt1 (E,J) mRNA in the normal healthy heart. Raldh1 (B), Raldh2 (C), Tbx18 (D) and Wt1 (E) are expressed in the epicardium and the adjacent subepicardial mesenchyme of the atrioventricular sulcus to various extends. Raldh1 (G) is expressed throughout the epicardium overlaying the atria. Raldh2 (H), Tbx18 (I) and Wt1 (J) are expressed in a small subset of the epicardial cells overlaying the atria. Panel K shows a representative four-chamber view of an adult heart one day post-MI; the absence of expression of cTnI mRNA demarcates the infarcted area. The boxes indicate the atrium (1), atrio-ventricular sulcus (2), infarcted area of left ventricular free wall (3), the apex (4), and the right ventricular free wall (5). In the subsequent panels we have indicated this number at the left top site to indicate the position the picture was taken. Wt1 mRNA is expressed in the epicardium covering the ischemic region three days post-MI (L), to a level comparable to the one in the epicardium covering the atria (M). At one week post-MI, Wt1 is expressed in the epicardium covering the apex and left ventricle (N). Tbx18 (O–R) is expressed throughout the epicardium of the atria (O), within the atrioventricular sulcus (P), and the epicardium overlaying the infarct (Q) and it border zone (R). Raldh1 (S–U) is expressed throughout the epicardium covering the ventricles after a myocardial infarction (S,T), and in the subepicardial mesenchyme that is found in the wide subepicardial space overlaying the border zone of the infarction (U). Raldh2 (V) is expressed throughout the epicardium at one week post-MI. To validate these data we performed a qPCR analysis (W). As expected cTnI mRNA is down-regulated immediately after the induction of the infarction. Wt1, Tbx18 and Raldh1 mRNA become transiently up-regulated, being most highly expressed at 3d post-MI. Abbreviations: LA; left atrium, LV: left ventricle, RV: right ventricle, AVS: atrioventricular sulcus, Epi: epicardium, Subepi: subepicardium. The bar is 100 µm.

Figure 2. Comparison of the expression pattern of Wt1 and Cre in the healthy and infarcted heart.

In the ventricles of control (A–C) Wt1^Cre mouse cTnI mRNA is expressed in all cardiomyocytes (A), whereas neither Wt1 (B) nor Cre (C) mRNA are detected in the ventricles. One day after LAD ligation (post-MI) (D–F), the expression level of cTnI mRNA has dropped below detection in the infarcted zone and is expressed at a lower level in the cardiomyocytes of the border zone than in remote healthy myocardium (D). At one day post-MI both Wt1 and Cre (E & F) are not detected. Three days post-MI (G–O), the expression of both Wt1 (H L) and Cre (I, N) are found in the epicardial cells covering the border of the infarcted area (G) and the infarct proper (J), but not covering remote, healthy myocardium (K, M & O). One week post-MI (P–U), the pattern of expression of Wt1 (R, S) and Cre (T, U) is essentially the same as at three days post-MI. Three months post-MI (U–W), Wt1 (W) and Cre (X) are, like in the control heart, not longer detectable. Abbreviations: LV: left ventricle, RV: right ventricle, Epi: epicardium. The bar is 100 µm.

Figure 3. Post ischemic epicardial lineage analysis.

In healthy Wt1^Cre X R26R hearts ß-Gal is detected in the entire epicardium covering the ventricles, atria and atrioventricular junction (A, B). βGal is also detected in cells of the coronaries and in inter-myocardial cells (A, A′). One day post-MI, no epicardial ß-Gal-expressing cells were observed to cover the infarcted area (C). In the epicardium and coronaries of the border zone ß-Gal was still present (D). Three days post-MI, ß-Gal-expressing epicardial cells covered the infarcted area (E). In the border zone a broadened (double arrow) subepicardial ß-Gal-expressing layer of cells appears (F,G). In the uninjured areas of the same heart ß-Gal expression was comparable to the control situation (H). The dotted line highlights the border between healthy and infarcted myocardium. One week post-MI the subepicardial layer has further broadened (double arrow, cf F & J) and the number of subepicardial ß-Gal-expressing cells has increased (J). One month post-MI the ß-Gal-expressing cells are present in the infarcted area and the epicardium covering the infarcted area (L–N). A subset of the ß-Gal-positive cells co-express cTnI (L), α-SMA (M) or PECAM (N). Three months post-MI the number of ß-Gal- and cTnI-co-expressing cells has increased and these cells are found throughout the infarcted area (O & P). Panel Q–S show consecutive sections showing the expression of cTnI, ß-Gal and SERCA2a. The arrow shows cardiomyocytes that are positive for all three markers. Abbreviations: LA: left atrium, LV: left ventricle, RV: right ventricle, Epi: epicardium, Subepi: subepicardium. The bar is 100 µm.

Figure 4. Proliferation of epicardial and subepicardial cells upon myocardial ischemia.

In control hearts BrdU was not detected in the epicardium covering the atrioventricular junction (A), left ventricle (B) and atrium (C). As a positive control, BrdU incorporation was confirmed in the crypts of the gut in each mouse (D). At two days post-MI BrdU was detected in Wt1-positive epicardial cells (arrows) and some Wt1-negative cells throughout the myocardium of the apex (E). The thin dotted line highlights the border between the epicardium and underlaying myocardium. At three days post-MI BrdU-positive cells are found within in the epicardium covering the infarcted area (F), atria (G), and in mesenchymal cells in the border zone of the infarct (H). At one week post-MI BrdU was detected in cells located in the subepicardial space (double arrow) covering the infarcted area (I, J). At two weeks post-MI, BrdU was detected in a few individual subepicardial cells of the atria (arrow) (K) and of the border zone of the infarct (L). At one and three months post-MI hardly any BrdU incorporation was detected in the heart (M–P). Abbreviations: LA; left atrium, LV: left ventricle, RV: right ventricle, AVS; atrioventricular junction, Epi:epicardium, Subepi: subepicardial mesenchyme. The bar is 100 µm.

Figure 5. Post-ischemic mesenchyme formation.

Snai1 mRNA is present from three days onwards in the subepicardial mesenchyme of the apex and LV covering the infarct (A), and border zone (B). qPCR analysis (C) showed that Snai1 is already induced at one day post-MI and remains high up to two weeks post-MI, after which it gradually returns to control levels. Its close relative Snai2 shows a similar activation pattern as Snai1, with the exception that Snai2 is already detected in the healthy control heart. Within the border zone (D) and infarcted area (E) the matricellular proteins Follistatin-like1 (E,H) and Periostin (F,I) are expressed at high levels in the non-myocardial cells (cf D,G). In control hearts, α-SMA is present in the smooth muscle layer of the coronaries (J). At one and two weeks post-MI α-SMA is detected in the subepicardial mesenchyme and in mesenchymal cells located in the myocardium adjacent to the epicardium overlaying the infarct (K,L). At one and two weeks post-MI α-SMA-postive subepicardial mesenchyme is also found in the atria (M). P-Smad is only found in a small minority of subepicardial mesenchymal cells (arrow) directly flanking myocardial cells and adjacent to the infarct (N). Whereas P-Erk is detected in the nucleus of a subset of epicardial cells in controls and after an infarction, as well as in the cytoplasm and nucleus of an extensive subset of the subepicardial mesenchyme (O–Q). The bar is 100 µm.

Figure 6. Expression of stem cell markers.

The different stem cell markers, CD34 (A–C), cKit (D–H) and PDGFRα (I–L) are shown in green, cTnI in red, and the nuclei in blue. In the healthy heart CD34-positive cells are found to be scattered through the epicardium covering the atria (A). After the induction of the ischemia CD34 is up-regulated and becomes highly expressed in the epicardium covering the infarct and its border zone (B,C), as well as in mesenchymal cells in the subepicardial space in the border zone (B). cKit is expressed in the epicardium and individual adjacent mesenchymal cells of the atria in control hearts (D). cKit becomes evident in scattered cells of the epicardium overlaying the border zone of the infarct at 3 days post-MI (E). At one week post-MI cKit is expressed throughout the epicardium overlaying the infarct and its border zone, as well as in a subset of immediately adjacent mesenchymal cells (G, H). Moreover, at this time point cKit is also expressed in the epicardium and a subset of adjacent subepicardial mesenchymal cells in the atrioventricular sulcus (F). In control heart PDGFRα is expressed in a small portion of the mesenchyme of the atrioventricular sulcus (I). At three days post-MI (J) and one week post-MI (L) PDGFRα is expressed in the epicardium overlaying the infarction. In the border zone PDGFRα is also expressed in mesenchymal cells within the subepicardial space (L). The dotted line highlights the border between infarcted tissue and healthy myocardium. The double arrow highlights the width of the subepicardial space. Abbreviations: LA: left atrium, LV: left ventricle, Epi: epicardium, AVS: atrioventricular sulcus. The bar is 100 µm.

Figure 7. Schematic overview of morphological and molecular changes upon myocardial ischemia.