Myocardial regeneration by activation of multipotent cardiac stem cells in ischemic heart failure

Abstract

In this study, we tested whether the human heart possesses a cardiac stem cell (CSC) pool that promotes regeneration after infarction. For this purpose, CSC growth and senescence were measured in 20 hearts with acute infarcts, 20 hearts with end-stage postinfarction cardiomyopathy, and 12 control hearts. CSC number increased markedly in acute and, to a lesser extent, in chronic infarcts. CSC growth correlated with the increase in telomerase-competent dividing CSCs from 1.5% in controls to 28% in acute infarcts and 14% in chronic infarcts. The CSC mitotic index increased 29-fold in acute and 14-fold in chronic infarcts. CSCs committed to the myocyte, smooth muscle, and endothelial cell lineages increased approximately 85-fold in acute infarcts and approximately 25-fold in chronic infarcts. However, p16(INK4a)-p53-positive senescent CSCs also increased and were 10%, 18%, and 40% in controls, acute infarcts, and chronic infarcts, respectively. Old CSCs had short telomeres and apoptosis involved 0.3%, 3.8%, and 9.6% of CSCs in controls, acute infarcts, and chronic infarcts, respectively. These variables reduced the number of functionally competent CSCs from approximately 26,000/cm3 of viable myocardium in acute to approximately 7,000/cm3 in chronic infarcts, respectively. In seven acute infarcts, foci of spontaneous myocardial regeneration that did not involve cell fusion were identified. In conclusion, the human heart possesses a CSC compartment, and CSC activation occurs in response to ischemic injury. The loss of functionally competent CSCs in chronic ischemic cardiomyopathy may underlie the progressive functional deterioration and the onset of terminal failure.

Fig. 1.

Heart failure and CSCs. Shown are the expression of surface epitopes (A) in CSCs and the number of CSCs per cm³ of viable myocardium (B). ^*, P < 0.05 vs. controls (C); ^**, P < 0.05 between the border and remote myocardium in acute myocardial infarction (MI). †, P < 0.05 between acute and chronic MI. (C) Expression of TERT in human cardiac protein lysates from control (C), acute (A), and chronic (Ch) infarcted hearts. (D) TERT activity measured by the telomeric repeat amplification protocol assay. Products of TERT activity start at 50 bp and display a 6-bp periodicity. Samples treated with RNase (+) were used as a negative control and HeLa cells as a positive control. Serial dilutions of proteins (0.5 and 1.0 μg) were used to confirm the specificity of the reaction. The band at 36 bp corresponds to an internal control for PCR efficiency. (E) Expression of phospho-Akt and total Akt; (F) unphosphorylated (filled arrowhead) and phosphorylated (open arrowhead) forms of TERT in an Akt kinase assay. (G) Expression of the telomere-related proteins, TRF-1, TRF-2, and DNA-PK (DNA-PK_cs, Ku86, and Ku70), and full-length and cleaved poly (ADP-ribose) polymerase. (H) Expression of cell cycle inhibitors and markers of cellular senescence. Optical density values are shown in Fig. 10.

Fig. 2.

Heart failure and CSC senescence. c-kit positive CSCs (green, A-F) express p16^INK4a (yellow, A and B) and p53 (magenta, C and D). The detection of telomeres in c-kit-positive CSCs is shown by small white fluorescence dots (E and F). Shown are control myocardium (A), acute infarcts (C and E), and chronic infarcts (B, D, and F). (Scale bars: 10 μm.)

Fig. 3.

Heart failure and CSC growth. (A-D) c-kit-positive CSCs (green, arrows) express telomerase (red dots, A and C) and MCM5 (white dots, B) or Ki67 (yellow dots, D). Myocytes are labeled by α-sarcomeric actin (red) and nuclei by DAPI (blue). (E) Percentage of telomerase competent CSCs in the cell cycle. (F-I) c-kit-positive CSCs (green) have nuclei labeled by phospho-H3 (red). (J) Mitotic index of CSCs. ^*, P < 0.05 vs. controls (C); ^**, P < 0.05 between the border and remote myocardium in acute MI; †, P < 0.05 between acute and chronic MI. A, B, and F-I are acute infarcts. (Scale bars: 10 μm.)

Fig. 4.

Heart failure and lineage commitment of CSCs. (A-C) Myocyte progenitors (A) express c-kit (green) and the myocyte transcription factor MEF2C (yellow dots); myocyte precursors (B) express c-kit (green), MEF2C (yellow dots) and have a thin layer of cytoplasm positive for α-sarcomeric actin (red). (C) Small developing myocytes (α-sarcomeric actin, red) express MEF2C (yellow dots) and have lost the stem cell surface antigen. Connexin 43 is detected between some of these maturing cells (green, arrows). (D) Myocyte progenitors and precursors. ^*, P < 0.05 vs. controls (C); ^**, P < 0.05 between the border and remote myocardium in acute MI; †, P < 0.05 between acute and chronic MI. Shown are acute infarcts (A and C) and a chronic infarct (B). (Scale bars: 10 μm.)

Fig. 5.

Heart failure and myocardial regeneration. (A) Area of regenerating myocardium within the infarct. Clusters of highly proliferating small developing myocytes are visible. Myocytes are labeled by cardiac myosin (red) and nuclei by DAPI (blue). Most of these cells are positive for MCM5 (white). Two dividing small myocytes are shown in Insets. (B) Cluster of cells included in a rectangle in the middle of an acute infarct. These cells are shown at higher magnification in C and D. They represent c-kit-positive cells (green, arrows) at times expressing cardiac myosin (red; D). (Scale bars: A and B, 100 μm; C and D, 10 μm.)

Fig. 6.

Myocardial regeneration and cell fusion. Regenerating myocytes in a male (A) and female (B) infarcted heart. In the male heart, only one X (magenta) and one Y (green) chromosome are detected. In the female heart, only two X chromosomes (magenta) are visible. (Scale bars: 10 μm.)