Challenges measuring cardiomyocyte renewal

Abstract

Interventions to effect therapeutic cardiomyocyte renewal have received considerable interest of late. Such interventions, if successful, could give rise to myocardial regeneration in diseased hearts. Regenerative interventions fall into two broad categories, namely approaches based on promoting renewal of pre-existing cardiomyocytes and approaches based on cardiomyogenic stem cell activity. The latter category can be further subdivided into approaches promoting differentiation of endogenous cardiomyogenic stem cells, approaches wherein cardiomyogenic stem cells are harvested, amplified or enriched ex vivo, and subsequently engrafted into the heart, and approaches wherein an exogenous stem cell is induced to differentiate in vitro, and the resulting cardiomyocytes are engrafted into the heart. There is disagreement in the literature regarding the degree to which cardiomyocyte renewal occurs in the normal and injured heart, the mechanism(s) by which this occurs, and the degree to which therapeutic interventions can enhance regenerative growth. This review discusses several caveats which are encountered when attempting to measure cardiomyocyte renewal in vivo which likely contribute, at least in part, to the disagreement regarding the levels at which this occurs in normal, injured and treated hearts. This article is part of a Special Issue entitled: Cardiomyocyte biology: Cardiac pathways of differentiation, metabolism and contraction.

Figure 1

Detection of cardiomyocyte nuclei in MHC-nLAC transgenic mice. A. Schematic depiction of the MHC-nLAC transgene. The MHC promoter consists of 4500 bp of 5′ flanking sequence plus Exons 1, 2 and the non-coding region of Exon 3. The protamine terminator was inserted down-stream of the beta-galactosidase sequence to ensure proper processing of transgene-encoded transcripts. B. Low power image of a section from an adult MHC-nLAC transgenic heart following reaction with X-GAL, a chromogenic beta-galactosidase substrate. Cardiomyocyte nuclei appear blue. C. Low power image of a section from an adult MHC-nLAC transgenic heart processed for beta-galactosidase immune reactivity. Cardiomyocyte nuclei are identified by green fluorescence.

Figure 2

Use of tritiated thymidine incorporation in conjuction with the MHC-nLAC reporter transgene to detect cardiomyocyte cell cycle activity in normal and injured hearts. A. Cardiomyocyte DNA synthesis in a normal heart from a mouse receiving a single injection of tritiated thymidine. The image shows a section following X-GAL reaction and autoradiography. An S-phase cardiomyocyte nucleus is identified by the presence of silver grains over blue signal. B. Detection of cardiomyocyte (arrow) and non-cardiomyocyte (arrowheads) DNA synthesis at the peri-infarct zone in a mouse carrying the MHC-nLAC reporter transgene following a single injection of tritiated thymidine. Insert shows the Hoechst epifluorescence signal from the same field.

Figure 3

Use of bromodeoxyuridine incorporation in conjuction with the MHC-nLAC reporter transgene to detect cardiomyocyte cell cycle activity in normal and injured hearts. A. Cardiomyocyte DNA synthesis in a normal heart from a mouse receiving a single injection of bromodeoxyuridine. The images show the same field of a section following processing for beta-galactosidase (green fluorescence, left panel) and bromodeoxyuridine (red fluorescence, middle panel) immune reactivity. The image in the right panel was captured using a dual-channel filter; the S-phase cardiomyocyte nucleus (indicated by the arrow in all three panels) appears yellow due to the overlay of green and red fluorescence. B. Detection of cardiomyocyte (yellow signal) and non-cardiomyocyte (red signal) DNA synthesis at the peri-infarct zone in a mouse carrying the MHC-nLAC reporter transgene following 7 days of bromodeoxyuridine infusion. Cardiomyocyte nuclei which did not enter S-phase during the labeling period appear green.