Do binucleate cardiomyocytes have a role in myocardial repair? Insights using isolated rodent myocytes and cell culture

Abstract

Neonatal and adult cardiomyocytes were isolated from rat hearts. Some of the adult myocytes were cultured to allow for cell dedifferentiation, a phenomenon thought to mimic cell changes that occur in stressed myocardium, with myocytes regressing to a fetal pattern of metabolism and stellate neonatal shape.Using fluorescence deconvolution microscopy, cells were probed with fluorescent markers and scanned for a number of proteins associated with ion control, calcium movements and cardiac function. Image analysis of deconvoluted image stacks and sequential real-time image recordings of calcium transients of cells were made.All three myocyte groups were predominantly comprised of binucleate cells. Clustering of proteins to a single nucleus was a common observation, suggesting that one nucleus is active in protein synthesis pathways, while the other nucleus assumes a 'dormant' or different role and that cardiomyocytes might be mitotically active even in late development, or specific protein syntheses could be targeted and regulated for reintroduction into the cell cycle.Such possibilities would extend cardiac disease associated stem cell research and therapy options, while producing valuable insights into developmental and death pathways of binucleate cardiomyocytes (word count 183).

Fig. (1)

Images of binucleate cardiomyocytes from the three isolates studied, acquired by deconvolution fluorescence microscopy as described (magnification of x600). The blue stain identifies nuclei (DAPI; two nuclei in each of the cells), the green is cardiac actin (Phallocidin) and the red are the protein, in these particular images, the alpha adrenoceptor. A=Neonate; B=Adult; C=Cultured (dedifferentiating) Adult (Mag. X 600).

Fig. (2)

Shows the IP31 receptors in a dedifferentiated cardiomyocyte (left panel, yellow) tightly clustered around one of the two nuclei while in the adult cardiomyocyte (right panel) however, the yellow Na⁺+K⁺ATPase pump protein does not demonstrate clustering Nuclei are blue, proteins are yellow and cardiac actin is green in the left panel and red in the right panel, due to different wavelength tags being used. (Mag. Ax900; Bx300).

Fig. (3)

Shows clustering of a number of probes for various proteins in binucleation. Panel A - freshly isolated adult cardiomyocytes and CGRP (red). Panel B - dedifferentiating myocyte with NFκB65. Panel C is NFκB50 in a dedifferentiating cell probed for alpha-adrenoreceptors. (Mag. X 600).

Fig. (4)

Model generated from multiple, stacked sections of fluorescence deconvolution acquisitions of a core sample of human tissue showing clustering of alpha-adrenoceptor protein around only one nucleus (single white arrow), and not with the other intramyocytic nucleus (double arrow).

Fig. (5)

Sequence of images from a cultured adult dedifferentiated cell using FLUO 4 as the calcium sensitive probe. Note the two nuclei indicated by the arrows in frame 1, the arrows show ‘hot spots’ or ‘sparks’ in frame 3. In frames 4 through 6, ‘firing’ of one of the nuclei can be seen. Frames 7 through 9 demonstrate the beginning of another cycle, with sparking in frame 8 and one nucleus firing in frame 9 and this is seen in binucleate neonatal cardiomyocytes as well.

Fig. (6)

Shows a sequence of images of freshly isolated adult cardiomyocyte, captured by real-time fluorescence spectrophotometry. The probe is FLUO 4. Highest concentrations of calcium are seen as bright white areas. Captures were made every 43 milliseconds.