A novel type of self-beating cardiomyocytes in adult mouse ventricles

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Abstract

This study was designed to investigate the presence of resident heart cells that are distinct from terminally-differentiated cardiomyocytes. Adult mouse heart was coronary perfused with collagenase, and ventricles were excised and further digested. After spinning cardiomyocyte-containing fractions down, the supernatant fraction was collected and cultured without adding any chemicals. Two to five days after plating, some of rounded cells adhered to the culture dish, gradually changed their shape and then started self-beating. These self-beating cells did not appreciably proliferate but underwent a further morphological maturation process to form highly branched shapes with many projections. These cells were mostly multinucleated, well sarcomeric-organized and expressed cardiac marker proteins, defined as atypically-shaped cardiomyocytes (ACMs). Patch-clamp experiments revealed that ACMs exhibited spontaneous action potentials arising from the preceding slow diastolic depolarization. We thus found a novel type of resident heart cells in adult cardiac ventricles that spontaneously develop into self-beating cardiomyocytes.

Introduction

Functional heart comprises heterogeneous cell lineages, including cardiomyocytes, endothelial cells and vascular smooth muscle cells. In recent years, a number of studies have demonstrated the existence of several kinds of stemlike cells in both fetal and adult hearts. Populations of cells expressing stem cell marker proteins, such as stem cell antigen-1 (Sca-1) [1], [2] and stem cell factor receptor (c-kit) [3], [4], have been identified as cardiac progenitor cells. Cell populations termed cardiac side population cells, showing stem cell characteristics, are identified by their ability to exclude Hoechst 33342 dye [5], [6]. These cells have been demonstrated to differentiate into cardiomyocytes in vitro in response to 5′-azacytidine [1], [2], oxytocin [2], [6] or trichostatin A [6]. LIM-homeobox transcription factor islet 1 (isl-1) has been identified in postnatal cardioblasts [7] as well as in embryonic myocardial precursors [8]. More recently, it has been demonstrated that cells derived from the cardiac epicardium contribute to the cardiomyocyte lineage [9], [10], [11]. Therefore, it is now generally accepted that the adult heart is not a rigidly terminally-differentiated organ.

Based on these studies, the question then arises as to whether the adult heart might contain resident cells distinct from terminally-differentiated cardiomyocytes. To examine this hypothesis, we prepared cardiac myocyte-depleted fraction (CMDF) from adult mouse ventricles and cultured them without adding any chemicals. We show here that some of the rounded cells that emerged from this CMDF culture spontaneously developed into self-beating cardiomyocytes with a peculiar morphology, defined as atypically-shaped cardiomyocyte (ACMs). This observation provides the evidence to suggest that the adult cardiac ventricles contain a novel type of heart cells that potentially express intrinsic pacemaker activity.

Section snippets

Materials and methods

Isolation and culture of CMDF cells. All animal experiments were performed in accordance with the guidelines of the institution’s Animal Care and Use Committee.

Adult heart of 8–14 weeks-old C57BL/6J mice (Charles River Japan) was enzymatically digested using a method similar to that described previously to dissociate viable cardiac ventricular myocytes [12]. The whole heart was coronary perfused at 37 °C for 10 min with the enzyme solution containing 0.1% collagenase, 0.006% trypsin and 0.006%

Rounded CMDF cells spontaneously develop into self-beating cardiomyocytes

As demonstrated in Fig. 1A, CMDF cells obtained from adult mouse heart appeared to be heterogenous in size (∅5–20 μm). These CMDF cells were directly plated in a semisolid methylcellulose-based culture medium without adding any hormones, growth factors, 5′-azacytidine or other chemicals. CMDF cells dispersed in the culture medium slowly adhered to the bottom of the culture dish and reconstructed the cell shapes depending on each cell type, such as large spreading cells, small cobble stone-like

Discussion

In this study, mouse cardiac ventricles were enzymatically digested and cardiomyocytes were pelleted by centrifugations. The remaining supernatant fraction contained smaller and lighter cells in addition to cell fragments. We cultured this fraction, CMDF, and found that some rounded CMDF cells spontaneously developed into ACMs that exhibited self-beating.

In addition to the fact that rounded CMDF cells spontaneously develop into ACMs without appreciably proliferating, it is noteworthy that

Acknowledgments

We appreciate the valuable assistance of Takefumi Yamamoto and Yasuhiro Mori with the confocal laser scanning microscopic experiments. This work was supported by Grants-in-Aid for Scientific Research from Japan Society for the Promotion of Science (No. 19590207 to M.O.-K. and No. 17590185 to H.M.).

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