Daisuke Matsuyama

Proliferation of neonatal cardiomyocytes by connexin43 knockdown via synergistic inactivation of p38 MAPK and increased expression of FGF1.

Gap junctions are intercellular channels that connect the cytoplasm of adjacent cell. Gap junctional intercellular communication has long been postulated to contribute to the maintenance of tissue homeostasis. Recent studies, however, have demonstrated that connexins, gap junction proteins, are involved in the regulation of a variety of cellular functions other than intercellular communication. Although, in neonatal rat ventricular myocytes, connexin-40, -43, and -45 are all expressed, connexin43 (Cx43) is the primary subtype. In this study, we examined whether and if so how the knockdown of a gap junction protein Cx43 with siRNA produced changes in the proliferative activity of neonatal cardiomyocytes. Cx43-knockdown resulted in a significant increase in the proliferation of cardiomyocytes. To clarify the mechanisms behind this increase, we investigated whether the activity of mitogen-activated protein kinases (MAPKs) changed on knockdown of Cx43. The knockdown decreased the expression of phosphorylated p38 (p-p38) MAPK. In addition, treatment of cardiomyocytes with a p38 MAPK inhibitor significantly increased the proliferative activity. Cultures were then co-treated with an inhibitor of p38 MAPK and fibroblast growth factor-1 (FGF1), since Cx43-knockdown significantly increased cytosolic FGF1 expression as well. The co-treatment enhanced the proliferation of cardiomyocytes compared with the treatment with the p38 MAPK inhibitor alone. Taken together, the present study demonstrated that Cx43-knockdown produced a significant increase in the proliferation of neonatal cardiomyocytes.

Negative-feedback regulation of ATP release: ATP release from cardiomyocytes is strictly regulated during ischemia.

Extracellular ATP acts as a potent agonist on cardiomyocytes, inducing a broad range of physiological responses via P2 purinoceptors. Its concentration in the interstitial space within the heart is elevated during ischemia or hypoxia due to its release from a number of cell types, including cardiomyocytes. However, the exact mechanism responsible for the release of ATP from cardiomyocytes during ischemia is not known. In this study, we investigated whether and how the release of ATP was strictly regulated during ischemia in cultured neonatal rat cardiomyocytes. Ischemia was mimicked by oxygen-glucose deprivation (OGD). Exposure of cardiomyocytes to OGD resulted in an increase in the concentration of extracellular ATP shortly after the onset of OGD (15 min), and the increase was reversed by treatment with blockers of maxi-anion channels. Unexpectedly, at 1 and 2h after the onset of OGD, the blocking of maxi-anion channels increased the concentration of extracellular ATP, and the increase was significantly suppressed by co-treatment with blockers of hemichannels, suggesting that ATP release via maxi-anion channels was involved in the suppression of ATP release via hemichannels during persistent OGD. Here we show the possibility that the release of ATP from cardiomyocytes was strictly regulated during ischemia by negative-feedback mechanisms; that is, maxi-anion channel-derived ATP-induced suppression of ATP release via hemichannels in cardiomyocytes.

Rhythmic Fluctuations in the Concentration of Intracellular Mg2+ in Association with Spontaneous Rhythmic Contraction in Cultured Cardiac Myocytes

Magnesium ions (Mg2+) play a fundamental role in cellular function, but the cellular dynamic changes of intracellular Mg2+ remain poorly delineated. The present study aims to clarify whether the concentration of intracellular Mg2+ possibly changes cyclically in association with rhythmic contraction and intracellular Ca2+ oscillation in cultured cardiac myocytes from neonatal rats. To do this, we performed a noise analysis of fluctuations in the concentration of intracellular Mg2+ in cardiac myocytes. The concentration was estimated by loading cells with either Mg‐fluo4/AM or KMG‐20/AM. Results revealed that the intensity of Mg‐fluo‐4 or KMG‐20 fluorescence fluctuated cyclically in association with the rhythmic contraction of cardiac myocytes. In addition, the simultaneous measurement of Fura2 and Mg‐fluo‐4 fluorescence revealed phase differences between the dynamics of the two signals, suggesting that the cyclic changes in the Mg‐fluo‐4 or KMG‐20 fluorescent intensity actually reflected the changes in intracellular Mg2+. The complete termination of spontaneous rhythmic contractions did not abolish Mg2+ oscillations, suggesting that the rhythmic fluctuations in intracellular Mg2+ did not result from mechanical movements. We suggest that the concentration of intracellular Mg2+ changes cyclically in association with spontaneous, cyclic changes in the concentration of intracellular Ca2+ of cardiac myocytes. A noise analysis of the fluctuation of subtle changes in fluorescence intensity could contribute to the elucidation of novel functional roles of Mg2+ in cells.

Maintenance and characterization of spontaneous contraction rhythm in cultured cardiac myocytes fused with cardiac fibroblasts

Cardiomyocytes (CMs) fuse with various cells including endothelial cells, cardiac fibroblasts (CFs). In addition, recent studies have shown that stem cells fuse spontaneously with cells remaining in the damaged tissues, and restore tissue functions after myocardial infarction. In this study, we investigated whether cultured cardiomyocytes fused with proliferative cardiac fibroblasts maintained the phenotype of functional myocytes by analyzing the spontaneous contraction rhythm after fusion with CFs lacking a beating capability. CMs and CFs cultured for 4 days in vitro were used in this study. The fusion of cultured CMs and CFs was achieved with polyethylene glycol (PEG) and hemagglutinating virus of Japan (HVJ). Analyses of CMs fused with CFs by using either PEG or HVJ to imitate spontaneous fusion in vivo demonstrated that CMs and CFs actually fused together and fused cells expressed lineage marker proteins of both CMs and CFs. In addition, fused cells reentered the G2-M phase of the cell cycle. Furthermore, fused cells retained the spontaneous contraction activity. The present study demonstrated that CMs fused with proliferative CFs showed the phenotype of both CMs and CFs and spontaneous rhythmic contraction.

Negative-Feedback Regulation of ATP Release During Ischemia in Cardiac Myocytes

Extracellular ATP acts as a potent agonist on cardiomyocytes, inducing a broad range of physiological responses via P2 purinoceptors. Its concentration in the interstitial space within the heart is elevated during ischemia or hypoxia due to its release from a number of cell types, including cardiomyocytes. However, the exact mechanism responsible for the release of ATP from cardiomyocytes during ischemia is not known. In this study, we investigated whether and how the release of ATP was strictly regulated during ischemia in cultured neonatal rat cardiomyocytes. Here we show the possibility that the release of ATP from cardiomyocytes was strictly regulated during ischemia by negative-feedback mechanisms; that is, maxi-anion channel-derived ATP-induced suppression of ATP release via hemichannels in cardiomyocytes.

Positive-Feedback Regulation Loop for the Loss of Cell Division and Binucleation Shortly after Birth in Cardiomyocytes

Shortly after birth, mammalian cardiomyocytes irreversibly exit from the cell cycle and become terminally differentiated. The cellular mechanisms responsible for the cessation of cell division and terminal differentiation of cardiomyocytes soon after birth have intrigued developmental biologists as well as cardiovascular physicians, but the genetic cues for the irreversible exit from the cell cycle soon after birth remain largely unknown. Here we examined whether and if so how oxidative stress to mammalian hearts during fetal-toneonatal transition produces changes in the proliferative activity and terminal differentiation of cardiomyocytes. Scavenging of reactive oxygen species (ROS) during fetal-to-neonatal transition, especially after birth, resulted in an increase in the proliferative activity and a decrease in the ratio of binucleated cardiomyocytes. Exposure to ROS in cultured cardiomyocytes increased the activity of p38 MAPK and the expression of connexin43 (Cx43). Not only down-regulation of Cx43 using siRNA but also the inhibition of p38 MAPK activity resulted in a significant decrease in the production of ROS in cardiomyocytes, suggesting that the signaling pathway ROS–p38 MAPK–Cx43 (especially, Cx43 at mitochondria, mtCx43) constituted a closed regulatory system with positive-feedback. In addition, continuous scavenging of ROS or suppression of p38 MAPK activity for 4 days after birth resulted in a significant decrease in the expression of mtCx43 and in the number of binucleated cardiomyocytes. This study demonstrated that the ROS-induced formation of a positive-feedback loop ROS–p38 MAPK–mtCx43 for the sustained activation of p38 MAPK soon after birth possibly contributes to the loss of cell division and binucleation in mammalian cardiomyocytes.

Rhythmic Fluctuations in Intracellular Mg2+ in Spontaneously Beating Cultured Cardiac Myocytes

Magnesium ions (Mg2+) play fundamental role in cellular function, but the cellular dynamic changes of intracellular Mg2+ remains poorly delineated. In this study, we performed noise analysis of the fluctuations in the concentration of intracellular Mg2+ in spontaneously beating cultured cardiac myocytes from neonatal rats. The concentration of intracellular Mg2+ was estimated by loading of cells with either Mg-fluo4/AM or KMG-20/AM. The results revealed that the intensity of Mg-fluo-4 or KMG-20 fluorescence cyclically fluctuated in association with rhythmic contraction of cardiac myocytes. In addition, the simultaneous measurement of Fura2 and Mg-fluo-4 fluorescence revealed that there were phase differences between the dynamics of two signals, suggesting that the cyclic changes in the Mg-fluo-4 or KMG-20 fluorescent intensity actually reflected the changes in intracellular Mg2+. Treatment of cultures with 2,3-butanedione monoxime (BDM), a reversible blocker of cardiac contraction, for 20 min resulted in the complete termination of spontaneous rhythmic contractions while both Ca2+ and Mg2+ oscillations were still observed, suggesting that the intracellular Mg2+ oscillation did not result from mechanical movements in association with the spontaneous rhythmic contraction of cardiac myocytes. The present study demonstrated that the concentration of intracellular Mg2+ changed in association with spontaneous, cyclic contractions and intracellular Ca2+ oscillation of cardiac myocytes. Noise analysis of the subtle changes in the fluorescence intensity may lead to the elucidation of novel functional roles played by changes in intracellular Mg2+ in cells.