Keiichi Fukuda

Calmodulin Kinases II and IV and Calcineurin Are Involved in Leukemia Inhibitory Factor–Induced Cardiac Hypertrophy in Rats

We recently reported that leukemia inhibitory factor (LIF) enhances Ca2+]i through an increase in L-type Ca2+ current (ICa,L) in adult cardiomyocytes. The aim of this study was to investigate whether LIF activates Ca2+-dependent signaling molecules, such as calcineurin and calmodulin kinases II and IV (CaMKII and CaMKIV), and, if so, whether these Ca2+-mediated signaling events contribute to LIF-mediated cardiac hypertrophy. We first confirmed that LIF increased ICa,L and [Ca2+]i in primary cultured rat neonatal cardiomyocytes. Calcineurin, CaMKII, and CaMKIV activities increased at 2 minutes and peaked by 1.6-, 2.2-, and 2.2-fold, respectively, at 15 minutes. Nicardipine or verapamil fully inhibited these activities. Autophosphorylation of CaMKII was also observed to parallel the timing of CaMKII activity, and this phosphorylation was blocked by nicardipine, verapamil, or EGTA. LIF treatment led to a 3-fold increase in nuclear factor of activated T cell–luciferase activity. To confirm that inositol triphosphate (IP3)-induced Ca2+ release from sarcoplasmic reticulum was not involved in this process, IP3 content and phosphorylation of phospholipase C&ggr; were investigated. LIF did not increase IP3 content or phosphorylate phospholipase C&ggr;. KN62 (an inhibitor of CaMKII and CaMKIV) attenuated c-fos, brain natriuretic peptide, &agr;-skeletal actin, and atrial natriuretic peptide expression. KN62 suppressed the LIF-induced increase in [3H]phenylalanine uptake and cell size. Cyclosporin A and FK506 slightly attenuated brain natriuretic peptide but did not affect c-fos or atrial natriuretic peptide expression. Cyclosporin A significantly reduced the LIF-induced increase in [3H]phenylalanine uptake. These findings indicated that LIF activated CaMKII, CaMKIV, and calcineurin through an increase in ICa,L and [Ca2+]i and that CaMKII, CaMKIV, and calcineurin are critically involved in LIF-induced cardiac hypertrophy.

Practical Application of Confocal Laser Scanning Microscopy for Cardiac Regenerative Medicine

Heart failure (HF) is an insidious disease in developed countries. Despite recent medical progress, the number of patients with HF continues to increase, with the mortality of HF as high as that of cancer. The only radical treatment for HF is cardiac transplantation, although the shortage of donor hearts poses a serious problem [1]. To overcome this unmet medical need, innovative technology is required. Specifically, cell transplantation therapy with regenerative cardiomyocytes is expected to eventually replace cardiac transplantation as the treatment for severe HF.

Induced Pluripotent Stem Cells for Clinical Use

Induced pluripotent stem cells (iPSCs) are expected to be a novel cell source for regenerative medicine. Although iPSCs represented a significant breakthrough, there were many initial obstacles for their clinical use such as exogenous sequence insertions, inefficient cell reprogramming, tumorigenic properties, and animal-derived culture components. However, much progress has been made in iPSC generation since their development. The first human trial of iPSC-derived cell transplantation was conducted in September 2014, in which iPSC-derived retinal pigment epithelial cells were transplanted to a patient with macular degeneration. Because multiple clinical trials using iPSCs are expected in the near future, preparation of guidelines for generating and selecting iPSC lines suitable for clinical application is a pressing issue. For clinical use of iPSCs, many examinations for evaluating iPSC lines must be conduct‐ ed before transplantation. Different combinations of reprogramming factors, gene derivation vehicles, and types of donor cells can affect the quality of iPSCs, and guidelines for selecting the most appropriate iPSC lines for clinical use are under development. Furthermore, development of timeand cost-effective selection methods is essential for expanding iPSC transplantation therapy. In this chapter, we review methods for preparing human iPSCs before clinical use and the issues that are important for defining standard‐ ization of clinical-grade iPSCs.