Research exchange with Cris: from fluorescence spectroscopy to human myocardium

I do not remember exactly when I got to know Cris (Fig. 1). The first contact may have been around the time of the connectin/titin issue. Cris reported in his doctoral thesis (dos Remedios 1965; dos Remedios & Gilmour 1978) that in addition to actin (thin) and myosin (thick) filaments, a third filament exists in the sarcomeres of striated muscle. I may have come to know Cris through the interaction with the late Dr. Koscak Maruyama, a discoverer of connectin (cf. Maruyama 1997).

Fig. 1.

Cris and Shin’ichi at the 62nd Annual Meeting of Biophysics USA in San Francisco (February 2018)

What I remember clearly is that I spent about a month in the summer of 1987 at Cris’s laboratory within the University of Sydney. This was due to the establishment of an exchange program for faculty members between Waseda University and the University of Sydney. I think it was Cris who was aware of the agreement, and I think the invitation came from Cris. Anyway, as the first faculty member to exchange research using the agreement, I decided to go to Sydney. One of the reasons I decided to go was that I was interested in the spectroscopic method of measuring the distance between proteins using the fluorescence resonance energy transfer (FRET) that was being advanced in Cris’s laboratory at that time. Using the method, I was interested in measuring the average distance between the heads of myosin molecules, especially when bound to actin filaments. Moreover, what made me want to go was because Masao Miki was staying as a postdoctoral fellow in Cris’s laboratory. Dr. Miki was a young graduate student at Nagoya University, Faculty of Science, Physics Department (of whom the big boss was the late Fumio Oosawa).

I received my Ph.D. in 1975 from the Oosawa laboratory, and from January 1976 to March 1979, I was a postdoctoral fellow at MIT (Dr. Toyoichi Tanaka, my classmate in high school) and BBRI (Drs. John Gergely and Jack Seidel) in Boston for a total of 3 years and 3 months. I had not studied abroad since returning to Japan in April 1979. In 1987, my laboratory in Japan was finally on track, and the results of research were beginning to come out, so it was a time when I was confident that I could leave the laboratory for a month or so.

However, when I went to Sydney, Cris decided to go to the USA during the summer vacation, and we ended up only overlapping for half of the month I was there. In addition, Dr. Miki said that his mother in Japan was feeling sick, so that he must return to Japan immediately from the Airport, where he welcomed me. Therefore, I could not meet Miki-san for the half a month that Cris was away. During their absence, my partner was Leo Phillips, a PhD student doing solid-state NMR studies of actin powder. The solid-state NMR that Leo was studying required huge amounts of protein. During my doctoral course, I was conducting research on the bending flexibility of actin filaments in a solution using the quasi-elastic light scattering method under the guidance of the late Dr. Satoru Fujime (cf. Ishiwata & Fujime, 1972). The purified actin used at that time was 3 mg/mL × 10 mL for each experiment. So, I always needed to prepare a large amount of actin (and myosin, tropomyosin, and troponin), but the amount of actin Leo used was far more than that. The research results were published in papers several years later (Phillips et al. 1991).

I was accustomed to performing fluorescence spectroscopy and was familiar with fluorescent labeling of proteins, so I was able to proceed with research as planned by myself. However, Leo, who cared about my unfamiliar dormitory life (a college dormitory on the University campus), took me to a pub in the city every weekend. The most memorable thing is that the pubs in the corners of each block have many different types of beer and I had quite a few tastings to choose which beer to drink that evening. By the time I finished the tasting and decided on a beer to drink, I was already in a good mood. In the first place, we drank it without eating, so I got drunk quickly.

Despite all the drinking on the weekend (or maybe some weekdays too), the research went smoothly. I was satisfied with the experimental results, but the publication of the paper was very late. It was published in Biophys. J. 10 years later in 1997 (Ishiwata et al. 1997). I did some additional experiments after returning to Tokyo, and it took time to make a model calculation, which was done by Kenji Yasuda (a graduate student at the time, now a professor at the Department of Physics, Waseda University). However, this paper, which I thought was (and still is) very good, seemed to be barely cited. The goal of this paper was to determine the distance between myosin heads when HMM and Subfragment-1 (S1) bind to F-actin under rigor condition. For that purpose, we selectively chose a fluorescent donor and an acceptor for labeling the SH group (the so-called SH1 group) existing in myosin heads. The results showed that the distances between the SH1 groups in HMM heads, between HMM and S1, and between S1 molecules bound to F-actin in the rigor state were 6.0–6.3 nm. On the other hand, the intramolecular distance between the bound heads of HMM was found to be significantly longer, about 8 nm.

My next close relationship with Cris resulted from his inspiration to commence a collaborative study using frozen myocardium prepared at the Sydney Human Heart Bank. We received thirty-one samples of human myocardial fragments preserved in liquid nitrogen including normal myocardium as well as pathological conditions (DCM (dilated cardiomyopathy)) and started the research at my laboratory in collaboration with Cris and Amy Li, a graduate student in Cris’s laboratory at that time. Although the research did not progress so much for several years, the research pace picked up significantly once Tatsuya Kagemoto (a graduate student of the master’s course) established a method for preparing single myofibrils from frozen myocardium. In particular, we were able to replace a mutated TnI in DCM sample with normal Tn of porcine heart in collaboration with Dr. Norio Fukuda (Jikei Medical School). As a result, we were able to show that the sarcomere length oscillation in spontaneous oscillatory contraction (SPOC) characteristics of DCM myofibrils became the normal myocardial type (Kagemoto et al. 2018).

Cris has kindly shown a long-time interest in our work on the dynamic properties of myofibrils, especially on the SPOC properties we have been studying for many years (Wolfe et al. 2011; Kagemoto et al. 2015). In particular, he was interested in how different the SPOC properties of the pathological myocardial (DCM) system were from those of normal myocardial system. Initially, we were able to put together a review article on SPOC in the Biophysical Reviews journal (Wolfe et al. 2011; Kagemoto et al. 2015). Cris also contributed a New and Notable article to the paper we published in Biophys. J. (Shimamoto et al. 2007; dos Remedios 2007).

Thus, I have had the opportunity to work with Cris for many years, starting from the problem of connectin/titin (dos Remedios 1965, 2018; dos Remedios & Gilmour 1978), the spectroscopic measurement of the intermolecular distance of myosin cross-bridges (Ishiwata et al. 1997), and the application of SPOC characteristics to the study of human myocardial system (Kagemoto et al. 2015, 2018). I am thankful that we have followed the same path of research and were able to continue our close joint research and close friendship. Both I and my current and former laboratory members thank Cris and his colleagues for their continued interests in our research, and we sincerely hope that the Cris-launched human myocardial tissue bank (dos Remedios et al. 2017) will be a major impetus for cardiac function research in the future.