The Louis N Katz prize

In 1971, I had the good fortune to be awarded the Louis N Katz Prize of the American Heart Association for Young Investigators for my first big manuscript (dos Remedios et al. 1972). Later, this manuscript was selected by the Editors of Journal of Muscle Research and Cell Motility inclusion in the list of 50 outstanding contributions to muscle research since the publications by AF Huxley and J Niedergerke and by HE Huxley and J Hanson in Nature in 1954. The news of this prize quickly spread back to Sydney, and in 1972 I was offered a Senior Lectureship in human anatomy at the University of New South Wales which I gratefully accepted. But then two years later, the head of the Department Professor Michael Blunt moved to the University of Sydney and invited me to follow him.

The Muscle Research Unit

In 1974, I established the Muscle Research Unit at the University of Sydney. I focused on the structure and function of the muscle protein actin. This ubiquitous protein is expressed in significant quantities (10% of total protein) in all cells and represents about 30% of the total contractile proteins in muscle. This research was initially funded (1975) by the National Heart Foundation and the ARC, but soon I had several concurrent project grants funded by the NHMRC that continued for three decades. I published the first images of actin monomer using two-dimensional crystals of actin (dos Remedios and Dickens, 1978), and over the next 20 years I published over 60 papers on actin and actin-binding proteins. Much of my focus was using fluorescence resonance energy transfer (FRET) spectroscopy to map intramolecular and intermolecular distances of actin of myosin. Sceptics of FRET believed that assuming the value of kappa (k2) to be 2/3 relied on the assumption that both donor and acceptor probes could freely precess. They argued that covalently binding these probes to surface amino acids would restrict probe movements; therefore, k2 was “unknown”. In 1995, we (dos Remedios and Pierre Moens) put that argument to rest, not only by citing scores of papers where k2 worked, but also by citing who demonstrated rotational freedom of peptide side chains (dos Remedios and Moens 1995). I also edited four books which arose from International Conferences I arranged devoted to advances in actin and actin-binding proteins. The last book (dos Remedios and Chhabra 2007) had 12,469 chapter downloads by 2017. However, my fascination with actin dwindled once the actin-DNaseI complex was crystallised; the atomic structure of actin was determined by Kabsch et al. in 1990.

Dr Victor Chang

In 1989 following a chance meeting, I began working with Dr Victor Chang, famous for his role in the development of human heart transplantation in Australia for which he was awarded the nation’s highest honour Companion of the Order of Australia in 1986, and in 2000 he was named Australian of the Century. Although we worked together for only two years before he was murdered, Victor changed the course of my life from a protein chemist to a 30-year research program with the aim of trying to understand the molecular basis for human heart failure. Tragically, Victor was murdered on July 4, 1991. By then, my students and I had gotten used to getting up in the small hours of the morning to collect the hearts at St Vincent’s Transplant Theaters. Also, I was travelling a lot in the US, UK, Japan and Europe developing close contacts with the muscle biophysics community through the Gordon Research conferences. My approach was simple. I believed that no one person could hope to get to the bottom of all forms of human heart failure, but as a community we could certainly make good progress.

Sydney Heart Bank

Today, the Sydney Heart Bank contains explanted hearts from over 350 patients. I am not overstating when I say the quality of the tissue from these patients was exceptional. All heart samples were snap-frozen in the theatres of the St Vincent’s Hospital in liquid nitrogen. All parts of the heart were collected within 40 minutes of the “cross-clamp” when the heart loses its supply of arterial blood. Initially, we collected only a few samples from each of the four chambers of the heart, but soon we realised that other researchers also needed tissue, particularly from the left ventricles. Victor Chang was very encouraging but real progress was inhibited by the paucity of healthy donor hearts with which we could compare with the failing hearts. Healthy non-failing hearts were primarily used as donors in the Heart & Lung Transplant Unit. However, every so often, a donor would become available with a tissue type that did not match any of the waiting patients. Gradually, we accumulated hearts that, for a variety of reasons, made them unsuitable as donors. Brain-dead donors often resulted from a motor vehicle accident (including motorcycles), but most arose from aneurisms or massive haemorrhages of the arteries that supply the brain. Others, particularly children, had drowned and been revived but with no brain wave activity. When there were no young patients waiting for a heart transplant, we collected the heart. Other donors sadly had taken their own lives. Over the decades, we have collected over 120 healthy donor hearts. None of these were post-mortem cases where tissue was not collected for 24 hours or more after death.

Sydney Heart Bank Logistics

Transportation of the heart samples around the world was achieved using nitrogen-vapour dewars. Airlines would not allow sufficient dry ice for a flight from Sydney to the West Coast of the USA. Our dewars could maintain – 190 °C for sufficient time to deliver samples from Sydney to researchers anywhere. When dewars returned to Sydney which sometimes created long delays, the Australian Quarantine Inspection Service (AQIS) even threatened to decontaminate dewars with liquid which would have destroyed a $2000 dewar. It took quite a lot of persistence to get access to the head of Australia’s AQIS and explain that our dewars were never used to transport animal tissue. That solved the problem. Collection, transportation and storage at temperatures completely prevented the degradation of sensitive biomolecules, such as RNA, and giant proteins like titin (the largest protein in the heart). Soon, reviewers of our manuscripts stopped assuming human tissue was degraded in any significant way. In fact, the tissue we collected in 1989 shows no sign of degradation in 2020. The collection process is really most efficient when two members of the team are involved. Usually, these were students working on various aspects of heart failure. I am grateful to Tom Yeoh, Toby Trahair (Trahair et al. 1990; 1993), Desiree Berry, Masako Tsubakihara, Sean Lal and Amy Li, to mention just a few, many of whom went on to do Medicine and a PhD. I could not have managed without their assistance. To date, we have collaboratively published 143 papers in high-quality journals.

What have we discovered?

The SHB has made many contributions to our understanding of the molecular basis of human heart failure. Here, I briefly outline three examples:

There has been a long-running polemic over whether post-natal human cardiomyocytes can proliferate. We sent 36 samples to Bernhard Kühn’s group (Harvard) of healthy SHB donors aged between 3 months and 65 years (Mollova et al. 2013; Polizzotti et al. 2015). They developed a novel method of isolating cardiomyocytes using a preliminary fixation followed by collagenase digestion. This yielded over 92% of cardiomyocytes. Using microscopy, they found that cardiomyocyte proliferation and growth contribute to heart development. They measured a 3.4-fold increase in the number of cardiomyocytes which was consistent with predictions based on cell cycle activity during childhood and young adolescence, suggesting that with treatment during this period it may be possible to stimulate cardiac repair.

The Sydney Heart Bank also contributed heart samples to the Jonas Frisén-Olaf Bergmann (Bergmann et al. 2015) laboratory in Stockholm, who took a very different approach to the question of proliferation. On April 26, 1986, the accident at the Chernobyl Nuclear Power Plant in northern Ukraine effectively resulted in pulse-labelling (14C) all people born after 1986. This was an extensive analysis of not only the left ventricle but of all chambers of the heart. We contributed samples from some of our younger donor hearts but over 50 human hearts were examined. They concluded that cardiomyocyte numbers do not appreciably change soon after birth and that changes in heart mass can be accounted for by hypertrophy remaining constant throughout the human lifespan, with a low turnover rate. Cardiac endothelial cell turnover was greater than 15% per year. Turnover of mesenchymal cells was also appreciable (< 4% year in adults), but the exchange rate for cardiomyocyte is highest in babies and children and their numbers increase into adulthood to < 1%. We will need new ideas and new techniques to determine which lab is correct.

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    The second example deals with a different but equally fundamental question. Hypertrophic cardiomyopathy (HCM) is the most common familial disease, and more than one-third of these patients carry a mutation in myosin heavy chain 7 (MYH7). While the severity of the phenotype depends on the location of the mutation in the MYH7 gene, it also depends on the fraction of mutated vs wild-type protein expressed. Theresia Kraft and her colleagues from the laboratory of the Late Bernard Brenner (Hannover) reported a surprising result. Using left ventricle samples from HCM patients, they measured contractile force in isolated single cardiomyocytes and found large cell-to-cell differences in Ca-induced contraction (Montag et al. 2018). They also characterised gene transcription by visualising active transcription of intronic and exonic sequences of MYH7-pre-mRNA (Montag et al. 2018). Some cardiomyocytes were essentially the same, as healthy donors suggested they expressed no mutated MYH7 RNA. In total, 27% of myocyte nuclei were without active transcription sites and this was inconsistent with a continuous transcription mechanism. However, it was consistent with a burst-like transcription of mutant where wild-type alleles result in some myocytes having a completely wild-type expression, while adjacent cells had a completely hypertrophic phenotype (Montag et al. 2018). These reports ring “alarm bells” when attempting to phenotype from analyses of bulk heart tissue.

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    My third example arose in a curious way. We had been collaborating with Michael Ackerman and Martijn Bos (Mayo Clinic) for nearly a decade but we puzzled over the meaning of the data, so we decided to not publish. Then, in February, when COVID-19 became hot news, they remembered that ACE2 was the single largest upregulated (×3.5) gene and was the most (×5) upregulated protein. The connection to COVID-19 and heart complications suddenly became clearer because ACE2 is the membrane receptor through which SARS-CoV-2 enters cardiomyocytes, thus conferring an increased risk of cardiac involvement in COVID-19. The paper (Bos et al. 2020) was quickly accepted, but it illustrates how good science is never wasted.

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