Recollections of my Ph. D. research in Professor Cris dos Remedios’ laboratory

Prof Cris dos Remedios congratulated after receiving his DSc by his son Oliveri.

Making contact

I had completed a Masters of Biomedical Engineering at the University of New South Wales and wished to continue the study of the protein, dystrophin. My assessment of dystrophin in muscle biopsy samples helped to classify the muscle samples waiting for the early classification between Duchenne and Becker’s muscular dystrophy.

The muscular dystrophy specialist, Prof Graham Morgan, advised me to contact Cris. I arranged an appointment to meet Cris at his laboratory and I explained I wished to continue my studies part time and complete a PhD. Cris listened, understood that I was working in pathology and that my previous postgraduate studies had all been completed part time. A meeting was arranged that afternoon with the Head of Department, Assoc. Prof Cedric Storey. My application was supported and a wonderful period of scientific discovery had begun.

Continuing postgraduate study

The Muscle Research Unit had a vast collection of human heart samples that enabled me to study the membrane and membrane-associated proteins in the two forms of striated muscle, cardiac and skeletal. They extend from the sarcolemma to the nuclear membrane in cardiomyocytes. Alterations to their structure and function can lead to dilated cardiomyopathy (DCM) in cardiac muscle and muscular dystrophy in skeletal muscle. Cardiac and skeletal muscle disease can both occur in the same patients.

During my PhD studies, Dr. Julian Barden supplied me with an exclusive antibody to human P2X₁ receptors. These specialized transmembrane sarcolemmal proteins are ligand-gated ion channels, and their structure and function were studied in relation to their possible role in dilated cardiomyopathy and muscular dystrophy. Today these ATP receptors continue to be important in regulating the flow of blood in the coronary arteries via the intrinsic sympathetic nerve endings in the heart.

St Vincent’s Hospital heart and lung transplant theatres

Only human tissue was examined in my experiments. Myocardium was obtained from patients undergoing cardiac transplantation (e.g. dos Remedios et al. 1996). The operations took place in the late evening/early morning because these were the only times when multiple, adjacent theatres were available for donors, heart and lung recipients.

The transplant coordinators would alert Cris that a heart would be available and I would arrive to find him always present to supervise and support. The precious samples were snap frozen in liquid nitrogen in the theatres and later transported to the laboratory, and there was always the realization that the important research would be another step in the understanding of the disease. The samples collected have gone on to many researchers all around the world.

The skeletal muscle samples were from open muscle biopsies of myopathic patients that were routinely screened for dystrophin. Electrophoresis using linear gradient SDS PAGE gels and western blotting was used to identify and quantify the membrane proteins in the tissue (Fig. 1).

Fig. 1.

Western blot of SDS-PAGE gel showing the P2X₁ protein bands at 45 kDa. Lane 1, DCM #6 (20% loading); 2, DCM #5 (20% loading); 3, DCM #4 (15% loading); 5, DCM #2 (25% loading); 6, DCM #1 (20% loading); 7, ND #6 (20%); 8, ND #5 (15%); 9, ND #4 (20%); 10, ND #3 (30%); 11, ND #2 (20%); 12–22, serial dilutions of ND #1 loading (30, 30, 25, 25, 20, 20 15, 15, 10, and 10%, respectively). Figure 1 is reproduced with permission of Wiley Press

P2X₁ receptors

The expression levels of the P2X₁ receptors were determined in samples from the atria (e.g. Berry et al. 1998) and left ventricles of patients with dilated cardiomyopathy and were compared with the levels in non-diseased donor hearts. Significant up-regulation of the P2X₁ receptor was detected in the atria of the DCM patients but at that stage not in their ventricles (Berry et al. 1999).

The ecto-ATPase, α-sarcoglycan, was also studied to determine alterations of this protein expression correlated with that of the P2X₁ receptors in left ventricle of DCM patients (Berry et al. 2000).

The same level of α-sarcoglycan is consistent with the status of the P2X₁ receptors in left ventricle of DCM patients.

The same proteins were then studied in myopathic skeletal muscle. The P2X₁ receptor expression was found to be up-regulated in end-stage muscle disease in Duchenne muscular dystrophy but was down-regulated in the early stages. This suggested that there may be a regulatory mechanism to prevent the entry of Ca²⁺ ions in the early stages of the disease.

Decreased dystrophin expression was detected in a single patient with McArdle’s disease, and this may be important in the understanding of the cytoskeletal organization and energy metabolism. New material will permit the extension of this work to other McArdle patients.

Dystrophin and Beta-dystroglycan

Dystrophin and Beta-dystroglycan are proteins associated with muscle sarcolemma, whereas emerin and lamin A/C are associated with the nuclear envelope (e.g. Berry et al. 2001). Expression levels of these proteins in samples from the terminally failing and non-diseased hearts were examined. No alterations in the expression of these proteins were found in the DCM hearts studied. The results stimulated the formulation of the hypothesis that changes in one of these proteins can affect the expression of the others that are linked or functionally associated with cellular membranes.

With thanks

The encouragement and support from Cris gave me the inspiration to continue and complete this sometimes difficult task.

His support extended beyond that of an academic teacher to that of a listener whose kindness in personal adversity will always be remembered.

I regard myself very privileged to have had Cris as a supervisor and thank Cris and the University of Sydney for the opportunity to achieve this major goal in my career.

Desiree Ann Berry

PhD 2001