A major interest of our lab is to investigate the cellular mechanisms involved in the regulation of calcium cycling at the level of sarcoplasmic reticulum and its role in cardiac disease. For these experiments, we apply conventional biochemical and molecular biology assays and digital confocal imaging. Model systems include established cell lines, primary cell cultures and transgenic animals. These techniques serve as valuable experimental tools to investigate PLN mutations, cellular protein sorting and Ca2+ cycling.
Above is a general overview of the major proteins involved in muscle excitation-contraction. Our research interests focus on proteins involved in Ca2+ release from the sarcoplasmic reticulum and its subsequent reuptake. In particular, we are interested in phospholamban, its skeletal muscle homologue sarcolipin, and SERCA.
1. Phospholamban (PLN) in Cardiac Muscle
The overall objectives of this project are to provide a clearer understanding of the role of phospholamban (PLN) in the normal working of the heart, and what are the cellular mechanisms in human PLN genetic diseases that result in dilated cardiomyopathy. Further studies of the formation of the PLN–SERCA2a complex might provide further clues as to the mechanisms that underlie PLN regulation and provide new insights into ways to design chemicals that would disrupt this interaction selectively. Our aim is to provide such novel approaches to aid in the diagnosis and treatment of cardiac diseases resulting from defects in the regulation of intracellular calcium concentrations.
Shown, at left, is a schematic diagram of the 52 amino acids that constitute PLN, together with the phosphorylation sites of PLN (Ser-16 and Thr-17)
These experiments are performed together with several international collaborative initiatives involving Dr. Litsa Kranias (U Cincinnati) , Dr Peter Backx (U Toronto), Dr. Jon Seidman (Harvard), Dr. Christine Seidman (Harvard), and Dr. David MacLennan (U Toronto).
2. Sarcolipin (SLN) in Skeletal and Cardiac Muscle
In collaborative studies with proteomic experts Dr. Thomas Kislinger (Ontario Cancer Institute, U of Toronto) and Dr. Andrew Emili (Donnelly Centre for Cellular and Biomolecular Research, U of Toronto) we have been developing methodologies for the large scale study of proteomic expression profiles in cardiovascular diseases. Together with Dr David MacLennan (Banting and Best Department of Medical Research, U of Toronto) and Dr Peter Liu (Toronto General Hospital, University Health Network) we are applying these techniques to study the mechanisms involved in the progression of dilated cardiomyopathy in mouse models, as well as in human patients. These experiments are undertaken with the aim of uncovering new cellular pathways that are involved in the disease in order to develop new therapeutic targets. As well, we have been attempting to identify new blood-borne biomarkers of cardiovascular disease to develop new screening tools.
The overall objectives of this project are to provide a clearer understanding of the role of the PLN homologue, sarcolipin, in the normal working of the heart, as well as skeletal muscle. We also investigate SLN interactions with SERCA since recent work has indicated that SLN and PLN can interact and bind with SERCA resulting in the superinhibition of SERCA.
Shown at left is a schematic diagram of the amino acids that constitute SLN (red, left panel) and PLN (blue, right panel), which highlights the similar identity between these two proteins.
We routinely perform in vitro experiments using HEK-293 transfected cells followed by cellular imaging and biochemical analyses. Below are cells transfected with flag-tagged SLN and flag-tagged PLN and visualized with confocal microscopy.
These experiments involved several international collaborators including Dr. Jens Andersen (U Aarhus), Dr. Muthu Periasamy (Ohio State) and Dr Russ Tupling (U Waterloo).
3. Targeted Transgenesis
In order to investigate the roles of several calcium regulatory proteins, we routinely generate mouse models with either transgenic overexpression of altered (mutant) or wildtype proteins . We have recently adopted a targeted transgenic approach to the overexpression of our constructs, with the strategy shown above (together with Dr Alan Peterson, McGill U). The cDNAs of interest are inserted into a HPRT targeting vector and eventually insert into the HPRT-null locus of mutant ES cells. The result is a targeted insertion of one copy of our cDNA. For our ongoing studies, we design our constructs with tissue-specific promoters (such as alpha-MHC) to achieve skeletal- and cardiac-specific expression.
Assistant Professor of Physiology, University of Toronto
Anthony Gramolini, PhD
Shown above is the gel-free, shotgun mass spectrometry, expression system employed
Ms Roxana Chis, Graduate Student (MSc) in Physiology
Dr Tetsuaki Miyake, PDF
Ms Melissa Noronha, Graduate Student (MSc) in Physiology
Mr Timothy Ryan, Graduate Student (MSc) in Physiology
Dr Nicolas Bousette, PDF
Dr Parveen Sharma, Research Associate
Ms Wenping Li, Tech II
Ms Sarah Elschenbroich, Visiting PhD Graduate Student, Food Chemistry
From time to time, we will have many interesting projects for enthusiastic graduate students and postdoctoral fellows.
For Post-doctoral Applicants, the University of Toronto must recognize your Ph.D. title. We also require proficiency in spoken and written English, and a demonstrated command of a relevant scientific discipline. Your resume should demonstrate a proven track record in terms of productivity and long-term independent research potential, as evidenced by first-author peer-reviewed publications and public presentations. Foreigners must be eligible for employment authorization in Canada; please inquire at your local Canadian Embassy or Consulate about the details for obtaining a suitable work-permit and allow sufficient time to get this organized. You should be acquainted with the general academic requirements, cost-of-living, and available funding sources such as relevant local, national and international Fellowships.
We have stimulating projects for Graduate Students, and I recommend that you look into the Department of Physiology website for information about the programs, as you must first be accepted into the Departmental graduate program (http://www.uoftphysiology.com). Prospective undergraduate or MSc students need a strong academic standing to be competitive.
A few competitive positions are available for highly motivated Undergraduate students with strong academic track records, and strong letters of recommendation. To be considered you should familiarize yourself with our work (e.g. browse our Website and recent publications) and understand the nature of our research publications before you apply. You should become familiar as to what we do and how this relates to your own academic training and interests. You should understand the requirements and demands of academic research, and be confident in your own strengths, potential for academic research, and your ability to communicate in spoken and written English.
Department of Physiology
112 College St, Rm 307
CH Best Institute
Toronto, ON Canada
Lab Phone: 416-978-1643
Fax: 416-978-8528 ANTHONY.GRAMOLINI@UTORONTO.CA