Cardiovascular proteomics

1. Cardiovascular proteomics • Jennifer Van Eyk MD • Associate Professor of Physiology • Queen’s University • Kingston, Ontario • Eric Topol MD • Provost and Chief Academic Officer • Chairman and Professor • Department of Cardiovascular Medicine • The Cleveland Clinic Foundation • Cleveland, Ohio Source:WITA Proteomics

2. Cardiovascular proteomics What is proteomics? Proteomics is the study of the proteins in a cell at a given time “Just right now, if we could capture the cells that were in your heart, or in your vasculature, or in your aorta — that’s the proteins we are after.” Van Eyk Looks at • expression of proteins by genes • posttranslational modification of proteins (eg phosphorylation and oxidation used for intracellular signaling)

3. Cardiovascular proteomics Proteomics vs genomics • Genomics goes after gene changes • eg in HF — upregulation of ANF • in hypertrophy — more myofilaments expressed • Proteomics looks at gene changes and posttranslational modifications • “When we study proteomics, we are really trying to capture all of the changes within the cell.” Van Eyk

4. Cardiovascular proteomics How big is the proteome? • 1 gene  1 protein • A protein may have 10–15 posttranslational modifications that are disease-induced • The proteome could be up to 10 times the size of the genome same protein, 2 different modifications Source: Incyte Genomics

5. Cardiovascular proteomics Proteomics to validate genomics • Changes in DNA or mRNA may not correlate with changes in protein expression • “Whatever you see at a genomic level…you really have to double-check and make sure that that is happening also at the protein level.” Van Eyk • Some of the disparities between the mRNA and protein levels could just reflect a time delay

6. Cardiovascular proteomics Proteomics and genomics today • Genomics and proteomics are currently uncoupled • Genomic researchers report SNPs (single nucleotide polymorphisms) with no data on the protein Mikkelsson J, et al. Circulation 2001;104(8):876-880 • “Has to be viewed as suspect.” • “Are we going to continue to see these isolated reports: here is a genomic finding with no protein correlation?” Topol

7. Cardiovascular proteomics Proteomics and genomics in 5 years • Genomics is a new trend and people are just trying to get the data out “My guess is within 5 years you will have to prove at the protein level as well.” Van Eyk • Proteomics is no different. Currently, can get away with lists of proteins without identifying the posttranslational modifications “That is soon going to change…. Proteomics is going to have to have the functional verification, with time.” Van Eyk

8. Cardiovascular proteomics Cost of proteomics • Proteomics is driven a lot by industry, capable of high-throughput • Proteomics is expensive • Cost of equipment and expertise Source: The Wistar Institute

9. Cardiovascular proteomics Academic proteomics • Broad-based screening • tries to see all the proteins • in individual labs Focused proteomics • looks at small group of proteins in theproteome (subproteome) • in core facilities

10. Cardiovascular proteomics Industry proteomics • Constructing databases • selling lists of the proteins in the heart vs the brain • Diagnostics and therapeutics • Drug development • compare effects of drug A vs drug B on the proteome

11. Cardiovascular proteomics Industry proteomics • “Small biotech companies are…either driven by the technology that they’ve made, and they are trying to sell technology that is very specific to proteomics, or they are trying to sell information from databases, or they’re trying to use that information, let’s say, for diagnostics.” • “Diagnostics are actually the first things I think that will be most influenced by proteomics.” Van Eyk

12. Cardiovascular proteomics Diagnostic proteomics • Troponin I for MI • TnI is degraded and modified in the myocardium during ischemia • TnI is released due to necrosis into the blood stream, either intact or with all these posttranslational modifications Source: Jennifer Van Eyk

13. Cardiovascular proteomics Diagnostic proteomics • “If you are having a heart attack and you have intact TnI, and I am having a heart attack and I have the degradation products that are linked to more severe ischemia, then I would predict that my heart is not going to be doing as well as yours.” Van Eyk

14. Cardiovascular proteomics Diagnostic proteomics • “I believe that any disease-induced modification that could be specific for a disease state…can be used as a biomarker, as long as it is there in enough abundance.” Van Eyk

15. Cardiovascular proteomics Therapeutic proteomics • Go after end-effectors of the disease process or beginning-effectors • ie proteins that you can change with a drug to stop the process • Requires knowledge of the proteome and the disease process • “I believe that is the only way we are going to get new drug targets.” Van Eyk

16. Cardiovascular proteomics Drug discovery • Past approach • go after favorite proteins (eg, TnI, beta-adrenergic 1 receptor) • if one turns out to be important in disease, create a drug against it • Proteomics approach • provides an immense amount of information on many, many proteins • have 100s and 100s of potential drug targets

17. Cardiovascular proteomics Drug discovery • “The big problem actually is that proteomics, and genomics also, will give us so much information. It’s being able to pull out what information really means and which piece of information is really important, and going after those.” Van Eyk

18. Cardiovascular proteomics CV applications of proteomics • “Do you think that most of the processes that are common, like HF from a dilated cardiomyopathy, idiopathic,…or decompensation of coronary disease,… are going to be advanced by the whole field over the years ahead?” • “It sounds like this…is really going to change our approach, not just perhaps to new diagnostics and drug discovery but to the understanding of the disease state in a more enhanced fashion.” Topol

19. Cardiovascular proteomics CV applications of proteomics • Dilated cardiomyopathies • mutations in different myofilament proteins can produce same disease phenotype • HF, stunning, systolic dysfunction • phenotypes can be caused by myofilament contractile defect, or calcium handling defect, or a combination of both • Using diagnostic proteomics, hopefully you will be able to stratify patients according to the cause of their diseases, and you might treat them differently

20. Cardiovascular proteomics CV applications of proteomics • “With well-done proteomic studies… you can define the protein changes around a disease phenotype. Then all those protein changes have to be analyzed independently. Because a protein change even in HF can actually be a good change…and one you want to promote.” Van Eyk

21. Cardiovascular proteomics Are we fooling ourselves? • Example of simple genetic diseases (such as Marfan syndrome) “Once you have the genetic and proteomic side delineated, can you really see your way through to the next step?” “Are we fooling ourselves?… Here we are, 12–13 years since the cystic fibrosis gene, and we have no new therapies, we have no new ways to prevent the disease, and we understand the gene and protein.” Topol

22. Cardiovascular proteomics Are we fooling ourselves? • These genetic diseases are more complex than expected • 1 gene product is mutated but many proteins are affected, and these are not necessarily known • a lot of these diseases are chronic; the body has been trying to compensate causing further changes in proteins

23. Cardiovascular proteomics Are we fooling ourselves? • Potential therapies for these diseases • replace the missing protein • inhibit the posttranslational changes that occur in acute disease (eg during CABG)

24. Cardiovascular proteomics On the horizon • “This is obviously very exciting. Perhaps in the future there is probably nothing that bubbles up to the top as having more promise.” Topol • “It is still a field in its infancy, even though people have been working on proteomics - on the technology - for 20 years.” • “It is going to take time to really see the potential of it.” Van Eyk

25. Cardiovascular proteomics Limitations • Rushed studies that are poorly designed will produce a lot of false information or information that doesn’t help • It may be hard to get funding when the initial excitement dies down • The studies do take a very long time, and the public may lose interest • “Although it’s not going to be a quick fix…the incubation phase is going to be well worth it.” • Topol

26. Cardiovascular proteomics Exciting findings • Already finding changes to proteins never expected • For example, myosin light chain 1 • studied for 20 years and known to be unphosphorylatable; in fact it is phosphorylated Arrell DK, et al. Circ Res 2001;89(6):480-487 • “We are seeing the world differently now at the protein level. And as soon as you find any protein that is changed, that just opens up so many doors and possibilities.” Van Eyk

27. Cardiovascular proteomics Recommended reading Cardiovascular proteomics:evolution and potential Arrell DK, Neverova I,Van Eyk JE. Circ Res 2001;88(8):763-773 Source: WITA Proteomics