Dependence of Longevity and Health on TRB3 Genotype

Anthony J. Muslin, M.D.


Project Overview:
Adult onset, type II diabetes mellitus is becoming increasingly prevalent and is frequently associated with cardiovascular disease. Type II diabetes mellitus is caused by the loss of responsiveness of skeletal muscle, adipose and cardiac tissue to insulin action. After release from the beta cells of the pancreas, insulin binds to cell surface receptors that activate an intracellular signaling cascade that includes the protein kinase Akt. Activation of this kinase leads to the translocation of glut4 containing vesicles to the cell surface, promoting glucose uptake by cells. Trb3 is an intracellular protein that binds to and inactivates Akt.

Polymorphisms in the TRB3 gene were discovered and our ongoing work suggests that the TRB3 genotype correlates with outcomes in patients with coronary artery disease. One polymorphism in the TRB3 gene (Q84R) results in a missense mutation that leads to the substitution of an arginine residue for a glutamine residue and results in a protein with increased ability to inhibit Akt. The Q84R polymorphism was found in a preliminary study by other investigators to be associated with an increased incidence of hypertriglyceridemia and also with an earlier age at first myocardial infarction (MI).

In our work over the past year, we investigated the Q84R TRB3 polymorphism in a population of patients from the INFORM trial who presented with acute MI or unstable angina. We found that the Q84R polymorphism was associated with a higher rate of re-hospitalization in the year following MI or unstable angina, and was also associated with increased responsiveness to β-blocker therapy in terms of hospitalization rates. In our planned studies, we will determine whether the Q84R SNP correlates with clinical outcomes, clinical responsiveness to β-blocker therapy, hyperglycemia, hypertriglyceridemia, and hypercholesterolemia in a second larger and completely separate population of patients from the TRIUMPH registry. In addition, we will determine whether the Q84R SNP is associated with an altered acute response to β-blocker therapy in healthy volunteers in a prospective clinical trial.  These studies will help to establish the utility of genomic studies in the clinical management of patients with cardiovascular disease.


Final Report Abstract:
Type II diabetes mellitus is a common condition that is thought to be caused by insulin resistance in muscle, fat and other tissues. In response to feeding, pancreatic beta cells release insulin that binds to cell surface receptors, resulting in the activation of an intracellular signal transduction cascade that include the protein kinase Akt2. TRB3 is an inducible pseudokinase that binds to and inhibits Akt2 function.  Polymorphisms in the TRB3 gene were identified several years ago. A major polymorphism in the TRB3 gene (Q84R) results in a missense mutation that encodes a protein with increased ability to inhibit Akt. The Q84R polymorphism was found in several studies by other investigators to be associated with an increased incidence of hypertriglyceridemia and myocardial infarction. In animal model work in our laboratory, we showed that TRB3 expression is murine heart cells and myocardium is increased in response to hyperglycemia, hyperlipidemia and ischemia. We also showed that overexpression of TRB3 in murine myocardium sensitized these animals to pathological cardiac remodeling after myocardial infarction. In clinical studies, we investigated the Q84R TRB3 polymorphism in a population of patients from the INFORM and TRIUMPH trials who presented with acute myocardial infarction (MI) or unstable angina. We found that the Q84R polymorphism was associated with a higher rate of re-hospitalization following MI or unstable angina, and was also associated with increased responsiveness to b-blocker therapy in terms of hospitalization rates. We also found that cardiac mortality was increased in the INFORM and TRIUMPH patients with Q84R TRB3 who were not treated with beta blockers.  These studies suggest that patients with the Q84R TRB3 should be aggressively treated with beta blockers and also show the utility of genomic studies in the clinical management of patients with cardiovascular disease.

Lay Summary:

The prevalence of adult-onset sugar diabetes is dramatically increasing. Diabetes is associated with the development of a serious disease of the blood vessels, atherosclerosis, which causes heart attacks and strokes. In adult-onset diabetes, insulin does not work properly, and muscle and fat do not take up sugar from the blood stream. A recently identified protein, called TRB3 (“trib three”) was found to inhibit the action of insulin, and this new protein may play a role in the development of diabetes and disease of the blood vessels. Chromosomal DNA contains genes that encode proteins that are made inside of cells.  The gene that encodes the TRB3 protein is changed in about 20-25% of people, and that change is called a single nucleotide polymorphism (SNP, “snip”). The SNP in the TRB3 gene causes the TRB3 protein to be more effective at blocking insulin action. Therefore, people with the TRB3 SNP are predicted to be more likely to develop diabetes and more likely to get atherosclerotic disease of blood vessels. In this project, we looked at the TRB3 SNP in patients that were admitted to the hospital with heart attacks. We obtained blood from these patients, and analyzed DNA in blood samples for the presence of the TRB3 SNP. We compared the DNA results with the results of the physical examination of the patients, their hospital course, and their response to medications such as beta-blockers. We determined that the TRB3 SNP is associated with a worse prognosis after heart attack and also showed that it is associated with a better response to beta-blocker therapy. In this way, we can now identify people that are at higher risk for these diseases, and we can enable effected individuals to be treated more aggressively with drugs at an earlier point in their life to prevent the development of serious complications.

Introduction:

Type II diabetes mellitus is characterized by reduced tissue responsiveness to insulin stimulation. Glucose uptake by insulin-stimulated cells is dependent on the translocation of glut4 glucose transporters from intracellular vesicles to the plasma membrane. Glut4 translocation to the plasma membrane is dependent on the insulin-stimulated activation of the intracellular protein kinase Akt2. The intracellular pseudokinase TRB3 is an inducible protein that binds to and inactivates Akt2 (3). A missense polymorphism in the TRB3 gene (Q84R) was previously identified and this polymorphism was associated with increased cardiovascular disease. Early identification of people harboring the Q84R TRB3 polymorphism, may allow for prompt intervention to prevent adverse outcomes and to optimize therapeutic response. In this project, we performed experiments to:

  1. Determine the factors that promote TRB3 expression and determine the consequences of increased TRB3 activity in murine cardiomyocytes and myocardium.
  2. Determine the association between the Q84R missense polymorphism in TRB3 and clinical outcomes, and the response to beta-blocker therapy, in patients with MI from the INFORM and TRIUMPH registries.
  3. Determine the association between the Q84R missense polymorphism in TRB3 and metabolic abnormalities, including serum lipid and blood sugar levels, in patients with MI from the TRIUMPH registry.

Methods
 
A. Analysis of TRB3 expression in cultured murine cardiomyocytes and myocardium. 
Cultured murine cardiomyocytes were subjected to various provocative stimuli such as hypoglycemia, palmitate treatment, ischemia by use of hypoxic bags, and thapsigargin (to induce endoplasmic reticulum stress). Mice were subjected to myocardial infarction and tissue samples were obtained from the left ventricle. Cells and tissues were used to analyze TRB3 mRNA levels by quantitative real-time PCR and protein levels by immunoblotting.

B. Generation of transgenic mice with cardiac-specific TRB3 overexpression and analysis of these mice.
Transgenic mice were generated by use of the alpha-myosin heavy chain promoter linked to a wild type form of TRB3. Multiple lines of transgenic mice were generated and they were bred into the C57Black genetic background. Transgenic TRB3 mice were subjected to experimental myocardial infarction by placing a suture around the left anterior descending coronary artery.

C. Analysis of Q84R Polymorphism in human blood samples.
lood specimens were processed in compliance with HIPPAA guidelines. Banked blood samples was used to obtain genomic DNA using an extraction kit from Gentra Systems. Pyrosequencing was performed with the PSQ HS96A system with MA v2.0 software.

D. Acute Coronary Syndrome Registry (INFORM Trial) 
Patients were prospectively enrolled into a registry of people with acute coronary syndrome (ACS) with either myocardial infarction (n=680) or unstable angina (n=519) after informed written consent was obtained at two hospitals in Kansas City, Missouri. The INFORM Trial enrolled patients between March 1, 2001 and October 31, 2002. Myocardial infarction (MI) was defined by the presence of an elevated troponin level in blood, and by the presence of ST-segment elevation or depression consistent with an MI. Unstable angina was present if the troponin level was normal with new onset of chest pain (class III by the Canadian Cardiovascular Society Classification), recent worsening of chest pain, or greater than 20 minutes of rest chest pain. Patients who had a diagnostic test that excluded significant coronary artery disease (e.g., cardiac catheterization) were removed from the study. 

Each patient was interviewed to determine health status, the medial record was carefully reviewed, laboratory results were obtained, and drug administration was reviewed. Blood samples were obtained for genetic analysis from every patient after separate written consent was obtained. Blood samples were stored at -80 degrees C for subsequent analysis. Approval from the appropriate institutional review boards was obtained prior to the performance of the study. 735 patients participated in the genetics subsection of the trial.

E. Acute MI Registry (TRIUMPH Trial) 
Patients were enrolled into this prospective registry of people with myocardial infarction (MI) after informed written consent was obtained at twenty hospitals throughout the United States. Approval from the appropriate institutional review boards was obtained prior to the initiation of the study. Over 4300 patients were enrolled into this trial as of October 2009. MI was defined by the presence of an elevated troponin level in blood, and by the presence of ST-segment elevation or depression. 
Each patient was interviewed to determine health status, the medial record was carefully reviewed, laboratory results were obtained, and drug administration was reviewed. Blood samples were obtained for genetic analysis from every patient after separate written consent was obtained. The clinical outcome for each patient was followed for 5 years after initial presentation with MI.
 
E. Statistical Analysis of Results
Patient characteristics were summarized and compared stratified by genotype and discharge beta-blocker therapy status. Continuous variables were reported as mean standard deviation and compared using t-tests. Categorical variable was  summarized by frequency and percent and compared with chi-square or Fisher’s exact tests. 
The primary outcome was time to rehospitalization for cardiac events through 12, 24 and 60 months.

Results:
 
1.  Laboratory animal studies of TRB3 function.
TRB3 is an inducible factor that is expressed in cardiomyocytes and myocardium in response to various stressful conditions. In cultured cardiomyocytes, we demonstrated that hyperglycemia, hyperlipidemia, ischemia, and other noxious stimuli promote the expression of TRB3. We also demonstrated that TRB3 expression resulted in significant inhibition of Akt2 signaling in cardiomyocytes. We performed experimental myocardial infarction (MI) in mice and found that TRB3 was induced in the infarct border zone after MI. We generated transgenic mice with cardiac-specific overexpression of TRB3. These transgenic mice were sensitized to the development of pathological cardiac remodeling after experimental MI (Figure 1). Therefore, TRB3 activity plays a deleterious role in cardiac remodeling and cardiomyocyte survival. These results were recently published in Circulation Research in 2010.

Figure 1.Pathological remodeling is increased after myocardial infarction in transgenic 2x-TRB3 mice. At 12 weeks of age, 2x-TRB3 mice (n=9) and their nontransgenic littermates (n=13) were subjected to experimental myocardial infarction surgery by ligation of the left anterior descending coronary artery. LV sections from 2x-TRB3 and nontransgenic littermates were stained with Masson’s trichrome to determine infarct size.

2. Clinical studies to examine Q84R TRB3 function. 
Our animal studies demonstrated that increased TRB3 function has a variety of maladaptive consequences on cardiac structure and function. The Q84R TRB3 polymorphism, found in 20-25% of the general population, is associated withincreased TRB3 activity and is therefore predicted to have a detrimental effect on cardiac function.

To investigate the relationship between the Q84R TRB3 genotype and cardiac events, we analyzed the genotype of several thousand patients in the TRIUMPH and INFORM trials (described above in Methods). Our results showed that percentage of patients enrolled in these trials after MI or ACS with the Q84R TRB3 genotype was not higher than a control population without ACS or MI. However, this was not a prospective observational analysis of these patients, which would be required to better assess the relative risks of the polymorphism. For example, Q84R TRB3 patients could have died at a high rate from myocardial infarction before they were entered into the TRIUMPH or INFORM trials, skewing the prevalence data.   In the future, analysis of the genotype of Framingham Study patients that are enrolled before they develop cardiac disease may be better suited to more definitely assess the relative risk of the Q84R TRB3 genotype.

The Q84R TRB3 polymorphism, however, was associated with a significantly higher rehospitalization rate after MI or ACS in the INFORM trial, in the absence of beta blocker therapy. Treatment of Q84R TRB3 patients with beta blockers markedly reduced the rehospitalization rate (Figure 2).

Figure 2.Increased rehospitalization rates in patients from the INFORM trial with the Q84R TRB3 genotype (GA/GG) not taking beta blockers. Rehospitalization rates were evaluated in patients taking or not taking beta blocker medications.

Analysis of diabetic patients with the Q84R TRB3 polymorphism showed that INFORM and TRIUMPH patients had a trend toward a higher mortality rate after MI or ACS in the absence of beta blocker therapy (Figure 3). We await further mortality data from the TRIUMPH trial, to see if this difference achieves statistical significance. The rehospitalization rate data from the TRIUMPH trial is not available yet, but should be obtained in the next year.

Figure 3.A trend towards an increased mortality rate in diabetic patients from the INFORM and TRIUMPH trials with the Q84R TRB3 genotype (G-Carrier) not taking beta blockers. Mortality rates were evaluated in patients taking or not taking beta blocker medications.

3. Publications.

a. Avery J, Etzion S, Debosch BJ, Jin X, Lupu TS, Beitinjaneh B, Grand J, Kovacs A, Sambandam N, Muslin AJ. TRB3 Function in Cardiac Endoplasmic Reticulum Stress. Circ Res. 2010 Apr 1. [Epub ahead of print]
b. Muslin AJ. New insights into cardiac aging. Circulation. 2009 Oct 27; 120(17):1654-6. Epub 2009 Oct 12. 
c. Muslin AJ. MAPK signalling in cardiovascular health and disease: molecular mechanisms and therapeutic targets. Clin Sci (Lond). 2008 Oct;115(7):203-18.

Discussion

TRB3 is an intracellular protein that binds to and inhibits the action of Akt2, a key participant in the insulin signaling cascade. TRB3 expression in cardiomyocytes is dynamic, and stressors such as hyperglycemia, hyperlipidemia and ischemia were found by my group to induce TRB3. Furthermore, TRB3 induction in heart cells dramatically antagonizes insulin signaling and results in reduced glucose uptake, and increased cell death. Experimental myocardial infarction in mice was performed and TRB3 expression increased in the infarct border zone, thought to be responsible for pathological cardiac remodeling. Transgenic mice with overexpression of TRB3 in myocardium were generated, and these animals were sensitized to pathological remodeling after MI. Taken together, these basic and translational studies support a model that TRB3 induction in heart is detrimental to cardiac recovery after injury. Furthermore, agents that inhibit TRB3 expression or activity are predicted to be beneficial for the treatment of patients with heart disease.

In humans, a naturally occurring polymorphism in TRB3, Q84R, is found in 20-25% of the general population. This polymorphism results in the production of a protein that has increased ability to inhibit Akt2 activity. The initial reports by another group suggested that Q84R TRB3 patients had an increased incidence of cardiovascular disease. In our analysis of the INFORM and TRIUMP Trials, our data shows that diabetic patients with Q84R TRB3 have an increased incidence of rehospitalization after MI or ACS, if they do not take beta blockers. Interestingly, these patients respond very well to beta blocker therapy, normalizing their prognosis. Furthermore, we showed a trend towards increased mortality of diabetic Q84R TRB3 patients who did not take beta blockers after MI or ACS.  While these data need to be revalidated, one can argue that Q84R TRB3 patients should be aggressively treated with beta blockers.

Future Plans including planned grant submissions

The investigation of the regulation of TRB3 expression and activity in cardiac disease is continuing in my laboratory. We are currently investigating whether TRB3 inhibits Akt2 activity by promoting its ubiquitination and degradation by the proteasome. Also, we recently obtained mice with targeted disruption of the TRB3 gene and are using these animals to determine their response to myocardial infarction. Our prediction is that TRB3-null mice will be resistant to pathological cardiac remodeling after myocardial infarction. These experiments were included in a recent R01 grant submission to the NIH.

The clinical investigation of TRB3 polymorphisms and their relationship to the development of prognosis of cardiovascular disease continues in my laboratory. We await additional mortality and rehospitalization data from the TRIUMPH Trial which did not complete enrollment until 2009. This data is expected in the next few months. Confirmation of increased rehospitalization and mortality rates in Q84R TRB3 patients will allow us to submit this data for publication. Furthermore, this additional data will form the basis for a future R01 grant submission from the NIH.