Goals

1,200,000 people suffer a (new or recurrent) myocardial infarction every year, and about 40% of them die as a result of the attack. Those who survive the heart attack usually find their hearts in a weakened condition, as many of the heart muscle cells have died or will die. The heart is one of the least regenerative tissues in the body. Unlike skeletal muscle, which can replace injured and dead cells, cardiac muscle cannot regenerate or replace a fraction of the heart cells that die as a result of a myocardial infarction.

Our goal is to create a procedure by which cells can be differentiated into cardiomyocytes, the precursors to cardiac muscle cells. From that point, we will transfect these cells with DNA which will allow them to more effectively bind to damaged hearts and replace injured tissue, restoring the heart to its pre-myocardial infarction condition.

Differentiation

We are currently using a line of mouse cancer cells called P19 cells, which are capable of changing into many different cell types (pluripotency). Because P19 cells bear a great deal of resemblance to embryonic stem cells, without the requirement of the rigorous attention that stem cells require, their use is one of convenience. Our hope is to build a working model in P19 cells, and then to test the model in cells ranging from mouse stem cells to human bone marrow cells. Our eventual goal is to produce a system that can turn a patient's own bone marrow into working cells for his/her heart, thus removing any chance of immune rejection.

The process of differentiating cells into cardiomyocytes is a complex one, requiring several proteins and chemicals to be present at differing times during the process. We will be attempting a highly efficient differentiation of our P19 cells using a combination of:

• Nkx2.5
• GATA4
• Bone Morphogenetic Protein 4 (BMP4)

Nkx2.5 and GATA4 are transcription factors noted to enhance cardiac differentiation.

• We will activate translation of these proteins by using a Tet inducible promoter. In the presence of Doxycycline, the promoter activates translation whereas in the absence of Doxycycline, the promoter becomes inactivated. This will allow us to kick start the circuit, yet once activated, will remain completely autonomous.

BMP4 proves absolutely essential for growing P19 cells in a monolayer. It also acts in a time dependent manner, inhibiting cardiac differentiation at an early stage yet promoting differentiation after the cells have reached a precursor state. We will build in the necessary time delay by utilizing the Wnt/beta-catenin canonical pathway. As cells reach the precursor state, beta-catenin will build up and bind with TCF. This TCF/beta-catenin dimer will act as a transcription factor for BMP4.

• Time dependent BMP4 Pathway.:
  

• Proposed pathway and use of our constructs.:
  

Selection

P19 and Human Stem Cells will not differentiate into cardiomyocytes with 100% efficiency. However, because we would like to test our cells in vivo, we need to select for only those cells which have differentiated into cardiomyocytes. Differentiated cardiac cells express Alpha-MHC. We will use an alpha-MHC promoter to induce protein for antibiotic resistance. By growing the cells in the antibiotic, only those cells which have gained the resistance will survive, and only those cells that have differentiated into cardiac cells will gain resistance to the antibiotic. We will then have near-100% cardiomyocytes.