Our project is broken up into three sections, each with a defined goal on the journey towards a therapeutic for cardiac muscle repair.
Aim #1: Determine the relationship between TGF-B and F1R1 in terms of cardiac cell proliferation.
Our hypothesis is that in the neonatal cell environment, F1R1 binds to TGF-B and promotes proliferation by inhibiting fibrosis. We hypothesize that introducing TGF-B to neonatal cardiac cells will result in an decrease of proliferation and an increase of differenationation compared to the control. Adding both F1R1 and TGF-B will lead to F1R1 inhibiting TGF-B and thus an increase of proliferation and decrease of differentiation compared to TGF-B alone and similar rates compared to the control. The control, F1R1, and both F1R1 and TGF-B groups are expected to have a proliferation rate of 14.8% of cells and a differentiation rate of 20% of cells. The TGF-B group is expected to have a proliferation rate of 9.5% of cells and a differentiation rate of 80% of cells. Ki67 stain (proliferation stain) will be used in conjunction with SMA stain (differentiation stain) to understand the trends of proliferation and scar tissue formation. F1R1 has been shown to promote proliferation in previous studies. Understanding the mechanisms of action through which F1R1 promotes proliferation will provide insight into the regenerative properties of the neonatal heart and will be essential for the potential application of the peptide in therapeutics. This aim will partially explain the absence of proliferation in mature cardiac cells and provide a basis for developing methods to restore proliferation. If our hypothesis is correct, scar tissue inhibition is one of the mechanisms by which neonatal cardiac cells proliferate after injury or malformation.
Aim #2: Identify cardiac cell signaling pathways that are significantly influenced by F1R1.
Our working hypothesis is that F1R1 treatment alone should have some effect on the differentiation pathways in cardiac fibroblasts. We would expect to see some sort of downregulation in proteins associated with myofibroblast development after F1R1 treatment of TGF-b-exposed fibroblasts, specifically the SMAD and MAPK protein families17. In the case of the former, TGF-b binding induces Smad 2, 3, and 4 to form a complex that then goes on to bind to promoter sequences associated with the myofibroblast phenotype, increased matrix deposition, and therefore increased fibrosis in the heart. TGF-B can also initiate SMAD-independent pathways like PI3K/Akt and MAPK cascade components like ERK 1/2 and p38 MAPKs, which drive fibroblast activation on their own or pass on their signal to the SMAD pathway2, 14, 4. Furthermore, differentiation-inducing signals can also be generated by fibroblast mechanosensing, highlighting proteins like RhoA/ROCK and YAP/TAZ as potential targets of F1R14, 5 As a result of these factors, our primary focus will be on SMADs 2 and 3, but proteins involved in non-canonical TGF-b signaling and mechanoregulation must also be considered. Therefore in a Western blot experiment with two sets of F1R1-treated, scrambled F1R1-treated, and untreated cardiac fibroblast lysate, one of which will be exposed to TGFb, the F1R1-treated groups should exhibit a lower intensity band for at least one of the previously mentioned signaling elements when compared to the control and scramble-treated groups not treated with F1R1. Including a scrambled F1R1 group will reveal if any cellular changes are bought on by protein treatment itself, rather than the native structure and properties of F1R1. This will give insight into how F1R1 treatment promotes an anti-fibrotic phenotype in both the absence and presence of exogenous TGF-B and which downstream signaling elements are hindered. Characterizing the mechanism of F1R1 action in cardiac cell cultures in terms of its downstream effects will help to translate its effects to mature tissue and eventually the clinical setting.
Aim #3: Determine the effect of increasing F1R1 concentration past physiological standards.
We hypothesize that the rate of proliferation will increase as the concentration of F1R1 increases. Based on past literature, a concentration of 50 ug/ml of F1R1 within a cardiac fibroblast culture accurately depicts a neonatal rat cECM environment13. Although 50 ug/ml is an accurate concentration for biomimicry, we expect that the effects of F1R1 do not falter at higher concentrations. For this experiment, we will use 3 variable groups and a control group. The 3 variable groups will contain varying concentrations of F1R1 (50 ug/ml, 100 ug/ml, and 200 ug/ml). All 3 variable groups and the control group will also contain TGF-B at a consistent concentration of 2.5 ng/ml. The 50 ug/ml F1R1 will represent the current standard and we expect to see similar results as with our previous aims. 100 ug/ml and 200 ug/ml will represent concentrations above the physiological range and will show us if F1R1 continues to increase proliferation as concentration increases. We expect that there will be a significant increase with respect to an increasing concentration between each variable group. From this aim, we hope to better establish a method for future testing of the impact of F1R1 on the rate of proliferation.