Abstract 3572: Enhancing HIF-1a by Double shRNA Knockdown in Murine Myocardial Infarction
Background: In this study, we utilized a non-viral minicircle vector carrying two shRNA targeting sites. We hypothesis that double shRNA knockdown of PHD and AsPHD2 can be used for treatment of IHD by enhancing several proangiogenic genes.
Method: The best shortairpin sequence for inhibiting PHD2 (shPHD2) and AsPHD (shAsPHD) was inserted into the minicircle vector with the H1 promoter. Another construct with SV40 promoter driving firefly luciferase was inserted coupled to hypoxia response elements (HRE) to allow molecular imaging (Figure 1⇓). This construct was used to transfect mouse C2C12 myoblast cell line along with the shRNA constructs. Afterwards, shRNA constructs were injected intramyocardially following LAD ligation in adult FVB mice. Animals were randomized into shAsPHD group (n=10), shPHD2 group (n=10), shAsPHD + shPHD2 (n=10) versus control group with scramble sequence injection (n=10). Cardiac function was assessed by echocardiography at weeks 1 and 4.
Result: For in vitro cell culture, molecular imaging showed double knockdown expressed >50% higher angiogenesis level than shPHD2 group and >80% than scramble and shAsPHD group. Results are confirmed by Q-PCR of 6 different angiogenesis gene. For in vivo study, echocardiography showed significant improvement of LVEF in the minicircle double knockdown group (45.2%±3.6%) compared to the single knockdown with shPHD2 (39.2%±4.1%) or shAsPHD (36.7%±2.8%) and scramble (35.1%±3.2%) groups at week 4.
Conclusion: Taken together, this is the first study to demonstrate that double knockdown significantly increases HIF-1a, which lead to enhanced angiogenic gene response and improved cardiac contractility.
This research has received full or partial funding support from the American Heart Association, Western States Affiliate (California, Nevada & Utah).