AAV9-medated cardiac expression of lncRNA CARDINAL ameliorate TAC induced hypertrophy

Pathological cardiac hypertrophy is featured by enhanced protein synthesis. Translation inhibition is effective in treating cardiac hypertrophy, yet with systematic side effect. We identified a cardiac-enriched LncRNA CARDINAL, when over-expressed in cardiomyocyte using AAV9 driven by cTNT promoter, ameliorate transaortic constriction (TAC) induced hypertrophy. Overall design: GFP and full-length CARDINAL were separately cloned into ITR-containing AAV plasmid (Penn Vector Core P1967) harboring the chicken cardiac TNT promoter to get pAAV9-cTnT-CARDINAL and pAAV9-cTnT-GFP.AAV9 was packaged in HEK293T cells with AAV9: Rep-Cap and pHelper (pAd deltaF6, Penn Vector Core), then purified and concentrated by gradient centrifugation36. AAV9 titer was determined by quantitative PCR. AAV9 virus (5×109 viral genomes [vg]/g) was injected into postnatal day 1 WT pups by subcutaneous injection. Cardiac hypertrophy was induced by transverse aortic constriction (TAC) surgery as described previously. Briefly, mice with a body weight of 25-30g were anesthetized with isoflurane (3%–4% isoflurane for induction, 1%–2% isoflurane for maintenance). The chest was shaved and disinfected with alcohol. The chest was opened by left second intercostal thoracotomy. A 26-gauge needle was put on the ascending aorta. The needle and the ascending aorta were tightly ligated together using a 7-0 nylon suture at the transverse aorta, and the 26-gauge needle was removed immediately after ligation. In the sham operation, all procedures were the same except that no ligation was performed. Dissected intercostal space and chest skin were closed using a 6-0 silk suture. Mice genotypes were blinded to the surgeon. Samples were collected 4 weeks post surgery. knockout mouse was generated with CRISPR/Cas9 strategy.