A modified apical resection model with high accuracy and reproducibility in neonatal mouse and rat hearts.

Neonatal mouse heart can regenerate after left ventricle (LV) apical resection (AR). Since current AR rodent method is accomplished by resecting LV apex until exposure of LV chamber, it is relatively difficult to operate reproducibly. We aimed to develop a modified AR method with high accuracy and reproducibility and to investigate whether cardiac regenerative capacity could be replicated in neonatal rats. For 15% AR of whole heart weight in 1-day-old (P1) neonatal mice, a modified 10 µL pipette tip cut to 0.48 mm in internal diameter was connected to a vacuum pump working at 0.06 ± 0.005 MPa and gently kept in touch with LV apex for nearly but no more than 12 s. LV apex was resected by a single incision adjacent to the pipette tip. The modified AR method in P1 mice achieved cardiac structural and functional recovery at 21 days post resection (dpr). Data from different operators showed smaller variation of resected LV apex and higher reproducibility using the modified AR method. Furthermore, we showed that 5% AR of whole heart weight in P1 neonatal rats using a modified 200 µL pipette tip cut to 0.63 mm in internal diameter led to complete regeneration of LV apex and full preservation of cardiac function at 42 dpr. In conclusion, the modified AR rodent model leads to accurate resection of LV apex with high homogeneity and reproducibility and it is practically convenient for the study of structural, functional, and molecular mechanisms of cardiac regeneration in both neonatal mice and rats.

Angiotensin II-induced muscle atrophy via PPARγ suppression is mediated by miR-29b.

The activation of the renin-angiotensin system (RAS) induced by increased angiotensin II (AngII) levels has been implicated in muscle atrophy, which is involved in the pathogenesis of congestive heart failure. Although peroxisome proliferator-activated receptor gamma (PPARγ) activation can suppress RAS, the exact role of PPARγ in AngII-induced muscle atrophy is unclear. Here we identified PPARγ as a negative regulator of miR-29b, a microRNA that is able to promote multiple types of muscle atrophy. Suppression of miR-29b could prevent AngII-induced muscle atrophy both in vitro and in vivo. IGF1, PI3K(p85α), and Yin Yang 1 (YY1) were identified as target genes of miR-29b, and overexpression of these targets could rescue AngII-induced muscle atrophy. Importantly, inhibition of PPARγ was sufficient to induce muscle atrophy, while PPARγ overexpression could attenuate that. These data indicate that the PPARγ/miR-29b axis mediates AngII-induced muscle atrophy, and increasing PPARγ or inhibiting miR-29b represents a promising approach to counteract AngII-induced muscle atrophy.

CRISPR/Cas9-Mediated miR-29b Editing as a Treatment of Different Types of Muscle Atrophy in Mice.

Muscle atrophy is the loss of skeletal muscle mass and strength in response to diverse catabolic stimuli. At present, no effective treatments except exercise have been shown to reduce muscle atrophy clinically. Here, we report that CRISPR/Cas9-mediated genome editing through local injection into gastrocnemius muscles or tibialis anterior muscle efficiently targets the biogenesis processing sites in pre-miR-29b. In vivo, this CRISPR-based treatment prevented the muscle atrophy induced by angiotensin II (AngII), immobilization, and denervation via activation of the AKT-FOXO3A-mTOR signaling pathway and protected against AngII-induced myocyte apoptosis in mice, leading to significantly increased exercise capacity. Our work establishes CRISPR/Cas9-based gene targeting on miRNA as a potential durable therapy for the treatment of muscle atrophy and expands the strategies available interrogating miRNA function in vivo.