YAP Promotes Microtubule Growth to Facilitate Sarcomere Disassembly in Adult Cardiomyocytes

Mature mammalian cardiomyocytes (CMs) develop compact sarcomeric structures that inhibit proliferation. Consequently, CMs must dedifferentiate to a fetus-like state, which is accompanied by sarcomere disassembly, to enable successful cytokinesis. However, the regulation and coordination of CM dedifferentiation, cell cycle progression, and sarcomere reorganization remain unclear.

We generated adenovirus and adeno-associated virus (MyoAAV) vectors expressing YAP5SA and YAP5SA-S94A under X^on control for LMI070-inducible protein expression. We also developed MyoAAV-cTnT-Tuba1b-shRNA-miR30 for cardiomyocyte-specific knockdown of Tuba1b. These tools were used to investigate CM dedifferentiation and proliferation and sarcomere disassembly. We also performed Cleavage Under Targets and Release Using Nuclease to map the genome-wide binding sites of YAP5SA and YAP5SA-S94A in combination with RNA sequencing to identify YAP target genes. In addition, time-course live-imaging analysis was used to evaluate microtubule and sarcomere dynamics in adult CMs.

We show that microtubule expression and network density decline with cardiac maturation. Overexpression of YAP5SA, a constitutively active YAP mutant, promotes microtubule growth by stabilizing microtubule dynamics, leading to CM dedifferentiation, cell cycle re-entry, and sarcomere disassembly. In contrast, colchicine blocks these processes and significantly attenuates YAP-induced cardiac regeneration. Live imaging reveals a distinct mode of sarcomere disassembly driven by enhanced microtubule polymerization, wherein microtubule plus-ends directly interact with α-actinin and displace α-actinin fragments, thereby facilitating sarcomere breakdown. Furthermore, the YAP5SA-S94A mutation, which disrupts the YAP and TEA domain interaction, significantly reduces YAP5SA-induced microtubule growth, sarcomere disassembly, and cell cycle activity. Mechanistically, cleavage under targets and release using nuclease combined with RNA sequencing identified direct YAP targets, including Ajuba and Tuba1b, which are critical for microtubule growth. CM-specific knockdown of Tuba1b attenuates YAP-driven sarcomere disassembly.

These findings identify microtubule networks as an essential regulator modulating CM dedifferentiation and sarcomere reorganization, which is critical for CM cytokinesis and cardiac regenerative repair.