Single-Cell Hypertrophy Promotes Contractile Function of Cultured Human Airway Smooth Muscle Cells via Piezo1 and YAP Auto-Regulation

Severe asthma is characterized by increased cell volume (hypertrophy) and enhanced contractile function (hyperresponsiveness) of the airway smooth muscle cells (ASMCs). The causative relationship and underlying regulatory mechanisms between them, however, have remained unclear. Here, we manipulated the single-cell volume of in vitro cultured human ASMCs to increase from 2.7 to 5.2 and 8.2 × 10³ μm³ as a simulated ASMC hypertrophy by culturing the cells on micropatterned rectangular substrates with a width of 25 μm and length from 50 to 100 and 200 μm, respectively. We found that as the cell volume increased, ASMCs exhibited a pro-contractile function with increased mRNA expression of contractile proteins, increased cell stiffness and traction force, and enhanced response to contractile stimulation. We also uncovered a concomitant increase in membrane tension and Piezo1 mRNA expression with increasing cell volume. Perhaps more importantly, we found that the enhanced contractile function due to cell volume increase was largely attenuated when membrane tension and Piezo1 mRNA expression were downregulated, and an auto-regulatory loop between Piezo1 and YAP mRNA expression was also involved in perpetuating the contractile function. These findings, thus, provide convincing evidence of a direct link between hypertrophy and enhanced contractile function of ASMCs that was mediated via Piezo1 mRNA expression, which may be specifically targeted as a novel therapeutic strategy to treat pulmonary diseases associated with ASMC hypertrophy such as severe asthma.