Edited by David A. Weitz, Harvard University, Cambridge, MA, and approved December 4, 2017 (received for review August 7, 2017)
Ultrasound-induced microbubble oscillation can lead to cell injury or mechanotransduction via calcium signaling processes such as intracellular calcium waves (ICWs). However, the mechanisms by which microbubbles stimulate ICWs remain unknown. Using a microfluidic system with highly controlled bubble−cell interaction, we identified two distinct types of ICWs: a fast response correlating with significant membrane poration, and a slow response triggered by calcium influx through stretch-activated ion channels. The fast ICWs, distinguished from those under physiological conditions, are associated with cell injuries. We further elicited ICWs without cell injury by displacing integrin-binding beads on the cell membrane under mild cavitation conditions. This study provides mechanistic insights into ICWs for guiding ultrasound therapy in tissue modification, drug delivery, and cell mechanotransduction.
One of the earliest events in cellular mechanotransduction is often an increase in intracellular calcium concentration associated with intracellular calcium waves (ICWs) in various physiologic or pathophysiologic processes. Although cavitation-induced calcium responses are believed to be important for modulating downstream bioeffects such as cell injury and mechanotransduction in ultrasound therapy, the fundamental mechanisms of these responses have not been elucidated. In this study, we investigated mechanistically the ICWs elicited in single HeLa cells by the tandem bubble-induced jetting flow in a microfluidic system. We identified two distinct (fast and slow) types of ICWs at varying degrees of flow shear stress-induced membrane deformation, as determined by different bubble standoff distances. We showed that ICWs were initiated by an extracellular calcium influx across the cell membrane nearest to the jetting flow, either primarily through poration sites for fast ICWs or opening of mechanosensitive ion channels for slow ICWs, which then propagated in the cytosol via a reaction−diffusion process from the endoplasmic reticulum. The speed of ICW (CICW) was found to correlate strongly with the severity of cell injury, with CICW in the range of 33 μm/s to 93 μm/s for fast ICWs and 1.4 μm/s to 12 μm/s for slow ICWs. Finally, we demonstrated that micrometer-sized beads attached to the cell membrane integrin could trigger ICWs under mild cavitation conditions without collateral injury. The relation between the characteristics of ICW and cell injury, and potential strategies to mitigate cavitation-induced injury while evoking an intracellular calcium response, may be particularly useful for exploiting ultrasound-stimulated mechanotransduction applications in the future.
Author contributions: F.L., F.Y., F.G., and P.Z. designed research; F.L., C.Y., F.Y., and T.L. performed research; F.L. contributed new reagents/analytic tools; F.L., D.L., T.L., and P.Z. analyzed data; and F.L., F.G., and P.Z. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1713905115/-/DCSupplemental.
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