Frequency sensitive mechanism in low-intensity ultrasound enhanced bioeffects

Abstract

This study presents two novel theoretical models to elucidate frequency sensitive nuclear mechanisms in low-intensity ultrasound enhanced bioeffects. In contrast to the typical 1.5 MHz pulsed ultrasound regime, our group previously experimentally confirmed that ultrasound stimulation of anchored chondrocytes at resonant frequency maximized gene expression of load inducible genes which are regulatory markers for cellular response to external stimuli. However, ERK phosphorylation displayed no frequency dependency, suggesting that the biochemical mechanisms involved in enhanced gene expression is downstream of ERK phosphorylation. To elucidate such underlying mechanisms, this study presents a theoretical model of an anchored cell, representing an in vitro chondrocyte, in an ultrasound field. The model results showed that the mechanical energy storage is maximized at the chondrocyte's resonant frequency and the energy density in the nucleus is almost twice as high as in the cytoplasm. Next, a mechanochemical model was developed to link the mechanical stimulation of ultrasound and the increased mechanical energy density in the nucleus to the downstream targets of the ERK pathway. This study showed for the first time that ultrasound stimulation induces frequency dependent gene expression as a result of altered rates of transcription factors binding to chromatin.

Fig 1. Model geometry.

Cell attached to a planar surface (polystyrene to mimic the properties of a 6-welled plate) immersed in fluid (growth media). The transducer (ultrasound source) is positioned at the top. The cell position changes so that it always lies at an antinode or node.

Fig 2. Segment of ERK pathway.

Ultrasound leads to the phosphorylation/activation of an unknown enzyme (MAPKKK) which kick starts the ERK signaling pathway resulting in gene expression.

Fig 3. Model of cell attached to a plane (90° with respect to the incident planar ultrasonic wave), the color represents displacement in nanometers.

A) Cell at anti-node in 5 MHz field. B) Cell at node in 8 MHz field.

Fig 4. Total mechanical energy density.

Total mechanical energy density in the cytoplasm and nucleus for cells attached to a plane located at a pressure anti-node and node.

Fig 5. c-Fos gene expression with modeled frequency sensitive nuclear transport using Eq 20.

A) c-Fos gene expression versus time for frequency dependent nuclear transport, B) c-Fos gene expression at a snapshot in time, 1.5 hours following the completion of ultrasound stimulation.

Fig 6. c-Fos gene expression with modeled frequency sensitive chromatin binding using Eq 22.

A) c-Fos gene expression versus time with frequency sensitive chromatin binding, B) c-Fos gene expression at 1.5 hours following the completion of ultrasound stimulations, C) experimental results from Louw et al. [11] and D) c-Fos gene expression at 1.5 hours following the completion of ultrasound stimulation for modeled frequency sensitivity for both nuclear transport and chromatin binding.