Focused ultrasound activates voltage-gated calcium channels through depolarizing TRPC1 sodium currents in kidney and skeletal muscle

Abstract

Pulsed focused ultrasound (pFUS) technology is being developed for clinical neuro/immune modulation and regenerative medicine. Biological signal transduction of pFUS forces can require mechanosensitive or voltage-gated plasma membrane ion channels. Previous studies suggested pFUS is capable of activating either channel type, but their mechanistic relationship remains ambiguous. We demonstrated pFUS bioeffects increased mesenchymal stem cell tropism (MSC) by altering molecular microenvironments through cyclooxygenase-2 (COX2)-dependent pathways. This study explored specific relationships between mechanosensitive and voltage-gated Ca2+ channels (VGCC) to initiate pFUS bioeffects that increase stem cell tropism.Methods: Murine kidneys and hamstring were given pFUS (1.15 or 1.125 MHz; 4MPa peak rarefactional pressure) under ultrasound or magnetic resonance imaging guidance. Cavitation and tissue displacement were measure by hydrophone and ultrasound radiofrequency data, respectively. Elastic modeling was performed from displacement measurements. COX2 expression and MSC tropism were evaluated in the presence of pharmacological ion channel inhibitors or in transient-receptor-potential-channel-1 (TRPC1)-deficient mice. Immunohistochemistry and co-immunoprecipitation examined physical channel relationships. Fluorescent ionophore imaging of cultured C2C12 muscle cells or TCMK1 kidney cells probed physiological interactions.Results: pFUS induced tissue deformations resulting in kPa-scale forces suggesting mechanical activation of pFUS-induced bioeffects. Inhibiting VGCC or TRPC1 in vivo blocked pFUS-induced COX2 upregulation and MSC tropism to kidneys and muscle. A TRPC1/VGCC complex was observed in plasma membranes. VGCC or TRPC1 suppression blocked pFUS-induced Ca2+ transients in TCMK1 and C2C12 cells. Additionally, Ca2+ transients were blocked by reducing transmembrane Na+ potentials and observed Na+ transients were diminished by genetic TRPC1 suppression.Conclusion: This study suggests that pFUS acoustic radiation forces mechanically activate a Na+-containing TRPC1 current upstream of VGCC rather than directly opening VGCC. The electrogenic function of TRPC1 provides potential mechanistic insight into other pFUS techniques for physiological modulation and optimization strategies for clinical implementation.

Focused ultrasound stimulates ER localized mechanosensitive PANNEXIN-1 to mediate intracellular calcium release in invasive cancer cells

“…This result is in contrast with previous studies that identified an important role for cytoskeletal networks in transducing US stimuli (De Cock et al, 2015). An important difference between our high frequency non-contact focused US stimulus and most other studies is that the latter uses low frequency US and requires physical contact with the PM through microbubbles (Burks et al, 2019;Carreras-Sureda et al, 2018;Clapham, 2007). This could explain why an intact cytoskeleton appeared necessary for internal Ca 2+ release in these other studies.…”

“…This raised the possibility that an internal mechanosensory event is present and coupled to Ca 2+ release from an internal storage site. Other work has also identified an ER dependent Ca 2+ response mechanism using more conventional US stimulation involving IP3R activation (Burks et al, 2019). This response required an intact cytoskeleton believed to be important for the mechanotransduction of the stimulus to the ER membrane localized IP3R .…”

“…cells. This new model can be compared to current working model largely derived from conventional US stimulation for comparison ( Figure 9B) (Burks et al, 2019;Carreras-Sureda et al, 2018;Clapham, 2007).…”

The Impact of Focused Ultrasound in Two Tumor Models: Temporal Alterations in the Natural History on Tumor Microenvironment and Immune Cell Response

“…Future studies will need to examine specific mechanisms of DNA damage by pFUS, which could include generation of reactive oxygen species (ROS) through pFUS-induced cytosolic Ca 2+ fluxes [57]. We have demonstrated that pFUS generates Ca 2+ transients by activating mechanically sensitive ion channels in the plasma membrane, which leads to depolarization of voltage-gated calcium channels [58]. While these exposures did not generate cavitation forces, intracellular Ca 2+ fluxes have also been reported from inertial cavitation mechanical forces [59].…”

Acoustic Radiation or Cavitation Forces From Therapeutic Ultrasound Generate Prostaglandins and Increase Mesenchymal Stromal Cell Homing to Murine Muscle

“…Specific microenvironmental changes resulting from mechanotransduction of US have received substantial attention, yet it remains unclear how the mechanical effects of US interact with tissues elicit these molecular responses. We previously demonstrated in skeletal muscle and kidney that pFUS at the above parameters mechanically activates the transient receptor potential channel 1 (TRPC1), which subsequently activates voltage gated calcium channels to initiate a calcium-dependent increase in COX2 expression (Burks et al, 2019). At those pFUS parameters, formation and cavitation of bubbles from dissolved tissue gases was not detected and viscoelastic modeling suggested that tissue deformations generated by acoustic radiation forces (ARF) were sufficient to increase COX2.…”