Response Of BK Channel In Ventricular Myocytes To Substrate Stiffness

Substrate stiffness regulates the growth,maturation and beating rate of ventricular myocytes.Ventricular myocytes generate contraction by sliding between actin and myosin and measure mechanical responsesof the substrate.This process depends primarily on intracellular Ca2+signaling.Opening of BK channel leads tohyperpolarization,thereby closing the voltage-gated caicium channes that control intracellular Ca2+levels.Our research aimed to reveal the potential role of BK channel in sensing substrate stiffness in ventricular myocytes.Our two types of polydimethylsiloxane substrate preparations had similarsurface topography but a 10-fold difference in Young's modulus.Firstly we showed that with the substrate stiffness increasing,ventricular myocytes spreading area increased and ventricular myocytes labeled for an F-actin had well-defined striations with more articulated stress fibers on stiffer substrate.Besides,though the protein expression of L-type voltage-gated Ca2+ channelexhibited the same,its current density was greater in myocytes cultured on stiff substrates.This difference could be abolished by blocking BK channel.Moreover,increased substrate stiffness significantly reduced the expression of total BK channel and STREX-BK channel while reduction of the latter was far more.It also inhibited the activity of the BK channel,including the current amplitude,open probability,voltage-dependence and calcium-dependence,both in primarily cultured myocytes andheterologously expressed HEK293 cells.However STREX-deleted BK channel function remained largely unaffected by substrate stiffness.Finally,when we expressed L-type Ca2+ channel alone in HEK293 cells,the Ca2+ current density was not different on the soft and stiff substrates.By co-expressing L-type Ca2+ channel and BK channel,the Ca2+ current density was greater in HEK 293 cells on stiff substrate.In contrast,when we expressed L-type Ca2+ channel and STREX-deleted BK channel together in HEK293 cells,substrate stiffness had no effect on the Ca2+ current density.Taken together,our results suggested that BK channel via its STREX sequence senses substrate stiffness and regulates the L-type Ca2+ channel activity.This provides new insight into the molecular mechanism by which ventricular myocytes sense substrate stiffness.