The effects of 2',5'-di(tert-butyl)-1,4-benzohydroquinone (TBQ) on calcium handling in rat ventricular myocytes

Greensmith, DJ; Miller, L; Eisner, D

Abstract

2',5'-di(tert-butyl)-1,4-benzohydroquinone (TBQ) is a reversible inhibitor of SERCA, potentially making it a useful tool to study the effects of SERCA inhibition. It has been used in a variety of cell types including ventricular myocytes (1). However, it is currently unknown if TBQ also has effects on other components of ventricular intracellular Ca handling. The aim of these experiments was therefore to characterise the effects of TBQ on Ca handling in the rat ventricular myocyte and assess its suitability as specific inhibitor of SERCA.Male Wistar rats were killed in accordance with the The Home Office Animal (Scientific Procedures) Act 1986 for enzymatic isolation of ventricular myocytes which were subsequently loaded with the Ca indicator Fluo-3AM to measure global intracellular Ca. Myocytes were voltage clamped via perforated patch and paced at 0.5 Hz to measure sarcolemmal Ca fluxes.TBQ produced a concentration dependent decrease of the rate of decay of systolic Ca, consistent with a decrease in SERCA activity. This was fully reversible at all concentrations tested. For most experiments we used 10 µM TBQ as this produced a large effect without completely abolishing the systolic Ca transient. 10 µM TBQ produced a 48 ± 5 and 54 ± 6 % decrease in the amplitude and the rate of decay of the systolic Ca transient respectively. However, at 10 µM, TBQ also produced a 23 ± 7 % decrease of the peak Ca current without altering the voltage dependence. SR Ca content was also reduced by 62 ± 4 %. Interestingly, at higher concentrations (100 µM), TBQ also activated an outward current that demonstrated a current-voltage relationship consistent with a potassium current. This outward current was sensitive to Glibenclamide. Thapsigargin (another inhibitor of SERCA) did not produce this outward current, suggesting its activation is TBQ dependent, rather than as a result of SERCA inhibition.These data show that in rat ventricular myocytes, TBQ can be used to reversibly inhibit SERCA. However, at concentrations that decrease SERCA activity, TBQ also decreases the Ca current, and, at higher concentrations, activates an outward current which appears to be an ATP dependent potassium current. We conclude that TBQ cannot be used as a specific inhibitor of SERCA in rat ventricular myocytes.

Citation

Greensmith, D., Miller, L., & Eisner, D. (2014). The effects of 2',5'-di(tert-butyl)-1,4-benzohydroquinone (TBQ) on calcium handling in rat ventricular myocytes. Proceedings of the Physiological Society (Online), 31, PCA042

• Journal Article Type: Article
• Publication Date: Jul 1, 2014
• Deposit Date: Jan 22, 2015
• Journal: Proceedings of The Physiological Society
• Publisher: Cambridge University Press (CUP)
• Peer Reviewed: Peer Reviewed
• Volume: 31
• Pages: PCA042
• Publisher URL: https://www.physoc.org