Abstract

In a recent study, 2,5-Di-(tert-butyl)−1,4-benzohydroquinone (TBQ) produced a concentration dependent and fully reversible inhibition of the sarcoplasmic reticulum Ca²⁺ ATPase (SERCA) in rat ventricular myocytes¹. While TBQ is a potentially useful research tool to study SERCA inhibition in cardiac cells, many additional effects were observed including production of an outward current consistent with activation of an ATP dependent potassium channel. The current study aims to determine the mechanisms underlying these effects.

Rat and sheep ventricular myocytes were isolated by enzymatic digestion. Intracellular ATP levels were measured using a ViaLight Plus Cell Proliferation Kit (Lonza). Mitochondrial oxygen consumption and levels of hydrogen peroxide were measured using an Oxygraph-2k high resolution respirometer (Oroboros Instruments).

In rat and sheep ventricular myocytes, TBQ produced a concentration dependent decrease of intracellular ATP where 100 μM TBQ decreased ATP levels to approximately 60% of control. Removal of glucose from the experimental solutions had no effect on the magnitude of effect. In rat homogenates, TBQ produced a concentration dependent decrease of mitochondrial oxygen uptake, with 100 μM TBQ decreasing rate to 85% of control. TBQ increased levels of hydrogen peroxide, however catalase, did not attenuate TBQs effect on mitochondrial oxygen uptake.

The current findings suggest TBQ decreases intracellular ATP, a phenomenon which may account for many of the effects observed previously, including activation of an ATP dependent potassium channel. The reduction in ATP appears to be associated with an effect on oxidative phosphorylation rather than glycolysis. While TBQ is associated with an increase in hydrogen peroxide, which may increase oxidative stress, the experiments carried out with catalase suggest that this does not contribute to TBQs effect on mitochondrial function.