Properties of blebbistatin for cardiac optical mapping and other imaging applications

Abstract

Blebbistatin is a recently discovered myosin II inhibitor. It is rapidly becoming a compound of choice to reduce motion artifacts during cardiac optical mapping, as well as to study cell motility and cell invasion. Although blebbistatin has a number of advantages over other electromechanical uncouplers, many of its properties have yet to be addressed. Here we describe several methodological issues associated with the use of blebbistatin, including its spectral properties, reversibility, and its effect on tissue metabolic state. We show that if precautions are not taken, perfusion with blebbistatin may result in blebbistatin precipitate that accumulates in the vasculature. Although such precipitate is fluorescent, it is not detectable within wavelength bands that are typically used for transmembrane voltage fluorescence imaging (i.e., emission wavelengths >600 nm). Therefore, blockage of the microcirculation by blebbistatin may cause data misinterpretation in studies that use voltage-sensitive dyes. Blebbistatin may also impact imaging of green fluorophores due to the spectral shift it causes in endogenous tissue fluorescence. 3D excitation–emission matrices of blebbistatin in precipitate form and in various solutions (DMSO, water, and 1 % aqueous albumin) revealed significant changes in the fluorescence of this molecule in different environments. Finally, we examined the reversibility of blebbistatin’s uncoupling effect on cardiac contraction. Our findings provide important new information about the properties of this myosin II inhibitor, which will aid in the proper design and interpretation of studies that use this compound.

The mechanical uncoupler blebbistatin is associated with significant electrophysiological effects in the isolated rabbit heart

“…Nevertheless, the energetic demand of the uncoupled heart is predictably less than that of a contracting heart, and it is possible that the decrease in flow is not sufficient to promote ischaemia. In support of this, Swift et al (2012) have demonstrated that NADH accumulation in ischaemic tissue is five times slower in the presence of blebbistatin than that seen in control conditions. It cannot be ruled out that vasoconstriction is a reflection of a reduction in energetic demand.…”

“…Exp Physiol 98.5 (2013) pp 1009-1027 the mitochondria and cytosol, reducing ATP availability despite inhibition of contractile activity and a reduction in net oxygen consumption (de Tombe et al 1992;Stapleton et al 1998). Swift et al (2012) have previously reported that blebbistatin can form a precipitate when mixed in solutions at room temperature and can accumulate within the coronary vasculature. This is unlikely to be a factor in the present experiments, because all solutions were heated to 37 • C and passed through a 5 μm micropore filter before perfusion.…”

“…Swift et al (2012) have previously reported that blebbistatin can form a precipitate when mixed in solutions at room temperature and can accumulate within the coronary vasculature. This is unlikely to be a factor in the present experiments, because all solutions were heated to 37°C and passed through a 5 μm micropore filter before perfusion.…”

Sensing Cardiac Electrical Activity With a Cardiac Myocyte–Targeted Optogenetic Voltage Indicator

“…This strategy cannot be applied in vivo because of the toxicity of the commonly used dyes and the incompatibility of the in vivo use of electromechanical uncouplers as well as their effects on AP morphology. 6,[44][45][46][47] Here, we provide first experimental evidence that a GEVI, expressed stably and homogeneously in cardiac myocyte membranes in vivo, can overcome these limitations. We first scrutinized the VSFP-imaging modality in Langendorff-perfused hearts and validated its use for the visualization of myocardial electric conduction under physiological and pathological heart rate and rhythm ( Figure 5).…”

Construction and use of a zebrafish heart voltage and calcium optical mapping system, with integrated electrocardiogram and programmable electrical stimulation

“…Stock solutions of 10 mM (-)-blebbistatin (Sigma-Aldrich, St. Louis, MO) and 5 mM RH 237 (Molecular Probes, Eugene, OR) dissolved in 100% DMSO were kept at Ϫ20°C and were used at 15 M (0.15% DMSO) and 8 M (0.16% DMSO) final concentrations, respectively. Warming of blebbistatin before dilution increased the consistency of inhibition of cardiac contraction (31). Fresh aliquots of Rhod-2 AM (Molecular Probes) gave the largest transient sizes with the lowest background levels.…”