Application of blebbistatin as an excitation–contraction uncoupler for electrophysiologic study of rat and rabbit hearts

Fedorov, Vadim V.; Lozinsky, Ilya; Sosunov, Eugene A.; Anyukhovsky, Evgeniy P.; Rosen, Michael R.; Balke, C. William; Efimov, Igor R.

doi:10.1016/j.hrthm.2006.12.047

Cited by 312 publications

(343 citation statements)

References 19 publications

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“…In the present study, we utilized BDM, which is known to change several membrane conductances (6,56) and thereby causes some electrophysiological effects (14,37,40), including flattening the APD restitution curve (3). To examine the potential effect of BDM on the dynamics of cardiac response, we conducted two additional experiments using a new excitation-contraction uncoupling agent, blebbistatin, which is considered to have no effects on the AP morphology in rabbit cardiac tissue (22). Similar to our experiments using BDM, we observed three concurrent dynamics, normal, 2:2, and 2:1 rhythm, when [K ϩ ] o was regionally elevated and PI was shortened to 140 ms.…”

Section: Discussionmentioning

confidence: 64%

Regional increase of extracellular potassium leads to electrical instability and reentry occurrence through the spatial heterogeneity of APD restitution

Sidorov¹,

Uzelac

Wikswo

2011

American Journal of Physiology-Heart and Circulatory Physiology

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Sidorov VY, Uzelac I, Wikswo JP. Regional increase of extracellular potassium leads to electrical instability and reentry occurrence through the spatial heterogeneity of APD restitution. Am J Physiol Heart Circ Physiol 301: H209 -H220, 2011. First published May 2, 2011; doi:10.1152/ajpheart.01141.2010.-The heterogeneities of electrophysiological properties of cardiac tissue are the main factors that control both arrhythmia induction and maintenance. Although the local increase of extracellular potassium ([K ϩ ]o) due to coronary occlusion is a well-established metabolic response to acute ischemia, the role of local [K ϩ ]o heterogeneity in phase 1a arrhythmias has yet to be determined. In this work, we created local [K ϩ ]o heterogeneity and investigated its role in fast pacing response and arrhythmia induction. The left marginal vein of a Langendorff-perfused rabbit heart was cannulated and perfused separately with solutions containing 4, 6, 8, 10, and 12 mM of K ϩ . The fluorescence dye was utilized to map the voltage distribution. We tested stimulation rates, starting from 400 ms down to 120 ms, with steps of 5-50 ms. We found that local [K ϩ ]o heterogeneity causes action potential (AP) alternans, 2:1 conduction block, and wave breaks. The effect of [K ϩ ]o heterogeneity on electrical stability and vulnerability to arrhythmia induction was largest during regional perfusion with 10 mM of K ϩ . We detected three concurrent dynamics: normally propagating activation when excitation waves spread over tissue perfused with normal K ϩ , alternating 2:2 rhythm near the border of [K ϩ ]o heterogeneity, and 2:1 aperiodicity when propagation was within the high [K ϩ ]o area. [K ϩ ]o elevation changed the AP duration (APD) restitution and shifted the restitution curve toward longer diastolic intervals and shorter APD. We conclude that spatial heterogeneity of the APD restitution, created with regional elevation of [K ϩ ]o, can lead to AP instability, 2:1 block, and reentry induction. restitution heterogeneity; regional perfusion; optical mapping THE STATIONARY AND DYNAMIC heterogeneities of electrophysiological properties of cardiac tissue play a key role in the induction and maintenance of cardiac arrhythmias (1,10,16,59). Cardiac tissue heterogeneities increase drastically when acute regional ischemia occurs. The early stage of acute ischemia is characterized by abrupt metabolic and electrophysiological changes in the affected area (11). Among them, the more prominent are increased extracellular potassium ([K ϩ ] o ) (25, 26), decreased creatine phosphate (43, 63), elevated intracellular inorganic phosphate (63), acidosis (27, 46), catecholamine release (39), and partial depolarization of transmembrane potential (V m ) (28,36).Since an early change in V m closely relates to [K ϩ ] o accumulation (34, 60), the K ϩ imbalance is considered to be the major factor affecting excitability and is responsible for slow impulse propagation and induction of reentry (51) Although numerous studies have been devoted to investigation ...

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Section: Discussionmentioning

confidence: 64%

Regional increase of extracellular potassium leads to electrical instability and reentry occurrence through the spatial heterogeneity of APD restitution

Sidorov¹,

Uzelac

Wikswo

2011

American Journal of Physiology-Heart and Circulatory Physiology

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“…The use of two excitation wavelengths for Fura-2/AM-based Ca 2+ measurements further compounds this. Also, uncoupling contraction from excitation and Ca 2+ release is necessary for the current protocols; achieved by BDM and blebbistatin, but this may confound the morphology of APs and CaTs, especially in the whole-heart preparation [29], although minimal effects of BDM [30] and blebbistatin [31] have also been reported. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 …”

Section: Strengthsmentioning

confidence: 99%

2‐photon excitation fluorescence microscopy enables deeper high‐resolution imaging of voltage and Ca²⁺ in intact mice, rat, and rabbit hearts

Ghouri

Kelly

Burton³

et al. 2013

Journal of Biophotonics

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We describe a novel two-photon (2P) laser scanning microscopy (2PLSM) protocol that provides ratiometric transmural measurements of membrane voltage (V m ) via Di-4-ANEPPS in intact mouse, rat and rabbit hearts with subcellular resolution. The same cells were then imaged with Fura-2/AM for intracellular Ca 2+ recordings. Action potentials (APs) were accurately characterized by 2PLSM vs microelectrodes, albeit fast events (<1ms) were sub-optimally acquired by 2PLSM due to limited sampling frequencies (2.6kHz). The slower Ca 2+ transient (CaT) time course (>1ms) could be accurately described by 2PLSM. In conclusion, V m -and Ca 2+ -sensitive dyes can be 2P excited within the cardiac muscle wall to provide AP and Ca 2+ signals to ~400μm.Abstract Figure: 2P-excited images of Di-4-ANEPPS-and Fura-2/AM-loaded myocardium including the resultant AP and CaT extracted from the presented protocols.

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“…Furthermore, selective treatment with blebbistatin has been demonstrated as a tool to synchronize mammalian culture cells during different stages of mitotic division (metaphase, anaphase, telophase), a task that proved difficult in the past due to the short duration of anaphase [72]. In addition, both BDM and blebbistatin have been used to eliminate the 'motion artifacts' caused by muscle contractions during live optical imaging of cardiac electrical activity [73,74]. On a more practical therapeutic level, treatment with BDM has been highlighted as a means of increasing the storage lifetime and efficiency of heart transplantation, as BDM treatment reduces stress and metabolic deregulation in donor hearts [46,83].…”

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confidence: 99%

Small-Molecule Inhibitors of Myosin Proteins

et al. 2012

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Advances in screening and computational methods have enhanced recent efforts to discover/ design small-molecule protein inhibitors. One attractive target for inhibition is the myosin family of motor proteins. Myosins function in a wide variety of cellular processes, from intracellular trafficking to cell motility, and are implicated in several human diseases (e.g., cancer, hypertrophic cardiomyopathy, deafness and many neurological disorders). Potent and selective myosin inhibitors are, therefore, not only a tool for understanding myosin function, but are also a resource for developing treatments for diseases involving myosin dysfunction or overactivity. This review will provide a brief overview of the characteristics and scientific/therapeutic applications of the presently identified small-molecule myosin inhibitors before discussing the future of myosin inhibitor and activator design.The identification and characterization of pharmacological compounds that inhibit the functional activity of one or more specific proteins or processes has been the subject of much scientific investigation. On a basic science level, these membrane-permeable compounds provide the scientific community with a tool for the targeted and functional inhibition of a given protein in the cell; a potent means of evaluating the intracellular functions of that protein [1,2]. From a biomedical standpoint, the characterization of these small-molecule inhibitors affords an opportunity for the development of novel disease treatments centering on the repression of an offensive molecule or the reversal of its downstream effects [3][4][5].At present, several complementary methods for obtaining suitable small-molecule inhibitors of specific proteins exist. Traditional methods in inhibitor discovery involve the systematic testing of a series of chemically synthesized or naturally occurring compounds. Advances in robotics and data processing have made it possible to use high-throughput screens to test libraries of thousands or even millions of potential drugs for their ability to inhibit the function of a specific protein in a targeted biochemical or cellular assay [6][7][8]. These inhibitor discovery processes are complemented by more precise methods in small-molecule inhibitor design. Structure-based methods rely on the use of x-ray crystallographic or NMRbased structures of a protein of interest to design small molecules likely to bind and inhibit protein function [9,10]. Computer-aided inhibitor design uses computational methods to optimize potential inhibitors identified by screening or structure-based methods, to virtually © 2013 Folma Buss * Author for correspondence: Tel.: +44 1223 763348, Fax: +44 1223 762640, fb207@cam.ac.uk. Financial & competing interests disclosureThe authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance w...

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Application of blebbistatin as an excitation–contraction uncoupler for electrophysiologic study of rat and rabbit hearts

Cited by 312 publications

References 19 publications

Regional increase of extracellular potassium leads to electrical instability and reentry occurrence through the spatial heterogeneity of APD restitution

Regional increase of extracellular potassium leads to electrical instability and reentry occurrence through the spatial heterogeneity of APD restitution

2‐photon excitation fluorescence microscopy enables deeper high‐resolution imaging of voltage and Ca²⁺ in intact mice, rat, and rabbit hearts

Small-Molecule Inhibitors of Myosin Proteins

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Application of blebbistatin as an excitation–contraction uncoupler for electrophysiologic study of rat and rabbit hearts

Cited by 312 publications

References 19 publications

Regional increase of extracellular potassium leads to electrical instability and reentry occurrence through the spatial heterogeneity of APD restitution

Regional increase of extracellular potassium leads to electrical instability and reentry occurrence through the spatial heterogeneity of APD restitution

2‐photon excitation fluorescence microscopy enables deeper high‐resolution imaging of voltage and Ca2+ in intact mice, rat, and rabbit hearts

Small-Molecule Inhibitors of Myosin Proteins

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2‐photon excitation fluorescence microscopy enables deeper high‐resolution imaging of voltage and Ca²⁺ in intact mice, rat, and rabbit hearts