Blebbistatin: use as inhibitor of muscle contraction

Farman, Gerrie P.; Tachampa, Kittipong; Mateja, Ryan D.; Cazorla, Olivier; Lacampagne, Alain; Tombe, Pieter P. de

doi:10.1007/s00424-007-0375-3

Cited by 100 publications

(110 citation statements)

References 36 publications

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“…6B shows total myocyte TnC fluorescence normalized to the short SL condition as a function of [Ca 2+ ]. These data were obtained in the presence of a high concentration of 2,3-butanedione monoxime (50 mM), an agent that blocks strongly bound, force-generating actin-myosin interactions (17). In WT, but not HM, myocytes, stretch induced a significant increase in TnC fluorescence at all [Ca 2+ ].…”

Section: Resultsmentioning

confidence: 94%

Titin strain contributes to the Frank–Starling law of the heart by structural rearrangements of both thin- and thick-filament proteins

Ait-Mou

Hsu

Farman

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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170

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The Frank-Starling mechanism of the heart is due, in part, to modulation of myofilament Ca 2+ sensitivity by sarcomere length (SL) [length-dependent activation (LDA)]. The molecular mechanism(s) that underlie LDA are unknown. Recent evidence has implicated the giant protein titin in this cellular process, possibly by positioning the myosin head closer to actin. To clarify the role of titin strain in LDA, we isolated myocardium from either WT or homozygous mutant (HM) rats that express a giant splice isoform of titin, and subjected the muscles to stretch from 2.0 to 2.4 μm of SL. Upon stretch, HM compared with WT muscles displayed reduced passive force, twitch force, and myofilament LDA. Time-resolved small-angle X-ray diffraction measurements of WT twitching muscles during diastole revealed stretch-induced increases in the intensity of myosin (M2 and M6) and troponin (Tn3) reflections, as well as a reduction in cross-bridge radial spacing. Independent fluorescent probe analyses in relaxed permeabilized myocytes corroborated these findings. X-ray electron density reconstruction revealed increased mass/ordering in both thick and thin filaments. The SL-dependent changes in structure observed in WT myocardium were absent in HM myocardium. Overall, our results reveal a correlation between titin strain and the Frank-Starling mechanism. The molecular basis underlying this phenomenon appears not to involve interfilament spacing or movement of myosin toward actin but, rather, sarcomere stretch-induced simultaneous structural rearrangements within both thin and thick filaments that correlate with titin strain and myofilament LDA. myofilament length-dependent activation | small-angle X-ray diffraction | rat | passive force | fluorescent probes T he Frank-Starling law of the heart describes a cardiac regulatory control mechanism that operates on a beat-to-beat basis (1). There is a unique relationship between ventricular endsystolic volume and end-systolic pressure that is determined by cardiac contractility. As a result, ventricular stroke volume is directly proportional to the extent of diastolic filling. In conjunction with heart rate and contractility, the Frank-Starling mechanism constitutes a major determinant of cardiac output. Although the Frank-Starling mechanism has been well established for well over a century, the molecular mechanisms underlying this phenomenon are not resolved (1). At the cellular level, an increase in sarcomere length (SL) results in an immediate increase in twitch force development. Existing data, mostly derived from permeabilized isolated myocardium, strongly support the notion that this phenomenon is due to an increase in the Ca 2+ responsiveness of the cardiac contractile apparatus, a phenomenon termed "myofilament lengthdependent activation" (LDA) (1).The mechanism by which the mechanical strain signal is transduced by the cardiac sarcomere is not known. We have recently demonstrated that LDA develops within a few milliseconds following a change in SL (2), a finding suggestive of a mole...

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Section: Resultsmentioning

confidence: 94%

Titin strain contributes to the Frank–Starling law of the heart by structural rearrangements of both thin- and thick-filament proteins

Ait-Mou

Hsu

Farman

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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170

231

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“…To determine whether piperine affects these activities, we used a specific myosin inhibitor, blebbistatin, which decreases myosin ATPase activity by about 90% (26,27). The ATPase activity of nonmyosin proteins accounts for about one third of the total, and the small increase seen with piperine may be due to blebbistatin-free myosin heads (Fig.…”

Section: Resultsmentioning

confidence: 99%

Piperine’s mitigation of obesity and diabetes can be explained by its up-regulation of the metabolic rate of resting muscle

Nogara

Naber

Pate

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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We identify a target for treating obesity and type 2 diabetes, the consumption of calories by an increase in the metabolic rate of resting skeletal muscle. The metabolic rate of skeletal muscle can be increased by shifting myosin heads from the super-relaxed state (SRX), with a low ATPase activity, to a disordered relaxed state (DRX), with a higher ATPase activity. The shift of myosin heads was detected by a change in fluorescent intensity of a probe attached to the myosin regulatory light chain in skinned skeletal fibers, allowing us to perform a highthroughput screen of 2,128 compounds. The screen identified one compound, which destabilized the super-relaxed state, piperine (the main alkaloid component of black pepper). Destabilization of the SRX by piperine was confirmed by single-nucleotide turnover measurements. The effect was only observed in fast twitch skeletal fibers and not in slow twitch fibers or cardiac tissues. Piperine increased ATPase activity of skinned relaxed fibers by 66 ± 15%. The K d was ∼2 μM. Piperine had little effect on the mechanics of either fully active or resting muscle fibers. Previous work has shown that piperine can mitigate both obesity and type 2 diabetes in rodent models of these conditions. We propose that the increase in resting muscle metabolism contributes to these positive effects. The results described here show that up-regulation of resting muscle metabolism could treat obesity and type 2 diabetes and that piperine would provide a useful lead compound for the development of these therapies.myosin | fluorescence | skeletal muscle | super-relaxed state | obesity A n epidemic of obesity and type 2 diabetes is currently affecting a large fraction of the world population (1, 2). This is primarily due to an overconsumption of food coupled with a reduction in physical activity. A natural antidote to both obesity and type 2 diabetes is to choose a healthy lifestyle, including a low-calorie diet and increased physical activity. However, many do not choose this option, and for some advanced patients, increased physical activity is not possible. The response to overfeeding in humans is diverse: some store the excess calories almost entirely, whereas others metabolize most of them (3). The variation is strongly dependent on both lifestyle and genetics. As strenuous activity was limited in some of these protocols, the diversity of weight gain was attributed to light activities, such as fidgeting (3, 4). Alternatively, the diversity may be due to variation in the metabolic rate of resting muscle and to how this responds to activity (5).A pharmacological approach to combating obesity has been of limited value, of the order of 5-10% weight loss when combined with lifestyle changes (for review, see refs. 6 and 7). Therapies for type 2 diabetes produce little effect, with the exception of metformin (for review, see ref. 8). Lifestyle changes, including better diets and increased activity levels, have a larger effect than pharmaceuticals. Here we suggest a target for combating obesity and...

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“…If cessation of contraction did not occur, an additional 0.5 mol was administered. Blebbistatin is an excitation-contraction uncoupler that eliminates contraction without affecting calcium cycling or electrical activity (11,12). No additional preparation was needed for Langendorff-perfused hearts.…”

Section: Heart Preparationmentioning

confidence: 99%

NADH changes during hypoxia, ischemia, and increased work differ between isolated heart preparations

Wengrowski

Kuzmiak-Glancy

Jaimes

et al. 2014

American Journal of Physiology-Heart and Circulatory Physiology

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Wengrowski AM, Kuzmiak-Glancy S, Jaimes R 3rd, Kay MW. NADH changes during hypoxia, ischemia, and increased work differ between isolated heart preparations. Am J Physiol Heart Circ Physiol 306: H529 -H537, 2014. First published December 13, 2013; doi:10.1152/ajpheart.00696.2013.-Langendorff-perfused hearts and working hearts are established isolated heart preparation techniques that are advantageous for studying cardiac physiology and function, especially when fluorescence imaging is a key component. However, oxygen and energy requirements vary widely between isolated heart preparations. When energy supply and demand are not in harmony, such as when oxygen is not adequately available, the imbalance is reflected in NADH fluctuations. As such, NADH imaging can provide insight into the metabolic state of tissue. Hearts from New Zealand white rabbits were prepared as mechanically silenced Langendorffperfused hearts, Langendorff-perfused hearts, or biventricular working hearts and subjected to sudden changes in workload, instantaneous global ischemia, and gradual hypoxia while heart rate, aortic pressure, and epicardial NADH fluorescence were monitored. Fast pacing resulted in a dip in NADH upon initiation and a spike in NADH when pacing was terminated in biventricular working hearts only, with the magnitude of the changes greatest at the fastest pacing rate. Working hearts were also most susceptible to changes in oxygen supply; NADH was at half-maximum value when perfusate oxygen was at 67.8 Ϯ 13.7%. Langendorff-perfused and mechanically arrested hearts were the least affected by low oxygen supply, with halfmaximum NADH occurring at 42.5 Ϯ 5.0% and 23.7 Ϯ 4.6% perfusate oxygen, respectively. Although the biventricular working heart preparation can provide a useful representation of mechanical in vivo heart function, it is not without limitations. Understanding the limitations of isolated heart preparations is crucial when studying cardiac function in the context of energy supply and demand.Langendorff; biventricular working; blebbistatin; nadh imaging; workload THE HEART IS RESPONSIBLE FOR pumping blood with vital supplies to the entire body and therefore requires a constant high rate of energy supply. Additionally, the heart has a large dynamic range with the capacity to increase its workload 10-fold and maintain this increase for extended periods of time. This metabolic requirement necessitates acute regulation of energy supply to match demand, as well as a constant adequate oxygen supply. Any limitation in energy supply is rapidly detrimental to cardiac function.The demand for ATP and oxygen varies, depending on the amount of work being performed (12a, 17). The amount of available ATP to the heart at any given time is enough for only a few beats and must constantly be resynthesized to keep up with utilization (16). In the heart, 80 -90% of energy production occurs via oxidative phosphorylation (25). Fats and carbohydrates are broken down and electrons are harvested, reducing NAD ϩ to NADH, (and, to a lesser exte...

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Blebbistatin: use as inhibitor of muscle contraction

Cited by 100 publications

References 36 publications

Titin strain contributes to the Frank–Starling law of the heart by structural rearrangements of both thin- and thick-filament proteins

Titin strain contributes to the Frank–Starling law of the heart by structural rearrangements of both thin- and thick-filament proteins

Piperine’s mitigation of obesity and diabetes can be explained by its up-regulation of the metabolic rate of resting muscle

NADH changes during hypoxia, ischemia, and increased work differ between isolated heart preparations

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