measuring sarcoplasmic calcium concentration in isolated mouse muscle fibers

Enzymatic Isolation and Characterization of Variable Length Mouse Muscle Fibers

The mature skeletal muscle of mammals is a multifunctional tissue. It heavily regulates metabolism, is the main source of heat production, and its dynamical properties confer upon it a key role in respiration, movement of body segments, or displacement from one point to another1,2,3. Skeletal muscle is also relevant for the pathophysiology of many illnesses, including inherited and chronic conditions, such as myopathies, dystrophies, or sarcopenia, as well as many non-muscle chronic conditions, such as cardiometabolic diseases3,4,5,6,7,8.
The ex vivo study of the structural and functional properties of mature skeletal muscle in the context of health and disease has been possible mainly through two experimental models: whole muscle and isolated fibers. In the 20th century, researchers exploited the properties of the whole, intact extensor digitorum longus (EDL), soleus, tibialis anterior, and gastrocnemius muscles of different small species as pivotal models to learn about motor units, fiber types, and dynamic properties such as force and kinetics of contraction and relaxation9,10,11,12,13,14,15,16. However, the advent of more refined cell biology studies moved the area toward the study of single muscle fibers. Pioneering work then enabled the isolation of intact flexor digitorum brevis (FDB) fibers of rats by enzymatic dissociation for subsequent characterization17,18,19. Although FDB fibers can also be obtained by manual dissection20, the ease and high throughput of enzymatic dissociation of murine muscles, in addition to their suitability for a variety of experimental approaches, have made the latter model widely used during the last two decades.
The short FDB fibers are suitable for electrophysiological and other biophysical studies, biochemical, metabolic, and pharmacological analyses, electron and fluorescence microscopy experiments, transfection for cell biology approaches, or as a source of stem cells in myogenesis studies5,21,22,23,24,25,26,27,28,29,30,31,32. However, using only FDB fibers in muscle experiments narrows the scope of research dealing with fiber types and limits the amount of biological material available for some methodological techniques or for gaining more information from one animal. These limitations hinder a clear correlation of cellular physiological phenomena with previous biochemical and dynamical studies performed in different whole, intact, muscles (e.g., EDL, soleus, peronei).
Overcoming these limitations, some groups succeeded in dissociating the longer EDL and soleus muscles24,33,34,35,36,37,38,39,40, opening the door to further extend the method to other relevant muscles. However, the use of EDL and soleus fibers is still scarce, likely due to the lack of methodological details for getting them as intact fibers. Here, we describe in detail how to isolate fibers of different lengths and types from six muscles: three of them already described (FDB, EDL, and soleus) and three of them successfully dissociated for the first time (extensor hallucis longus [EHL], peroneus longus [PL], and peroneus digiti quarti [PDQA]). The results of the present work confirm that the model of enzymatically dissociated fibers is apt for a wide range of studies and future correlations with previously published data, thus increasing the availability of models for mature skeletal muscle studies.

Author Spotlight: Isolation of Long Muscle Fibers from Mouse Hindlimb Muscles for Studying Excitation-Contraction Coupling Across Fiber Types

Intact Short, Intermediate, and Long Skeletal Muscle Fibers Obtained by Enzymatic Dissociation of Six Hindlimb Muscles of Mice: Beyond Flexor Digitorum Brevis

In this study, we describe a method to isolate fibers of different lengths and types from six hindlimb muscles from mice. Also, we tested the suitability of the fibers for physiological experiments dealing with the study of the excitation-contraction coupling mechanism. For years, obtaining long muscle fibers was challenge, limiting the scope of many studies in the field.Now we show that it is possible to obtain lean fibers with mouse flexors, extensors, and evertors with lengths of up to six millimeters. As an example, obtaining fibers from six different muscles has allowed us to show that the calcium transient kinetics known as morphology type II, can be generalized to type IIB and IIX regardless of the location or function of their muscle source. We believe that the model of enzymatically isolated fibers is suitable for a variety of experimental approaches with different types of technologies that allow us to answer mechanistic questions within the range of biochemistry, physiology, biophysics, cell biology, and molecular biology.

Measuring Sarcoplasmic Calcium Concentration in Isolated Mouse Muscle Fibers

To begin, mount a clean glass slide on the experimental bath chamber. Coat the slide with two to three microliters of laminin and let it dry for 30 seconds. Then, pour approximately 400 microliters of the muscle fiber suspension onto the slide and allow the fibers to adhere to the laminin for 10 to 15 minutes.Place the experimental chamber onto the stage of an inverted microscope equipped for epifluorescence. To verify the viability of the fibers, place two platinum electrodes on either side of the experimental chamber. After applying rectangular current pulses for 0.8 to 1.2 milliseconds, observe the muscle fiber contractions at the extremes.Load the fibers with 3.5 to 4.5 micromolars of the fast calcium dye Mag-Fluo-4 AM for four to five minutes in Tyrode solution. After washing the slide with Tyrode solution, let the intracellular dye de-esterify for 15 to 20 minutes in the dark. During the acquisition of calcium transients under dimmed illumination, collect and save the light signals with an oil immersion 40X objective and a photomultiplier tube connected to a digitizer.In the acquisition software, ensure a scale of zero to 200 arbitrary units. Then, adjust the size of the excitation spot and the gain of the photomultiplier tube to set the resting fluorescence, or F-rest, to 10 arbitrary units. For analyzing the recordings, set a low-pass filter to the whole trace at one kilohertz.Then, adjust the peak to calculate the F-rest in one second of the trace. Adjust the F-rest to zero and measure the peak sarcoplasmic calcium transient's amplitude. Measure the rise time from 10%to 90%of the amplitude, the duration at half-maximum, and the decay time from 90%to 10%of the amplitude.Then, estimate the decay kinetics according to a fit with the bi-exponential function. Calculate the peak calcium concentration in micromolar. Finally, save the values of the time constants of decay tau 1 and tau 2, and amplitudes A1 and A2.The FDB and EDL muscles showed calcium kinetics known as morphology type II Soleus muscles displayed morphology type I calcium transients.Fibers from PDQA, PL, and EHL muscles shared the type II morphology. The calcium transient's kinetic signals of PDQA, PL, and EHL were similar to the signals of FDB EDL muscles, but differed from soleus muscle signals.

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Research

JoVE Journal

Biology

77 조회수.  University of Antioquia. 시작하려면 실험용 목욕실에 깨끗한 유리 슬라이드를 설치합니다. 슬라이드에 2-3 마이크로 리터의 라미닌을 바르고 30 초 동안 말리십시오. 그런 다음 약 400 마이크로 리터의 근섬유 현탁액을 슬라이드에 붓고 섬유가 10-15 분 동안 라미닌에 부착되도록합니다.실험 챔버를 에피형광을 위해 장착된 도립 현미경의 s에 놓습니다. 섬유의 생존력을 확인하려면 실험 챔버의 양...