rat gastrocnemius muscle fiber bundle dissection

Confocal Microscopic Analysis of Rat Muscle Mitochondria

Mitochondria form highly dynamic networks that are mainly regulated by the balance between its elongation (fusion) and fragmentation (fission)1,2, which are modulated by the expression and activity of proteins such as Mitofusin 1 and 2 (Mfn1 and Mfn2), and Optic protein atrophy 1 (Opa1), which regulate the fusion of the outer mitochondrial membrane and inner membrane, respectively1,2. Dynamin-related protein (Drp1) predominantly regulates mitochondrial fission when it is phosphorylated in the Ser6163.
In skeletal muscle, it has been well established that the mitochondrial network is arranged in structurally well-defined subpopulations based on their proximity to different cell regions (myofibrils, sarcolemma, and nuclei)4,5. Those mitochondria located right beneath the sarcolemma are called subsarcolemmal mitochondria (SSM), those located between the contractile filaments are called intermyofibrillar mitochondria (IMF), and the mitochondrial subpopulation around the nuclei are called perinuclear mitochondria network (PMN). Moreover, it has been suggested that these mitochondrial subpopulations have region-specific functions and are metabolically specialized4,5.
The maintenance of cellular energy homeostasis, which allows metabolic and contractile function, depends to a large extent on the interaction and communication on specific sites through the mitochondrial network (e.g., IMF and SSM interaction)4,6. In addition to mitochondria network interactions, the mitochondrion can also interact with other organelles, forming structural and functional complexes. In this regard, it has been shown that IMF can be located adjacent to the sarcoplasmic reticulum (SR) and in proximity to the Ca2+ release units (CRU), formed by the transverse tubules (TT)7. This fact is relevant due to the role of mitochondrial Ca2+ uptake in regulating ATP synthesis and apoptosis. Recently, a potential role in regulating cytosolic Ca2+ transients has also been suggested8.
TT are invaginations of sarcolemma that have a periodic distribution along the longitudinal axis of cardiomyocytes and skeletal muscle fibers9,10, similar to the IMF distribution5. Changes in the distribution of TT have important physiological implications, given their role in contractile function. However, these changes have been mainly evaluated in cardiomyocytes. Using Fast Fourier Transform (FFT) analysis allows the conversion of periodic signals from the distance domain to the frequency domain, resulting in an FFT spectrum that indicates the frequency and the regularity of the signal11,12,13. Although there is evidence that the organization of the mitochondrial network in skeletal muscle fibers is essential for adaptation to different metabolic conditions, as during regeneration after muscle injury14,15, most analyses are performed qualitatively.
Additionally, given that mitochondrial dysfunction has been associated with several skeletal muscle-related (e.g., disuse atrophy)2 and non-muscle diseases, particularly metabolic diseases, and the associated loss of muscle mass (i.e., atrophy)16, the quantitative evaluation of the mitochondrial network and distribution in skeletal muscle takes relevance. Recently, a significant difference in the longitudinal distribution of mitochondria of gastrocnemius muscle fibers between an obese group (Ob; Zucker fa/fa rats), and a lean group (Lean; Zucker +/+ rats) was identified through FFT17. This study demonstrated the usefulness of the FFT in analyzing the mitochondrial distribution. Therefore, this protocol presents a methodology to study mitochondria in live-skeletal muscle fibers from images obtained by fluorescence confocal microscopy. Mitochondrial density is quantified by background thresholding, and the analysis of longitudinal mitochondrial distribution by FFT analysis is also described. A workflow scheme is presented in Figure 1.

Author Spotlight: Mitochondrial Remodeling in Skeletal Muscle 

共焦点顕微鏡によるラット生体骨格筋線維のミトコンドリア密度と縦方向分布の解析

Watch this Scientific Journal Video about Rat Gastrocnemius Muscle Fiber Bundle Dissection at JoVE.com

Rat Gastrocnemius Muscle Fiber Bundle Dissection  

ラット腓腹筋繊維束解剖

まず、ラットの切除後肢を、3ミリリットルの氷冷リラックス液を含む60ミリリットルのペトリ皿に移します。次に、鉗子でアキレス腱を慎重に持ち上げます。虹彩はさみを使用して、骨から離れた筋肉を解剖します。実体顕微鏡で、腓腹筋全体を識別して分離します。先端の細い鉗子を使用して、外側の筋肉の頭を氷冷リラックス液を入れた新しいペトリ皿に移します。片方の端に鉗子で筋肉をそっと持ち、マイクロハサミを使用して筋肉を束に分けます。最後に、繊維束を2ミリリットルの氷冷リラックス液を含む新しいペトリ皿に移します。

rat-gastrocnemius-muscle-fiber-bundle-dissection

Research

JoVE Journal

Biology

Rat Gastrocnemius Muscle Fiber Bundle Dissection

556 ビュー. まず、ラットの切除後肢を、3ミリリットルの氷冷リラックス液を含む60ミリリットルのペトリ皿に移します。次に、鉗子でアキレス腱を慎重に持ち上げます。虹彩はさみを使用して、骨から離れた筋肉を解剖します。実体顕微鏡で、腓腹筋全体を識別して分離します。先端の細い鉗子を使用して、外側の筋肉の頭を氷冷リラックス液を入れた新しいペトリ皿に移し?...

ビデオ: Rat Gastrocnemius Muscle Fiber Bundle Dissection  

Analysis of the Mitochondrial Density and Longitudinal Distribution in Rat Live-Skeletal Muscle Fibers by Confocal Microscopy

The mitochondrion is an organelle that can be elongated, fragmented, and renovated according to the metabolic requirements of the cells. The remodeling of the mitochondrial network allows healthy mitochondria to meet cellular demands; however, the loss of this capacity has been related to the development or progression of different pathologies. In skeletal muscle, mitochondrial density and distribution changes are observed in physiological and pathological conditions such as exercise, aging, and obesity, among others. Therefore, the study of the mitochondrial network may provide a better understanding of mechanisms related to those conditions.
Here, a protocol for mitochondria imaging of live-skeletal muscle fibers from rats is described. Fibers are manually dissected in a relaxing solution and incubated with a fluorescent live-cell imaging indicator of mitochondria (tetramethylrhodamine ethyl ester, TMRE). The mitochondria signal is recorded by confocal microscopy using the XYZ scan mode to obtain confocal images of the intermyofibrillar mitochondrial (IMF) network. After that, the confocal images are processed by thresholding and binarization. The binarized confocal image accounts for the positive pixels for mitochondria, which are then counted to obtain the mitochondrial density. The mitochondrial network in skeletal muscle is characterized by a high density of IMF population, which has a periodic longitudinal distribution similar to that of T-tubules (TT).&#160;The Fast Fourier Transform (FFT) is a standard analysis technique performed to evaluate the distribution of TT that allows finding the distribution frequency and the level of their organization. In this protocol, the implementation of the FFT algorithm is described for the analysis of the longitudinal mitochondrial distribution in skeletal muscle.

Here, we present a protocol to analyze changes in mitochondrial density and longitudinal distribution by live-skeletal muscle imaging using confocal microscopy for mitochondrial network scanning.

The mitochondrion is an organelle that can be elongated, fragmented, and renovated according to the metabolic requirements of the cells. The remodeling of ...