Mitochondrial morphology and function varies across diaphragm muscle fiber types

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Highlights

  • Mitochondrial volume density is higher in type I and IIa than type IIx/IIb fibers.

  • Mitochondria in type I and IIa fibers are more filamentous than type IIx/IIb fibers.

  • SDHmax per unitary mitochondrial volume is higher in type I and IIa than type IIx/IIb fibers.

Abstract

In diaphragm muscle (DIAm), type I and IIa fibers are recruited to accomplish breathing, while type IIx/IIb fibers are recruited only during expulsive/straining behaviors. Thus, type I and IIa DIAm fibers are much more active (duty cycle of ∼40 %) than type IIx/IIb fibers (duty cycle of <1%), which we hypothesized underlies intrinsic differences in mitochondrial structure and function. MitoTracker Green labeled mitochondria were imaged in 3-D using confocal microscopy. Mitochondrial volume density (MVD, per muscle fiber volume) was higher, and mitochondria were more filamentous in type I and IIa DIAm compared to type IIx/IIb fibers. The maximum velocity of the succinate dehydrogenase reaction (SDHmax), measured using a quantitative histochemical technique was found to be higher in type I and IIa DIAm fibers compared to type IIx/IIb fibers with and without normalizing for MVD. These results are consistent with fiber type differences in the intrinsic structural and functional properties of DIAm fibers and closely match differences in energetic demands.

Introduction

The diaphragm muscle (DIAm) in mammals is the primary muscle involved in inspiration, although it also contributes to higher force airway protective and straining behaviors. Neural control of the DIAm during inspiration involves recruitment of fatigue resistant slow and fast motor units comprising type I and IIa fibers (Sieck and Fournier, 1989; Sieck, 1991; Sieck et al., 1996; Fogarty and Sieck, 2019). In contrast, higher force airway protective and expulsive/straining behaviors of the DIAm, require recruitment of more fatigable fast motor units comprising type IIx/IIb fibers (Sieck and Fournier, 1989; Sieck, 1991; Sieck et al., 1996; Fogarty and Sieck, 2019). Because of their recruitment during breathing, type I and IIa fibers are much more active with a duty cycle of ∼40 %, whereas type IIx/IIb fibers are infrequently activated with a duty cycle of <1% (Sieck and Fournier, 1989; Sieck, 1991; Fogarty and Sieck, 2019). The varying levels of activity have markedly different energetic requirements for mitochondrial support.

Previously, we used 2-D electron microscopy (EM) to estimate mitochondrial volume densities in type identified DIAm fibers and found that type I and IIa DIAm fibers have higher mitochondrial volume densities than type IIx/IIb fibers (Sieck et al., 1998). These estimates of mitochondrial volume densities were based on a very small sample of type-identified fibers per DIAm due to the limitations of EM. In the present study, we developed a technique to label mitochondria in DIAm fibers using MitoTracker, followed by 3-D imaging and reconstruction using confocal microscopy. Using this technique, we were able to evaluate mitochondrial volume density and morphology in a much larger sample of type identified DIAm fibers. Furthermore, in the same DIAm fibers we were able to determine mitochondrial function by measuring the maximum velocity of the succinate dehydrogenase reaction (SDHmax) using a quantitative histochemical technique (Sieck et al., 1986; Blanco et al., 1988). In previous studies using this technique, we found that type I and IIa DIAm fibers have higher SDHmax than IIx/IIb fibers (Sieck et al., 1986; Enad et al., 1989; Sieck et al., 1989, 1995; Sieck et al., 1996; Lattari et al., 1997; Fogarty et al., 2020). However, in these previous studies, we did not assess whether fiber type differences in SDHmax were associated with mitochondrial volume density or morphology.

In skeletal muscles in mice, mitochondria in type I and IIa fibers are more filamentous compared to type IIx/IIb fibers (Mishra et al., 2015). In primary human skeletal muscle cells (hSkMCs), we found that mitochondrial fusion induced by vitamin D (1α,25(OH)2D3) is associated with increased O2 consumption rate determined by respirometry (Ryan et al., 2016). Recently, in human airway smooth muscle cells exposed to tumor necrosis factor α (TnFα), we found that mitochondrial fragmentation is associated with reduced maximum O2 consumption rate even when normalized for changes in mitochondrial volume (Delmotte et al., 2017; Delmotte and Sieck, 2019). These results suggest there are intrinsic differences in mitochondrial function that depend on mitochondrial morphology. The purpose of the present study was to determine mitochondrial volume, mitochondrial morphology, and SDHmax in the same type identified DIAm fibers. We hypothesized that in type I and IIa DIAm fibers: 1) mitochondrial volume densities are higher, 2) mitochondrial morphology is more filamentous, and 3) intrinsic SDHmax (normalized for mitochondrial volume) is higher as compared to type IIx/IIb fibers, due to increased fusion of mitochondrial networks.

Section snippets

Ethical approval

All procedures were performed in accordance with the American Physiological Society's Animal Care Guidelines, the National Institutes of Health (NIH) guide for the use and care of laboratory animals (NIH Publications #8023, revised 1978) and were approved by the Institutional Animal Care and Use Committee (IACUC) at Mayo Clinic.

Experimental animals

In the present study, 6 pathogen-free Sprague-Dawley rats (300−370 g) (3 males and 3 females) were obtained from Envigo (Indianapolis, IN) and allowed at least 1 week to

DIAm fiber type classification

Classification of type I and IIa DIAm fibers in the rat was unambiguous with clear immunoreactivity for the anti-MyHCSlow and anti-MyHC2A antibodies, respectively (Fig. 1A). However, a clear distinction between type IIx and IIb DIAm fibers was not possible based on immunoreactivity for the anti-MyHC2X and anti-MyHCAll-but-2X antibodies. We were unable to validate an anti-MyHC2B antibody for use in rat DIAm fibers. Some DIAm fibers that displayed immunoreactivity for the anti-MyHC2X antibody

Discussion

The major novel findings of the present study are: i) mitochondrial volume density is higher in type I and type IIa compared to type IIx/IIb fibers, ii) mitochondria in type I and IIa fibers are more filamentous than in type IIx/IIb fibers, iii) SDHmax reflects the maximum respiratory capacity of DIAm fibers and is higher in type I and type IIa fibers compared to type IIx/IIb fibers, iv) SDHmax per mitochondrial volume is higher in type I and IIa fibers than type IIx/IIb fibers, reflecting

Funding

This work was supported by National Institutes of Health grants R01-AG044615 (GCS), R01-HL146114 (GCS) and T32-HL105355 (ADB).

CRediT authorship contribution statement

Alyssa D. Brown: Conceptualization, Methodology, Formal analysis, Investigation, Writing - original draft. Matthew J. Fogarty: Conceptualization, Methodology, Formal analysis, Investigation, Writing - review & editing. Gary C. Sieck: Conceptualization, Methodology, Formal analysis, Investigation, Writing - review & editing.

Declaration of Competing Interest

None of the authors has any conflicts of interest, real nor perceived, to disclose.

Acknowledgements

We would like to thank Rebecca Macken, Yun-Hua Fang, Dr. Philippe Delmotte, and Dr. Wen-Zhi Zhan for their assistance in this project.

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