Short communicationCharacterization of neutral sphingomyelinase activity and isoform expression in rodent skeletal muscle mitochondria
Introduction
Skeletal muscle is a highly dynamic and metabolically active organ. It is composed of various muscle fibers, which differ in oxidative capacity, mitochondrial content (Delp and Duan, 1996, Schiaffino and Reggiani, 2011, Schiaffino and Serrano, 2002, Tavi and Westerblad, 2011), and more importantly, susceptibility to apoptosis (Ciciliot et al., 2013). Skeletal muscle can undergo apoptosis as a result of aging (Dirks and Leeuwenburgh, 2002), prolonged periods of disuse (Powers et al., 2012), denervation, cancer, cachexia, starvation (Schwartz, 2008), and muscle myopathies such as Duchenne muscular dystrophy (Imbertet al., 1995, Millayet al., 2009, Robertet al., 2001).
There are two forms of apoptosis, extrinsic and intrinsic, with activation originating at the cell surface or the mitochondria, respectively (D'Arcy, 2019). The intrinsic pathway of apoptosis can be triggered by a variety of signals, such as high mitochondrial calcium, oxidative stress, and mitochondrial membrane permeability (D'Arcy, 2019). Ceramides, a central sphingolipid, have been extensively linked to increased mitochondrial permeability prior to apoptosis (Novgorodovet al., 2005, Regoet al., 2012, Siskindet al., 2006, Turpinet al., 2006). The hydrolysis of sphingomyelin is believed to be a primary way of generating ceramides, which is facilitated by neutral sphingomyelinase (nSMase).
Currently, four main isoforms of nSMase have been identified, nSMase1, 2, 3, and mitochondrial-associated (MA)-nSMase (Airola and Hannun, 2013). nSMase1 does not appear to posses in vivo SMase activity (Sawaiet al., 1999, Tomiuket al., 2000) and nSMase1 KO mice have no apparent metabolic or phenotypic defects or abnormalities (Zumbansen and Stoffel, 2002). nSMase2 has been the most studied isoform and previous research has demonstrated that nSMase2 is primarily found on the Golgi which translocates to the inner leaflet of the plasma membrane in response to oxidative stress (Airola and Hannun, 2013, Levyet al., 2006) and is highly expressed in the brain with limited mRNA and protein expression in skeletal muscle (Hofmannet al., 2000, Wuet al., 2010). Interestingly, despite limited expression in skeletal muscle, its absence (nSMase2 knockout) attenuated muscle degeneration and improved muscle function in the rodent model of Duchenne muscular dystrophy (Matsuzaka et al., 2020). In contrast, nSMase3 mRNA is highly expressed in skeletal muscle (Krutet al., 2006, Moylanet al., 2014) and appears to be involved in oxidant signalling (Ferreiraet al., 2010, Moylanet al., 2014). Finally, MA-nSMase is a mitochondrial specific nSMase highly expressed in brain, testis, pancreas, and epididymis but not skeletal muscle (Wu et al., 2010b) and contains a specific sequence of amino acids which tethers the protein to the outer mitochondrial membrane, and the catalytic subunit of the enzyme is on the cytosolic side, which suggests that this enzyme plays a role in generating ceramides on the outer mitochondrial membrane (Rajagopalan et al., 2015).
Fiber type isoform expression and mitochondrial localization of nSMases can serve as an important factor in ROS mediated signalling and apoptotic cell death. Importantly, different skeletal muscle types have been shown to differ in their ability to defend against oxidative stress, as slow oxidative muscles are equipped with a better antioxidant defense system to counteract ROS compared to fast glycolytic (Loureiro et al., 2016, Pinhoet al., 2017). Previous research has demonstrated that total nSMase in skeletal muscle at the whole tissue level did not differ between muscle types (Błachnio-Zabielskaet al., 2011, Moylanet al., 2014) and nSMase 2 and 3 mRNA were detected in mixed skeletal muscle, but nSMase activity and isoform expression had not been examined at the level of the mitochondria. This leads to question whether ceramide generating enzymes such as nSMase in the mitochondria of skeletal muscle play a role in the differential susceptibility to apoptosis experienced by muscle with varying fiber type compositions. Thus, the purpose of the study was to characterize total nSMase activity and individual isoform content, with a focus on nSMase2 and 3 given their putative roles in skeletal muscle, in mitochondria isolated from skeletal muscle that represent the spectrum of fiber type compositions.
Section snippets
Animals, tissue collection, and mitochondrial isolation
Nine male Wistar rats were purchased from Charles River (14 weeks of age). Animals were housed in a climate and temperature-controlled room with access to food and water ad libitum. This study was approved by the Animal Care Committee at Brock University. Animals were anaesthetized using isoflurane (2–5% at 1–2 L/min flow rate). Soleus, plantaris, extensor digitorum longus muscles were dissected from each hind limb, and diaphragm was removed after all other muscle were dissected. Subsarcolemmal
Results and discussion
To our knowledge, this is the first study to examine neutral sphingomyelinase activity and protein content in SS mitochondria isolated from skeletal muscle of varying fiber type compositions. Due to intrinsic differences between muscle fiber types, specifically type II fibers having greater sensitivity to apoptosis and oxidative stress, it was hypothesized that nSMase content and activity would be highest in SS mitochondria isolated from predominantly type II fibers compared to predominantly
Conclusion
This study is the first to report total nSMase activity and nSMase2 protein expression in SS mitochondria isolated from skeletal muscle. The role that nSMase plays in mitochondrial mediated apoptosis through its ability to generate ceramides is still largely unknown, however this study provides a foundation for future research. Further studies are needed to fully elucidate the specific contributions of nSMase2&3 isoforms in generating mitochondrial ceramides in skeletal muscle, and its
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgement
Funding for this research was supported by the Natural Sciences and Engineering Research Council (grant number 327015–06).
Authors contributions
SS performed the experiments, analyzed the data, and wrote the manuscript. JAW assisted in working up the peroxidase activity assay and edited the manuscript. PJL developed the overall study, analyzed the data, and wrote and edited the manuscript.
References (58)
- et al.
Structural basis of the sphingomyelin phosphodiesterase activity in neutral sphingomyelinase from Bacillus cereus
J. Biol. Chem.
(2006) - et al.
Neutral sphingomyelinase 2 activity and protein stability are modulated by phosphorylation of five conserved serines
J. Biol. Chem.
(2012) - et al.
Neutral sphingomyelinase 2 (nSMase2) is a phosphoprotein regulated by calcineurin (PP2B)
J. Biol. Chem.
(2010) - et al.
Calsarcin-3, a novel skeletal muscle-specific member of the calsarcin family, interacts with multiple Z-disc proteins
J. Biol. Chem.
(2002) - et al.
Abnormal calcium homeostasis in Duchenne muscular dystrophy myotubes contracting in vitro
Cell Calcium
(1995) - et al.
Oxidative stress and apoptosis
Pathophysiology
(2000) - et al.
Novel tumor necrosis factor-responsive mammalian neutral sphingomyelinase-3 is a C-tail-anchored protein
J. Biol. Chem.
(2006) - et al.
nSMase2 activation and trafficking are modulated by oxidative stress to induce apoptosis
Biochem. Biophys. Res. Commun.
(2006) - et al.
Biochemical properties of mammalian neutral sphingomyelinase 2 and its role in sphingolipid metabolism
J. Biol. Chem.
(2003) - et al.
Neutral sphingomyelinase-3 mediates TNF-stimulated oxidant activity in skeletal muscle
Redox Biol.
(2014)