NOX2-dependent ROS is required for HDAC5 nuclear efflux and contributes to HDAC4 nuclear efflux during intense repetitive activity of fast skeletal muscle fibers

Abstract

Reactive oxygen species (ROS) have been linked to oxidation and nuclear efflux of class IIa histone deacetylase 4 (HDAC4) in cardiac muscle. Here we use HDAC-GFP fusion proteins expressed in isolated adult mouse flexor digitorum brevis muscle fibers to study ROS mediation of HDAC localization in skeletal muscle. H(2)O(2) causes nuclear efflux of HDAC4-GFP or HDAC5-GFP, which is blocked by the ROS scavenger N-acetyl-l-cysteine (NAC). Repetitive stimulation with 100-ms trains at 50 Hz, 2/s ("50-Hz trains") increased ROS production and caused HDAC4-GFP or HDAC5-GFP nuclear efflux. During 50-Hz trains, HDAC5-GFP nuclear efflux was completely blocked by NAC, but HDAC4-GFP nuclear efflux was only partially blocked by NAC and partially blocked by the calcium-dependent protein kinase (CaMK) inhibitor KN-62. Thus, during intense activity both ROS and CaMK play roles in nuclear efflux of HDAC4, but only ROS mediates HDAC5 nuclear efflux. The 10-Hz continuous stimulation did not increase the rate of ROS production and did not cause HDAC5-GFP nuclear efflux but promoted HDAC4-GFP nuclear efflux that was sensitive to KN-62 but not NAC and thus mediated by CaMK but not by ROS. Fibers from NOX2 knockout mice lacked ROS production and ROS-dependent nuclear efflux of HDAC5-GFP or HDAC4-GFP during 50-Hz trains but had unmodified Ca(2+) transients. Our results demonstrate that ROS generated by NOX2 could play important roles in muscle remodeling due to intense muscle activity and that the nuclear effluxes of HDAC4 and HDAC5 are differentially regulated by Ca(2+) and ROS during muscle activity.

Redox-mediated signal transduction by cardiovascular Nox NADPH oxidases

“…Nox2-derived ROS in muscle fibers facilitate the export of the histone deacetylases HDAC4 and HDAC5 [129] and the subsequent increase in histone acetylation is thought to promote gene transcription. Some HDACs contain cysteines in their active center rendering them sensitive to NO and H 2 O 2 .…”

Reactive oxygen species generation is not different during isometric and lengthening contractions of mouse muscle

“…A second implication of our finding is that the sources responsible for contraction-induced ROS generation are not sensitive to stretch. Although we did not examine the source of ROS generation in the present study, others have suggested that nonmitochondrial sources (26,47) and specifically, NADPH oxidase (21,40), contribute to ROS generation during muscle contractions. While NADPH oxidase appears to be stretch sensitive in cardiac muscle cells (34), this may not be the case in skeletal muscle.…”

“…ROS oxidize CM-DCFH to form the fluorescent product CM-DCF, and the net increase in ROS over time is indicated by the increase in CM-DCF fluorescence over time (21,29,42). Therefore, we determined the "CM-DCF slope" (i.e., slope of the CM-DCF fluorescence-versus-time plot) during a resting period and during IC.…”

“…Contraction-induced ROS generation has been attributed to nonmitochondrial sources (26,47) and specifically, NADPH oxidase has been implicated (21,40). ROS production has also been reported during passive stretches of skeletal muscle fibers and cardiac myocytes (7,28,34).…”

ROS and RNS Signaling in Skeletal Muscle: Critical Signals and Therapeutic Targets

“…The transcriptional regulators histone deacetylase 4 and 5 (HDAC4, 5) are important regulating muscle plasticity and metabolism with activity. Recently, Nox2 dependent ROS has been shown to regulate the nuclear effluxes of the HDAC4, 5 after intense muscle activity(Liu, Hernandez-Ochoa, Randall, & Schneider, 2012). Finally, nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a transcription factor that regulates the transcription of over 200 genes responsible for antioxidant defenses in cell and antioxidant enzymes.…”