Unique hexosaminidase reduces metabolic survival signal and sensitizes cardiac myocytes to hypoxia/reoxygenation injury - PubMed (original) (raw)

Unique hexosaminidase reduces metabolic survival signal and sensitizes cardiac myocytes to hypoxia/reoxygenation injury

Gladys A Ngoh et al. Circ Res. 2009.

Abstract

Metabolic signaling through the posttranslational linkage of N-acetylglucosamine (O-GlcNAc) to cellular proteins represents a unique signaling paradigm operative during lethal cellular stress and a pathway that we and others have recently shown to exert cytoprotective effects in vitro and in vivo. Accordingly, the present work addresses the contribution of the hexosaminidase responsible for removing O-GlcNAc (ie, O-GlcNAcase) from proteins. We used pharmacological inhibition, viral overexpression, and RNA interference of O-GlcNAcase in isolated cardiac myocytes to establish its role during acute hypoxia/reoxygenation. Elevated O-GlcNAcase expression significantly reduced O-GlcNAc levels and augmented posthypoxic cell death. Conversely, short interfering RNA directed against, or pharmacological inhibition of, O-GlcNAcase significantly augmented O-GlcNAc levels and reduced posthypoxic cell death. On the mechanistic front, we evaluated posthypoxic mitochondrial membrane potential and found that repression of O-GlcNAcase activity improves, whereas augmentation impairs, mitochondrial membrane potential recovery. Similar beneficial effects on posthypoxic calcium overload were also evident. Such changes were evident without significant alteration in expression of the major putative components of the mitochondrial permeability transition pore (ie, voltage-dependent anion channel, adenine nucleotide translocase, cyclophilin D). The present results provide definitive evidence that O-GlcNAcase antagonizes posthypoxic cardiac myocyte survival. Moreover, such results support a renewed approach to the contribution of metabolism and metabolic signaling to the determination of cell fate.

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Conflict of interest statement

Disclosures: None.

Figures

Figure 1

Figure 1

Myocytes (n>/=5/group) were infected with AdGFP or Ad_O_-GlcNAcase (0 or 100 MOI) 48 hours prior to protein isolation or hypoxia-reoxygenation. A) Representative immunoblot of _O_-GlcNAcase protein shows significant elevation in _O_-GlcNAcase levels following Ad_O_-GlcNAcase infection. Representative immunoblot (B) and Densitometric analysis (C) of _O_-GlcNAc levels. Ad_O_-GlcNAcase significantly reduced _O_-GlcNAc levels. As expected, multiple immunopositive bands appear because the _O_-GlcNAc modification occurs on numerous proteins throughout the cell. D) _O_-GlcNAcase overexpression exacerbated post-hypoxic cardiac myocyte damage according to LDH release. E) _O_-GlcNAcase overexpression aggravated post-hypoxic injury according to propidium iodide positivity (n=4/group). *p<0.05 vs. 0 MOI Ad_O_-GlcNAcase or 100 MOI AdGFP.

Figure 2

Figure 2

NRCMs were subjected to pharmacologic repression of O-GlcNAcase activity. A) Representative immunoblots for _O_-GlcNAc levels following PUGNAc treatment (n=6/group) show a significant increase in _O_-GlcNAc levels compared to Vehicle. Multiple bands occur because _O_-GlcNAc is a post-translational modification. B) Densitometric analyses of _O_-GlcNAc western blots show significantly elevated _O_-GlcNAc levels compared to Vehicle. C) _O_-GlcNAcase inhibition with PUGNAc diminished post-hypoxic injury in NRCMs (according to LDH release) compared with Vehicle. D) _O_-GlcNAcase inhibition with PUGNAc reduced post-hypoxic injury (per PI positivity) compared with Vehicle. *p<0.05 vs. Vehicle.

Figure 3

Figure 3

A) _O_-GlcNAcase message knockdown (RNAi) significantly reduced _O_-GlcNAcase protein levels compared with Scr RNAi. B) Representative immunoblot for lysates from Scr vs. _O_-GlcNAcase RNAi NRCMs showing augmented _O_-GlcNAc levels compared to Scr. C) Densitometric analysis of _O_-GlcNAc immunoblots showed significant increase in _O_-GlcNAc levels for _O_-GlcNAcase RNAi compared with Scr RNAi. _O_-GlcNAcase RNAi-treated NRCMs (n=6/group) were more resistant to hypoxia-induced injury according to LDH release (D) and, PI positivity (E) compared to Scr RNAi. *p< 0.05 vs. Scr RNAi.

Figure 4

Figure 4

Assessment of sensitivity to loss of mitochondrial membrane potential in NRCMs overexpressing _O_-GlcNAcase (Ad_O_-GlcNAcase) or with inhibition of _O_-GlcNAcase (PUGNAc) following hypoxia. TMRM fluorescence was used to indicate mitochondrial membrane potential (n>/=6/group). A) In post-hypoxic myocytes, _O_-GlcNAcase overexpression (Ad_O_-GlcNAcase) exacerbated mitochondrial membrane potential loss. B) Quantification of the average relative fluorescence intensity for AdGFP and _O_-GlcNAcase overexpression (Ad_O_-GlcNAcase) showed significantly reduced recovery of mitochondrial membrane potential for Ad_O_-GlcNAcase-treated compared to AdGFP-treated cells. C) _O_-GlcNAcase inhibition (PUGNAc) attenuated the loss of mitochondrial membrane potential compared to Vehicle. D) Quantification of the average relative fluorescence intensity for Vehicle and _O_-GlcNAcase inhibition (PUGNAc) showed significant recovery of mitochondrial membrane potential for _O_-GlcNAcase inhibitor (PUGNAc) compared to Vehicle. Similarly, RNAi against _O_-GlcNAcase improved post-hypoxic mitochondrial membrane potential recovery (E&F).

Figure 5

Figure 5

Expression of putative molecular components of mitochondrial permeability transition pore (mPTP). Expression of CypD (A), ANT (B), and VDAC (C) were not significantly affected by genetic overexpression, pharmacologic inhibition, or RNAi.

Figure 6

Figure 6

Evaluation of calcium overload in post-hypoxic cardiac myocytes using Rhod-2AM ((n>/=6/group). Myocytes were treated with AdGFP or Ad_O_-GlcNAcase (A&B), or, Vehicle or PUGNAc (C&D). Following hypoxia myocytes undergo progressive calcium overload. Genetic overexpression of _O_-GlcNAcase exaggerates, while pharmacologic inhibition of _O_-GlcNAcase attenuates post-hypoxic calcium overload.

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