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Removal of Abnormal Myofilament O -GlcNAcylation Restores Ca 2+ Sensitivity in Diabetic Cardiac Muscle

View Article: PubMed Central - PubMed

ABSTRACT

Contractile dysfunction and increased deposition of O-linked β-N-acetyl-d-glucosamine (O-GlcNAc) in cardiac proteins are a hallmark of the diabetic heart. However, whether and how this posttranslational alteration contributes to lower cardiac function remains unclear. Using a refined β-elimination/Michael addition with tandem mass tags (TMT)–labeling proteomic technique, we show that CpOGA, a bacterial analog of O-GlcNAcase (OGA) that cleaves O-GlcNAc in vivo, removes site-specific O-GlcNAcylation from myofilaments, restoring Ca2+ sensitivity in streptozotocin (STZ) diabetic cardiac muscles. We report that in control rat hearts, O-GlcNAc and O-GlcNAc transferase (OGT) are mainly localized at the Z-line, whereas OGA is at the A-band. Conversely, in diabetic hearts O-GlcNAc levels are increased and OGT and OGA delocalized. Consistent changes were found in human diabetic hearts. STZ diabetic hearts display increased physical interactions of OGA with α-actin, tropomyosin, and myosin light chain 1, along with reduced OGT and increased OGA activities. Our study is the first to reveal that specific removal of O-GlcNAcylation restores myofilament response to Ca2+ in diabetic hearts and that altered O-GlcNAcylation is due to the subcellular redistribution of OGT and OGA rather than to changes in their overall activities. Thus, preventing sarcomeric OGT and OGA displacement represents a new possible strategy for treating diabetic cardiomyopathy.

No MeSH data available.


Removing abnormal O-GlcNAcylation restores myofilament Ca2+ sensitivity in diabetic cardiac muscle. A: O-GlcNAc Western blots (WB) demonstrate increased O-GlcNAcylation on STZ diabetic heart homogenates. B: Steady-state force-[Ca2+]o relationship in skinned diabetic and control cardiac STZ diabetic muscles pre-CpOGA (n = 5) and STZ post-CpOGA (n = 4), control pre-CpOGA (n = 6) and control post-CpOGA (n = 5). Comparison of ECa2+50 for all groups (lower panel). Maximal force (Fmax) and Hill coefficient (n). C: Proteomic identification and quantification of site-specific O-GlcNAc changes that are statistically significant (generalized linear model approach) on CpOGA treatment of STZ diabetic myofilaments but not on CpOGA treatment of control myofilaments. Highlighted in red rectangles are the sites that are significantly more O-GlcNAcylated in diabetic hearts. The horizontal line in the middle of each box indicates the median; the top and bottom borders of the box mark the 75th and 25th percentiles, respectively, the whiskers mark the 90th and 10th percentiles, and the • indicate outliers. Statistical analysis used moderated t-statistics, log-odds ratios of differential expression (B statistics), and raw and adjusted P values (FDR control by the Benjamini and Hochberg method). Con, control.
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Figure 1: Removing abnormal O-GlcNAcylation restores myofilament Ca2+ sensitivity in diabetic cardiac muscle. A: O-GlcNAc Western blots (WB) demonstrate increased O-GlcNAcylation on STZ diabetic heart homogenates. B: Steady-state force-[Ca2+]o relationship in skinned diabetic and control cardiac STZ diabetic muscles pre-CpOGA (n = 5) and STZ post-CpOGA (n = 4), control pre-CpOGA (n = 6) and control post-CpOGA (n = 5). Comparison of ECa2+50 for all groups (lower panel). Maximal force (Fmax) and Hill coefficient (n). C: Proteomic identification and quantification of site-specific O-GlcNAc changes that are statistically significant (generalized linear model approach) on CpOGA treatment of STZ diabetic myofilaments but not on CpOGA treatment of control myofilaments. Highlighted in red rectangles are the sites that are significantly more O-GlcNAcylated in diabetic hearts. The horizontal line in the middle of each box indicates the median; the top and bottom borders of the box mark the 75th and 25th percentiles, respectively, the whiskers mark the 90th and 10th percentiles, and the • indicate outliers. Statistical analysis used moderated t-statistics, log-odds ratios of differential expression (B statistics), and raw and adjusted P values (FDR control by the Benjamini and Hochberg method). Con, control.

Mentions: Using an MS approach, we have previously identified specific O-GlcNAcylation sites on five major cardiac myofilament proteins on normal hearts (14). We also showed that incubation of skinned cardiac muscles with GlcNAc reduces myofilament Ca2+ sensitivity, thus reproducing a hallmark of diabetic cardiac muscle (7,14). Yet, site-specific O-GlcNAcylation changes on diabetic hearts remain unknown. Nor is it clear whether removing endogenous O-GlcNAc from diabetic skinned cardiac muscles is sufficient to restore myofilament function. To address these questions, we first confirmed that O-GlcNAcylation is enhanced in protein extracts from STZ diabetic hearts (Fig. 1A). Next, we used CpOGA to remove O-GlcNAc from diabetic skinned cardiac muscles (Fig. 1B). Finally, we used a refined global quantitative proteomic technique that combines TMT labeling with β-elimination/Michael addition to quantify the O-GlcNAcylation changes in diabetic hearts and to identify the key amino acid residues where O-GlcNAc is reduced after CpOGA in STZ diabetic hearts but not in controls (Fig. 1C).


Removal of Abnormal Myofilament O -GlcNAcylation Restores Ca 2+ Sensitivity in Diabetic Cardiac Muscle
Removing abnormal O-GlcNAcylation restores myofilament Ca2+ sensitivity in diabetic cardiac muscle. A: O-GlcNAc Western blots (WB) demonstrate increased O-GlcNAcylation on STZ diabetic heart homogenates. B: Steady-state force-[Ca2+]o relationship in skinned diabetic and control cardiac STZ diabetic muscles pre-CpOGA (n = 5) and STZ post-CpOGA (n = 4), control pre-CpOGA (n = 6) and control post-CpOGA (n = 5). Comparison of ECa2+50 for all groups (lower panel). Maximal force (Fmax) and Hill coefficient (n). C: Proteomic identification and quantification of site-specific O-GlcNAc changes that are statistically significant (generalized linear model approach) on CpOGA treatment of STZ diabetic myofilaments but not on CpOGA treatment of control myofilaments. Highlighted in red rectangles are the sites that are significantly more O-GlcNAcylated in diabetic hearts. The horizontal line in the middle of each box indicates the median; the top and bottom borders of the box mark the 75th and 25th percentiles, respectively, the whiskers mark the 90th and 10th percentiles, and the • indicate outliers. Statistical analysis used moderated t-statistics, log-odds ratios of differential expression (B statistics), and raw and adjusted P values (FDR control by the Benjamini and Hochberg method). Con, control.
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Related In: Results  -  Collection

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Figure 1: Removing abnormal O-GlcNAcylation restores myofilament Ca2+ sensitivity in diabetic cardiac muscle. A: O-GlcNAc Western blots (WB) demonstrate increased O-GlcNAcylation on STZ diabetic heart homogenates. B: Steady-state force-[Ca2+]o relationship in skinned diabetic and control cardiac STZ diabetic muscles pre-CpOGA (n = 5) and STZ post-CpOGA (n = 4), control pre-CpOGA (n = 6) and control post-CpOGA (n = 5). Comparison of ECa2+50 for all groups (lower panel). Maximal force (Fmax) and Hill coefficient (n). C: Proteomic identification and quantification of site-specific O-GlcNAc changes that are statistically significant (generalized linear model approach) on CpOGA treatment of STZ diabetic myofilaments but not on CpOGA treatment of control myofilaments. Highlighted in red rectangles are the sites that are significantly more O-GlcNAcylated in diabetic hearts. The horizontal line in the middle of each box indicates the median; the top and bottom borders of the box mark the 75th and 25th percentiles, respectively, the whiskers mark the 90th and 10th percentiles, and the • indicate outliers. Statistical analysis used moderated t-statistics, log-odds ratios of differential expression (B statistics), and raw and adjusted P values (FDR control by the Benjamini and Hochberg method). Con, control.
Mentions: Using an MS approach, we have previously identified specific O-GlcNAcylation sites on five major cardiac myofilament proteins on normal hearts (14). We also showed that incubation of skinned cardiac muscles with GlcNAc reduces myofilament Ca2+ sensitivity, thus reproducing a hallmark of diabetic cardiac muscle (7,14). Yet, site-specific O-GlcNAcylation changes on diabetic hearts remain unknown. Nor is it clear whether removing endogenous O-GlcNAc from diabetic skinned cardiac muscles is sufficient to restore myofilament function. To address these questions, we first confirmed that O-GlcNAcylation is enhanced in protein extracts from STZ diabetic hearts (Fig. 1A). Next, we used CpOGA to remove O-GlcNAc from diabetic skinned cardiac muscles (Fig. 1B). Finally, we used a refined global quantitative proteomic technique that combines TMT labeling with β-elimination/Michael addition to quantify the O-GlcNAcylation changes in diabetic hearts and to identify the key amino acid residues where O-GlcNAc is reduced after CpOGA in STZ diabetic hearts but not in controls (Fig. 1C).

View Article: PubMed Central - PubMed

ABSTRACT

Contractile dysfunction and increased deposition of O-linked β-N-acetyl-d-glucosamine (O-GlcNAc) in cardiac proteins are a hallmark of the diabetic heart. However, whether and how this posttranslational alteration contributes to lower cardiac function remains unclear. Using a refined β-elimination/Michael addition with tandem mass tags (TMT)–labeling proteomic technique, we show that CpOGA, a bacterial analog of O-GlcNAcase (OGA) that cleaves O-GlcNAc in vivo, removes site-specific O-GlcNAcylation from myofilaments, restoring Ca2+ sensitivity in streptozotocin (STZ) diabetic cardiac muscles. We report that in control rat hearts, O-GlcNAc and O-GlcNAc transferase (OGT) are mainly localized at the Z-line, whereas OGA is at the A-band. Conversely, in diabetic hearts O-GlcNAc levels are increased and OGT and OGA delocalized. Consistent changes were found in human diabetic hearts. STZ diabetic hearts display increased physical interactions of OGA with α-actin, tropomyosin, and myosin light chain 1, along with reduced OGT and increased OGA activities. Our study is the first to reveal that specific removal of O-GlcNAcylation restores myofilament response to Ca2+ in diabetic hearts and that altered O-GlcNAcylation is due to the subcellular redistribution of OGT and OGA rather than to changes in their overall activities. Thus, preventing sarcomeric OGT and OGA displacement represents a new possible strategy for treating diabetic cardiomyopathy.

No MeSH data available.