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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.


Related in: MedlinePlus

Differential OGT and OGA subcellular localization and myofilament interactions in control and diabetic myocardium. Representative transmission electron microscopy images of control (A) and STZ diabetic rat myocardium (B). Ultrathin sections were examined with immunoelectron microscopy with primary antibodies (anti-OGT AL-28 and anti-OGA), and secondary antibodies gold-labeled (anti-rabbit, 12 nm; anti-chicken, 6 nm). Z-line, green arrowhead; OGT, purple circles; OGA, red circles; OGT and OGA in close vicinity, pink circles; OGT, purple arrowhead; and OGA, red arrowhead. Quantification of OGT (C) and OGA (D) number of particles/field in nine fields shows an increase for OGT (2 ± 0.6 vs. 8.6 ± 2.6, *P ≤ 0.028) and OGA (9.4 ± 2.9 vs 37.6 ± 12.6, *P ≤ 0.05) immunoelectron microscopy in STZ diabetic myocardium. Images were analyzed in ImageJ software (NIH). Representative coimmunoprecipitations (co-IP) of OGT (E) and OGA (F), followed by Western blots (WB) for α-sarcomeric actin, Tm, and MLC 1. A fraction of the inputs from controls (C1, C2), STZ (S1, S2), and agarose beads with no antibody (M) or isotype-specific normal antibody + agarose beads (1°) were included. G: Analysis of integrated signal density of myofilament immunoreactivity normalized to total IP OGT displayed a trend toward increased interactions with Tm and MLC 1. H: OGA co-IP shows that diabetic STZ rats display several fold increased associations with α-actin, α-Tm, and MLC 1. *P ≤ 0.05.
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Figure 5: Differential OGT and OGA subcellular localization and myofilament interactions in control and diabetic myocardium. Representative transmission electron microscopy images of control (A) and STZ diabetic rat myocardium (B). Ultrathin sections were examined with immunoelectron microscopy with primary antibodies (anti-OGT AL-28 and anti-OGA), and secondary antibodies gold-labeled (anti-rabbit, 12 nm; anti-chicken, 6 nm). Z-line, green arrowhead; OGT, purple circles; OGA, red circles; OGT and OGA in close vicinity, pink circles; OGT, purple arrowhead; and OGA, red arrowhead. Quantification of OGT (C) and OGA (D) number of particles/field in nine fields shows an increase for OGT (2 ± 0.6 vs. 8.6 ± 2.6, *P ≤ 0.028) and OGA (9.4 ± 2.9 vs 37.6 ± 12.6, *P ≤ 0.05) immunoelectron microscopy in STZ diabetic myocardium. Images were analyzed in ImageJ software (NIH). Representative coimmunoprecipitations (co-IP) of OGT (E) and OGA (F), followed by Western blots (WB) for α-sarcomeric actin, Tm, and MLC 1. A fraction of the inputs from controls (C1, C2), STZ (S1, S2), and agarose beads with no antibody (M) or isotype-specific normal antibody + agarose beads (1°) were included. G: Analysis of integrated signal density of myofilament immunoreactivity normalized to total IP OGT displayed a trend toward increased interactions with Tm and MLC 1. H: OGA co-IP shows that diabetic STZ rats display several fold increased associations with α-actin, α-Tm, and MLC 1. *P ≤ 0.05.

Mentions: Next, we analyzed OGT and OGA by double immunoelectron microscopy in hearts from STZ diabetic and control rats. Immunoelectron microscopy data fully corroborated the evidence obtained with the immunofluorescence approach, confirming the redistribution of both enzymes within the sarcomere. Indeed, similar to O-GlcNAc, OGT immunogold-labeled particles were mainly located at the Z-disk in normal hearts (Fig. 5A, top panel), whereas they were more diffused along the A-band in diabetic hearts (Fig. 5B, top panel). Instead of being localized mainly at the A-band (normal hearts), OGA formed sizable clusters in the vicinity of the Z-disk (diabetic hearts; Fig. 5B, bottom panel). Finally, we determined the frequency of OGT and OGA immunogold-labeled particles, examining 9–10 random fields of normal or diabetic myocardium and quantifying OGT (12 nm, purple circle) and OGA (6 nm, red circle) immunogold-labeled particles confined to the myofilament apparatus. Our approach revealed that both O-GlcNAc cycling enzymes were detected at higher frequency in diabetic myocardium (Fig. 5C and D). Taken together, these data suggest that, in analogy to cardiac kinases and phosphatases (39), mislocalized OGT and OGA activities can affect the function of contractile or regulatory proteins, or both.


Removal of Abnormal Myofilament O -GlcNAcylation Restores Ca 2+ Sensitivity in Diabetic Cardiac Muscle
Differential OGT and OGA subcellular localization and myofilament interactions in control and diabetic myocardium. Representative transmission electron microscopy images of control (A) and STZ diabetic rat myocardium (B). Ultrathin sections were examined with immunoelectron microscopy with primary antibodies (anti-OGT AL-28 and anti-OGA), and secondary antibodies gold-labeled (anti-rabbit, 12 nm; anti-chicken, 6 nm). Z-line, green arrowhead; OGT, purple circles; OGA, red circles; OGT and OGA in close vicinity, pink circles; OGT, purple arrowhead; and OGA, red arrowhead. Quantification of OGT (C) and OGA (D) number of particles/field in nine fields shows an increase for OGT (2 ± 0.6 vs. 8.6 ± 2.6, *P ≤ 0.028) and OGA (9.4 ± 2.9 vs 37.6 ± 12.6, *P ≤ 0.05) immunoelectron microscopy in STZ diabetic myocardium. Images were analyzed in ImageJ software (NIH). Representative coimmunoprecipitations (co-IP) of OGT (E) and OGA (F), followed by Western blots (WB) for α-sarcomeric actin, Tm, and MLC 1. A fraction of the inputs from controls (C1, C2), STZ (S1, S2), and agarose beads with no antibody (M) or isotype-specific normal antibody + agarose beads (1°) were included. G: Analysis of integrated signal density of myofilament immunoreactivity normalized to total IP OGT displayed a trend toward increased interactions with Tm and MLC 1. H: OGA co-IP shows that diabetic STZ rats display several fold increased associations with α-actin, α-Tm, and MLC 1. *P ≤ 0.05.
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Figure 5: Differential OGT and OGA subcellular localization and myofilament interactions in control and diabetic myocardium. Representative transmission electron microscopy images of control (A) and STZ diabetic rat myocardium (B). Ultrathin sections were examined with immunoelectron microscopy with primary antibodies (anti-OGT AL-28 and anti-OGA), and secondary antibodies gold-labeled (anti-rabbit, 12 nm; anti-chicken, 6 nm). Z-line, green arrowhead; OGT, purple circles; OGA, red circles; OGT and OGA in close vicinity, pink circles; OGT, purple arrowhead; and OGA, red arrowhead. Quantification of OGT (C) and OGA (D) number of particles/field in nine fields shows an increase for OGT (2 ± 0.6 vs. 8.6 ± 2.6, *P ≤ 0.028) and OGA (9.4 ± 2.9 vs 37.6 ± 12.6, *P ≤ 0.05) immunoelectron microscopy in STZ diabetic myocardium. Images were analyzed in ImageJ software (NIH). Representative coimmunoprecipitations (co-IP) of OGT (E) and OGA (F), followed by Western blots (WB) for α-sarcomeric actin, Tm, and MLC 1. A fraction of the inputs from controls (C1, C2), STZ (S1, S2), and agarose beads with no antibody (M) or isotype-specific normal antibody + agarose beads (1°) were included. G: Analysis of integrated signal density of myofilament immunoreactivity normalized to total IP OGT displayed a trend toward increased interactions with Tm and MLC 1. H: OGA co-IP shows that diabetic STZ rats display several fold increased associations with α-actin, α-Tm, and MLC 1. *P ≤ 0.05.
Mentions: Next, we analyzed OGT and OGA by double immunoelectron microscopy in hearts from STZ diabetic and control rats. Immunoelectron microscopy data fully corroborated the evidence obtained with the immunofluorescence approach, confirming the redistribution of both enzymes within the sarcomere. Indeed, similar to O-GlcNAc, OGT immunogold-labeled particles were mainly located at the Z-disk in normal hearts (Fig. 5A, top panel), whereas they were more diffused along the A-band in diabetic hearts (Fig. 5B, top panel). Instead of being localized mainly at the A-band (normal hearts), OGA formed sizable clusters in the vicinity of the Z-disk (diabetic hearts; Fig. 5B, bottom panel). Finally, we determined the frequency of OGT and OGA immunogold-labeled particles, examining 9–10 random fields of normal or diabetic myocardium and quantifying OGT (12 nm, purple circle) and OGA (6 nm, red circle) immunogold-labeled particles confined to the myofilament apparatus. Our approach revealed that both O-GlcNAc cycling enzymes were detected at higher frequency in diabetic myocardium (Fig. 5C and D). Taken together, these data suggest that, in analogy to cardiac kinases and phosphatases (39), mislocalized OGT and OGA activities can affect the function of contractile or regulatory proteins, or both.

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.


Related in: MedlinePlus