Nitroxyl (HNO) targets phospholamban cysteines 41 and 46 to enhance cardiac function

Figures

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  Figure 1.. HNO-induced cysteine modifications. (a) The reaction of HNO with thiols. (b) Potential HNO-induced modifications of PLN: inter- or intramolecular disulfide and/or sulfinamide formation.

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  Figure 2.. Sulfinamide signals on WT-PLN and its variants. (a–c) Selected region of 15N-edited 1H 2D-NMR spectra showing C41,46A-PLN (a; 60 µM, single Cys at position 36), C36,46A-PLN (b; 60 µM, single Cys at position 41; red), and C36,41A-PLN (c; 60 µM, single Cys at position 46; blue) upon treatment with the H15NO donor, 15N-2-MSPA (1 mM) in phosphate buffer containing DPC (pH 7.4) at 37°C for 30 min. (d) Overlay of H15NO-derived sulfinamide signals. (e–g) Selected region of 15N-edited 1H 1D-NMR spectra showing C41,46A-PLN (e; 60 µM, single Cys at position 36), C36,46A-PLN (f; 60 µM, single Cys at position 41), and C36,41A-PLN (g; 60 µM, single Cys at position 46) upon treatment with the H15NO donor, 15N-2-MSPA (1 mM) in phosphate buffer containing DPC (pH 7.4) at 37°C for 30 min. (h–j) Selected region of 15N-edited 1H 2D-NMR spectra showing C36A-PLN (h; 60 µM, Cys at positions 41 and 46; green), C46A-PLN (i; 60 µM, Cys at positions 36 and 41; blue), and WT-PLN (j; 60 µM; red) upon treatment with the H15NO donor, 15N-2-MSPA (1 mM) in phosphate buffer containing DPC (pH 7.4) at 37°C for 30 min. (k) Overlay of H15NO-derived sulfinamide signals. (l–n) Selected region of 15N-edited 1H 1D-NMR…

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  Figure 3.. Sulfinamide formation on WT-PLN at different concentrations of HNO donor. (a and b) Comparison of the sulfinamide signals generated on WT-PLN (60 µM) upon treatment with 1 mM (red) or 100 µM (blue) 15N-2-MSPA by 15N-edited 1H 2D-NMR (a) and 15N-edited 1H 1D-NMR analysis (b).

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  Figure 4.. Sulfinamide formation on C36,41A-PLN at different concentrations of HNO donor. (a and b) Comparison of the sulfinamide signals generated on C36,41A-PLN (60 µM, single Cys at position 46) upon treatment with 1 mM (red) or 100 µM (blue) 15N-2-MSPA by 15N-edited 1H 2D-NMR (a) and 15N-edited 1H 1D-NMR analysis (b).

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  Figure 5.. Sulfinamide formation on C36,46A-PLN at different concentrations of HNO donor. (a and b) Comparison of the sulfinamide signals generated on C36,46A-PLN (60 µM, single Cys at position at 41) upon treatment with 1 mM (red) or 100 µM (blue) 15N-2-MSPA by 15N-edited 1H 2D-NMR (a) and 15N-edited 1H 1D-NMR analysis (b).

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  Figure 6.. Sulfinamide formation on C36A-PLN at different concentrations of HNO donor. (a–d) Comparison of the sulfinamide signals generated on C36A-PLN (60 µM, Cys at positions 41 and 46) upon treatment with 1 mM (red) or 100 µM (blue) 15N-2-MSPA by 15N-edited 1H 2D-NMR (a) and 15N-edited 1H 1D-NMR analysis (b). Comparison of the sulfinamide signals generated on C46A-PLN (60 µM, Cys at positions 36 and 41) upon treatment with 1 mM (red) or 100 µM (blue) 15N-2-MSPA by 15N-edited 1H 2D-NMR (c) and 15N-edited 1H 1D-NMR analysis (d).

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  Figure 7.. PLN cysteine residues 41 and 46 are important for the functional uncoupling of PLN from SERCA2a by HNO. High Five insect cell microsomes containing SERCA2a coexpressed with PLN single cysteine constructs (C41,46A-PLN [single Cys at position at 36; left], C36,46A-PLN [single Cys at position at 41; middle], C36,41A-PLN [single Cys at position at 46; right]) were suspended (0.2 mg total protein/ml) in 250 mM sucrose and 10 mM imidazole, pH 7.0, treated with either vehicle (squares), anti-PLN monoclonal antibody 2D12 (circles), or 100 µM AS (triangles) and incubated at room temperature for 10 min, after which the treated microsomes were assayed for [Ca2+]-dependent ATPase activity at 37°C. The data are shown normalized to their respective maximas to better illustrate the AS/HNO-dependent shift in the [Ca2+]-dependent activity curve. Symbols are the average of three experiments, and the error bars represent the SD.

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  Figure 8.. Blocking cysteines prevents HNO-induced SERCA2a activation and abates increase in sarcomere shortening in cardiomyocytes. (a) Cardiomyocytes resuspended in Ca2+-free assay buffer at pH 7.0 (0.5 mg total protein/ml), were incubated with vehicle or CPM (5 µM) for 15 min at room temperature. Following treatment with 0 or 100 µM AS/HNO, the samples were assayed for [Ca2+]-dependent ATPase activity at 1 µM [Ca2+]free at 37°C. The data were normalized with respect to the activity of control samples (n = 4; *, P < 0.05). (b–d) Summary data of the impact of (b) AS/HNO (500 µM; n = 5 cells for each group; *, P < 0.05), (c) CPM (20 nM) followed by AS/HNO (500 µM; n = 9–14 cells for each group obtained from four to five different mice; P = NS), and (d) CPM (20 nM) followed by ISO (10 nM; n = 10 cells for each group obtained from four different mice; ***, P < 0.001 versus basal, ***, P < 0.001 versus CPM alone) on fractional sarcomere shortening (FS) expressed as fractional increase from diastolic levels for WT cardiomyocytes.