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

Abstract

Nitroxyl (HNO) positively modulates myocardial function by accelerating Ca²⁺ reuptake into the sarcoplasmic reticulum (SR). HNO-induced enhancement of myocardial Ca²⁺ cycling and function is due to the modification of cysteines in the transmembrane domain of phospholamban (PLN), which results in activation of SR Ca²⁺-ATPase (SERCA2a) by functionally uncoupling PLN from SERCA2a. However, which cysteines are modified by HNO, and whether HNO induces reversible disulfides or single cysteine sulfinamides (RS(O)NH₂) that are less easily reversed by reductants, remain to be determined. Using an ¹⁵N-edited NMR method for sulfinamide detection, we first demonstrate that Cys46 and Cys41 are the main targets of HNO reactivity with PLN. Supporting this conclusion, mutation of PLN cysteines 46 and 41 to alanine reduces the HNO-induced enhancement of SERCA2a activity. Treatment of WT-PLN with HNO leads to sulfinamide formation when the HNO donor is in excess, whereas disulfide formation is expected to dominate when the HNO/thiol stoichiometry approaches a 1:1 ratio that is more similar to that anticipated in vivo under normal, physiological conditions. Thus, ¹⁵N-edited NMR spectroscopy detects redox changes on thiols that are unique to HNO, greatly advancing the ability to detect HNO footprints in biological systems, while further differentiating HNO-induced post-translational modifications from those imparted by other reactive nitrogen or oxygen species. The present study confirms the potential of HNO as a signaling molecule in the cardiovascular system.

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.

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 spectra showing C36A-PLN (l; 60 µM, Cys at positions 41 and 46), C46A-PLN (m; 60 µM, Cys at positions 36 and 41), and WT-PLN (n; 60 µM) 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.

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).

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).

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).

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).

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.

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.