The denitrosylase SCoR2 controls cardioprotective metabolic reprogramming
- Medical Scientist Training Program, Case Western Reserve University School of Medicine, United States
- Institute for Transformative Molecular Medicine, Department of Medicine, Case Western Reserve University School of Medicine, United States
- Cardiovascular Research Institute, Case Western Reserve University School of Medicine, United States
- Department of Pharmacology, Weill Cornell Medicine, United States
- Department of Surgery, Duke University School of Medicine, United States
- Department of Medicine, Duke University School of Medicine, United States
- Harrington Discovery Institute, University Hospitals Cleveland Medical Center, United States
Figures
Figure 1 with 1 supplement
Knockout of SCoR2 protects from myocardial injury.
(A) Representative myocardial infarct staining after ischemia–reperfusion (I/R) in SCoR2+/+ (+/+) and SCoR2-/- (-/-) mice, taken from the same anatomical plane. Infarcted necrotic tissue is white, the area at risk is red, and tissue with normal perfusion is dark blue. (B) Quantification of the myocardial infarct size after I/R injury (24 hr reperfusion). AON (area of necrosis) is expressed as a percentage of the LV (left ventricle) and AAR (area at risk). (C) Quantification of left ventricular function after I/R injury (24 hr reperfusion). Ejection fraction (EF) and fractional shortening (FS) were determined by echocardiography. (D) Left ventricular internal diameter at end systole (LVID-s) was measured in SCoR2+/+ and SCoR2-/- mice after I/R injury (24 hr reperfusion). (A–D) N = 5 +/+, 6 -/- mice. (E) Serum troponin-1 concentration in +/+ and -/- mice after I/R injury (4 hr reperfusion); N = 3–4 mice/condition. (F) Serum lactate dehydrogenase (LDH) concentration in +/+ and -/- mice after I/R injury (4 hr reperfusion) normalized to +/+ sham; N=4–5 mice/condition. (G) Quantification of post-MI survival at 4 hr post-reperfusion in -/- (N = 27) and +/+ (N = 30) mice. 12/30 +/+ mice and 5/27 -/- mice did not survive at 4 hr post-reperfusion. (H, I) Quantification of TUNEL+ (apoptotic) nuclei in post-MI +/+ versus -/- myocardium at 4 hr post-reperfusion (N = 3 each), with representative images in (I). Red asterisks indicate TUNEL+ nuclei; scale bar = 50 µm. Significance in (B–D) assessed by two-tailed Student’s t-test, in (E) by one-tailed Mann–Whitney test, in (F) by one-tailed Student’s t-test, in (G) by one-sided chi-squared test, and in (H) by two-tailed Student’s t-test; *p < 0.05, **p < 0.01, ****p<0.0001, ns = not significant.
Figure 1—figure supplement 1
Characterization of SCoR2-/- mice at baseline and 24 hr post-MI.
Quantification of left ventricular contractile function (A–F), blood pressure (G, H), heart rate (I), and weight (J, K) in uninjured SCoR2+/+ (+/+) and SCoR2-/- (-/-) mice at baseline. Ejection fraction (EF (A)), fractional shortening (FS (B)), left ventricular diameter at end systole (LVID-s (C)), maximum LV pressure (D), left ventricular end diastolic pressure (LV EDP (E)), maximum change in LV pressure over time (LV dP/dt (F)), systolic blood pressure (SBP (G)), diastolic blood pressure (DBP (H)) and heart rate (HR (I)) were determined by echocardiography in N = 3–7 uninjured +/+ and -/- mice. Body weight and ratio of left ventricle (LV), right ventricle (RV), and lung dry weight to body weight were quantified in N = 4–5 +/+ and -/- mice in (J) and (K), respectively. At 24 hr post-MI, heart rate (HR (L)) and left ventricular parameters (stroke volume (SV (M)), cardiac output (CO (N)), left ventricular posterior wall at end systole/diastole (LVPW-s (O), LVPW-d (P)) and interventricular septal at end systole/diastole (IVS-s (Q), IVS-d (R))) were determined by echocardiography in N = 5–6 +/+ and -/- mice. Statistical significance in (A–R) was determined by two-tailed Mann–Whitney test; ns = not significant.
Figure 2 with 1 supplement
SCoR2 regulates protein S-nitrosylation in the mouse heart.
(A) cGMP (pmol/mg total protein) in SCoR2+/+ (+/+) and SCoR2-/- (-/-) mouse heart lysate post-I/R (1 hr reperfusion) as assessed by ELISA; N = 3 mouse hearts per group. (B) SNO-CoA (60 μM) added to +/+ mouse heart lysate (1 mg/ml) for 10 min increases protein S-nitrosylation as assessed by SNORAC and Coomassie blue staining, shown as fold increase relative to control after normalization to total protein; N = 10 +/+ mouse hearts per group. (C) In the presence of 100 μM NADPH (required for SCoR2 activity), cardiac protein S-nitrosylation is reduced, shown as fold decrease relative to SNO-CoA normalized to total protein; N = 10 +/+ mouse hearts per group. (D) Representative Coomassie-stained SDS/PAGE gel displaying SNO-proteins isolated by SNORAC following incubation of mouse heart extract with SNO-CoA alone or in combination with NADPH, as quantified in (B) and (C). Significance in (A) assessed by two-tailed Mann–Whitney test, and in (B, C) by ratio paired one-tailed Student’s t-test. **p ≤ 0.01, ****p ≤ 0.0001.
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Figure 2—source data 1
Original western blots for Figure 2D, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/106601/elife-106601-fig2-data1-v1.zip
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Figure 2—source data 2
Original files for western blot analysis displayed in Figure 2D.
- https://cdn.elifesciences.org/articles/106601/elife-106601-fig2-data2-v1.zip
Figure 2—figure supplement 1
Characterization of SCoR2 and protein S-nitrosylation in SCoR2-/- mice.
(A) Protein S-nitrosylation is regulated by four distinct classes of enzymes: NOS enzymes synthesize NO from l-arginine (Tsao et al., 2023), SNO synthase enzymes produce S-nitrosothiols (SNOs) from NO (Stone et al., 2016), S-nitrosylase (aka transnitrosylase) enzymes transfer SNO to target proteins to regulate activity, interactions, localization or stability (Johansson et al., 2017), denitrosylase enzymes (such as SCoR2) remove SNO from substrate proteins (Hausenloy and Yellon, 2013). This panel was created using Biorender.com. (B) Representative western blot showing expression of SCoR2 in hearts of +/+ and –/– mice after sham or ischemia–reperfusion (I/R) surgery. N = 4 mice per genotype. (C) NADPH-dependent SNO-CoA reductase activity measured in heart extracts from healthy untreated +/+ and -/- mice; N = 4 mice per genotype. (D, E) SNO-proteins identified via SNORAC from heart tissue isolated from healthy untreated +/+ (N = 3) and -/- (N = 4) mice. SNO-proteins shown in +Asc gel (D), total protein identified via Coomassie staining in input gel (E). Statistical significance in (C) determined by two-tailed Mann–Whitney test; *p ≤ 0.05.
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Figure 2—figure supplement 1—source data 1
JPEG file containing original western blots for Figure 2—figure supplement 1B, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/106601/elife-106601-fig2-figsupp1-data1-v1.zip
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Figure 2—figure supplement 1—source data 2
Original files for western blot analysis displayed in Figure 2—figure supplement 1B.
- https://cdn.elifesciences.org/articles/106601/elife-106601-fig2-figsupp1-data2-v1.zip
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Figure 2—figure supplement 1—source data 3
Original western blots for Figure 2—figure supplement 1D, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/106601/elife-106601-fig2-figsupp1-data3-v1.zip
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Figure 2—figure supplement 1—source data 4
Original files for western blot analysis displayed in Figure 2—figure supplement 1D.
- https://cdn.elifesciences.org/articles/106601/elife-106601-fig2-figsupp1-data4-v1.zip
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Figure 2—figure supplement 1—source data 5
Original western blots for Figure 2—figure supplement 1E, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/106601/elife-106601-fig2-figsupp1-data5-v1.zip
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Figure 2—figure supplement 1—source data 6
Original files for western blot analysis displayed in Figure 2—figure supplement 1EFigure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/106601/elife-106601-fig2-figsupp1-data6-v1.zip
Figure 3
A combined multi-omics approach identifies SCoR2-regulated SNO-proteins and metabolic pathways responsible for widespread metabolic reprogramming.
(A) S-Nitrosylated proteins in heart of SCoR2+/+ (+/+) and SCoR2-/- (-/-) mice post-I/R versus sham. Representative Coomassie-stained SDS/PAGE gel displaying SNO-proteins isolated by SNORAC using hearts of +/+ and -/- mice subjected to either sham operation or I/R (4 hr reperfusion). Ascorbate was omitted from the SNORAC assay (-Asc) as a specificity control. (B) Three coordinated screens in +/+ versus -/- mouse heart tissue, that is (1) SNORAC/MS (SCoR2-dependent S-nitrosoproteome 4 hr after I/R, elevated >1.2-fold in -/- vs. +/+) (Supplementary file 1; N = 3), (2) SCoR2 co-IP interactome (Supplementary file 1; N = 4), and (3) untargeted metabolomic screening in heart and plasma (Supplementary file 2; N = 5 per condition), converge on the proteins BDH1 and PKM2 as SCoR2 substrate SNO-proteins in the heart which alter relevant cardioprotective metabolic pathways. (C) Full list of 31 overlapping proteins identified in both screens (1) and (2), that is, the cardiac SCoR2-dependent S-nitrosoproteome and the cardiac SCoR2 interactome. Panels B and C were created using Biorender.com.
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Figure 3—source data 1
Original western blots for Figure 3A, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/106601/elife-106601-fig3-data1-v1.zip
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Figure 3—source data 2
Original files for western blot analysis displayed in Figure 3A.
- https://cdn.elifesciences.org/articles/106601/elife-106601-fig3-data2-v1.zip
Figure 4 with 1 supplement
SCoR2 regulates S-nitrosylation of BDH1 at Cys115, impacting ketolytic energy availability.
Quantification of SNO-BDH1 (A) and total BDH1 (B), relative to p97 ATPase loading control and to each other (C), in mouse heart (4 hr reperfusion); N = 8 SCoR2+/+ (+/+), N = 6 SCoR2-/- (-/-). Representative western blots shown in Figure 4—figure supplement 1A. Quantification of SNO-BDH1 (D) and total BDH1 (E), relative to p97 ATPase loading control and to each other (F), in mouse liver (1 hr reperfusion); N = 4 each. (G) HEK293 cells transfected with empty vector (EV), WT, or C115S BDH1 were treated with 250 μM SNO-CoA or vehicle for 10 min, followed by SNORAC and blotted for SNO-BDH1 and BDH1, together with loading controls SNO-p97 ATPase and input p97 ATPase. Representative of four independent experiments. (H, I) CHX pulse-chase assay, in which HEK293 cells transfected with V5-tagged BDH1 were treated with 100 μM ECNO or vehicle, in addition to 100 μg/ml CHX for 6–24 hr. BDH1 protein expression was quantified relative to t=0 (pre-CHX) in (H), and area under the curve (AUC) quantified in (I); N = 3 samples/group. (J–M) Metabolites corresponding to ketolytic energy availability in mouse heart and plasma subjected to sham or I/R injury (1 or 4 hr reperfusion), quantified as ion abundance by LC/MS-based untargeted metabolite profiling: (J) plasma acetoacetic acid, (K) heart acetoacetic acid, (L) heart acetyl-CoA, (M) heart phosphocreatine. N = 5 mice per condition per genotype. (N) Model depicting effect of SCoR2 deletion on cardiac ketone body metabolism in SCoR2-/- mice. (O–R) Human heart samples (IRB# Pro00005621, N = 13 with diagnosis of non-ischemic cardiomyopathy (NICM) and N = 13 without known cardiac pathophysiology (healthy)) subjected to SNORAC measuring SNO-BDH1 expression relative to loading control (SNO-p97 ATPase), representative SNORAC shown in (O), quantification in (P). (Q, R) SCoR2 protein expression in human heart samples (N = 7 healthy, N = 10 NICM from same cohort), as determined by western blot relative to p97 ATPase (representative image in (Q), quantified in (R)). (S–U) Human heart samples (IRB# Pro00005621, N = 8–9 with diagnosis of ischemic cardiomyopathy (ICM; pink) and N = 10 without known cardiac pathophysiology (healthy; black)) subjected to SNORAC measuring SNO-BDH1 (S) or western blot measuring SCoR2 relative to loading control (SNO-p97 ATPase or p97, respectively). Representative SNORAC/western blot gels shown in Figure 4—figure supplement 1G, H. Correlation of SNO-BDH1 (normalized to SNO-p97) versus BDH1 (normalized to p97) expression in healthy (N = 10) and ICM (N = 8) heart was assessed by simple linear regression in (U). Statistical significance in (A–C) determined by two-tailed Student’s t-test; (D–F) determined by two-tailed Mann–Whitney test; (H) determined by two-way ANOVA with Tukey’s multiple comparisons test; (I) determined by one-way ANOVA with Tukey’s multiple comparisons test; (J–L) determined by multiple independent Student’s t-tests performed between genotypes in each condition; (M, P) determined by two-tailed Mann–Whitney test; (R–T) determined by Student’s t-test; (U) determined by simple linear regression performed to identify SNO-BDH1 versus BDH1 relationship. R2 and p-value show the goodness of fit of the regression model and significance of slope difference from zero, respectively. *p ≤ 0.05, **p ≤ 0.01, ns = not significant.
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Figure 4—source data 1
Original western blots for Figure 4G, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/106601/elife-106601-fig4-data1-v1.zip
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Figure 4—source data 2
Original files for western blot analysis displayed in Figure 4G.
- https://cdn.elifesciences.org/articles/106601/elife-106601-fig4-data2-v1.zip
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Figure 4—source data 3
Original western blots for Figure 4O, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/106601/elife-106601-fig4-data3-v1.zip
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Figure 4—source data 4
Original files for western blot analysis displayed in Figure 4O.
- https://cdn.elifesciences.org/articles/106601/elife-106601-fig4-data4-v1.zip
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Figure 4—source data 5
Original western blots for Figure 4Q, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/106601/elife-106601-fig4-data5-v1.zip
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Figure 4—source data 6
Original files for western blot analysis displayed in Figure 4Q.
- https://cdn.elifesciences.org/articles/106601/elife-106601-fig4-data6-v1.zip
Figure 4—figure supplement 1
Characterization of BDH1 S-nitrosylation at conserved SNO site Cys115.
(A) Representative gel from SNORAC measuring SNO-BDH1 and western blot measuring total BDH1, relative to p97 ATPase loading control, in mouse heart (4 hr reperfusion), quantified in Figure 4A–C. (B) SNO-BDH1 assessed by SNORAC in SCoR2+/+ (+/+) and SCoR2-/- (-/-) liver tissue (N = 4 each) from mice (1 hr reperfusion), quantified in Figure 4D–F. (C) Representative Western blot, quantified in Figure 4H, I, denoting a CHX pulse-chase assay, in which HEK293 cells transfected with V5-tagged BDH1 were treated with 100 μg/ml CHX to block new protein synthesis, together with 100 μM ECNO or vehicle, with samples collected at 6–24 hr. Duplicates are shown by treatment condition. (D, E) Beta-hydroxybutyrate (β-HB) quantified as ion abundance in +/+ and -/- mouse heart and plasma by LC/MS-based untargeted metabolite profiling, N = 5 mice per condition per genotype. (F) Sequences of BDH1, or protein product of closest homology, from selected species aligned with constraint-based multiple alignment tool (COBALT); red color shows differences from the H. sapiens BDH1 sequence. Cys115 is indicated by a black arrow. T. nigroviridis protein product ID is CAG04267. Representative gel from SNORAC assessing SNO-BDH1 and SNO-PKM2 expression relative to SNO-p97 ATPase loading control (G) and from Western blot assessing BDH1, PKM2, and SCoR2 expression relative to p97 ATPase loading control (H) in human hearts without (N = 10) and with (N = 8–9) diagnosis of ischemic cardiomyopathy (ICM) (IRB # Pro00005621). Statistical significance in (D, E) determined by independent Student’s t-tests performed between genotypes at each time point.
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Figure 4—figure supplement 1—source data 1
Original western blots for Figure 4—figure supplement 1 (panel A), indicating the relevant bands.
- https://cdn.elifesciences.org/articles/106601/elife-106601-fig4-figsupp1-data1-v1.zip
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Figure 4—figure supplement 1—source data 2
Original files for western blot analysis displayed in Figure 4—figure supplement 1A (panel A).
- https://cdn.elifesciences.org/articles/106601/elife-106601-fig4-figsupp1-data2-v1.zip
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Figure 4—figure supplement 1—source data 3
Original western blots for Figure 4—figure supplement 1 (panel B), indicating the relevant bands.
- https://cdn.elifesciences.org/articles/106601/elife-106601-fig4-figsupp1-data3-v1.zip
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Figure 4—figure supplement 1—source data 4
Original files for western blot analysis displayed in Figure 4—figure supplement 1 (panel B).
- https://cdn.elifesciences.org/articles/106601/elife-106601-fig4-figsupp1-data4-v1.zip
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Figure 4—figure supplement 1—source data 5
Original western blots for Figure 4—figure supplement 1 (panel C), indicating the relevant bands.
- https://cdn.elifesciences.org/articles/106601/elife-106601-fig4-figsupp1-data5-v1.zip
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Figure 4—figure supplement 1—source data 6
Original files for western blot analysis displayed in Figure 4—figure supplement 1 (panel C).
- https://cdn.elifesciences.org/articles/106601/elife-106601-fig4-figsupp1-data6-v1.zip
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Figure 4—figure supplement 1—source data 7
Original western blots for Figure 4—figure supplement 1 (panel G), indicating the relevant bands.
- https://cdn.elifesciences.org/articles/106601/elife-106601-fig4-figsupp1-data7-v1.zip
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Figure 4—figure supplement 1—source data 8
Original files for western blot analysis displayed in Figure 4—figure supplement 1 (panel G).
- https://cdn.elifesciences.org/articles/106601/elife-106601-fig4-figsupp1-data8-v1.zip
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Figure 4—figure supplement 1—source data 9
Original western blots for Figure 4—figure supplement 1 (panel H), indicating the relevant bands.
- https://cdn.elifesciences.org/articles/106601/elife-106601-fig4-figsupp1-data9-v1.zip
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Figure 4—figure supplement 1—source data 10
Original files for western blot analysis displayed in Figure 4—figure supplement 1 (panel H).
- https://cdn.elifesciences.org/articles/106601/elife-106601-fig4-figsupp1-data10-v1.zip
Figure 5
SCoR2 regulates carbohydrate metabolism, including polyols, to provide antioxidative protection from injury via the pentose phosphate shunt pathway (PPP).
(A) Human heart samples (IRB# Pro00005621, N = 9 with diagnosis of ischemic cardiomyopathy (ICM) and N = 10 without known cardiac pathophysiology (healthy)) subjected to SNORAC measuring SNO-PKM2 relative to loading control (SNO-p97 ATPase). Representative SNORAC/Western blot gels shown in Figure 4—figure supplement 1G, H. (B–J) Metabolites quantified by ion abundance in SCoR2+/+ (+/+) and SCoR2-/- (-/-) mouse heart and plasma by LC/MS-based untargeted metabolite profiling; N = 5 each condition. (B) Heart lactate. (C–K) Metabolites organized by relationship to the PPP, as inputs (C, D), products (E–H), or polyol compounds that are categorized as downstream end products of the PPP (I, J). NADPH measured in heart lysate after 2 hr reperfusion, erythrose 4-phosphate measured after 4 hr reperfusion. (K) Recombinant SCoR2 activity quantified via NADPH consumption by spectrophotometer in the presence of canonical substrate (100 μM SNO-CoA) or carbohydrates (1 mM); [SCoR2]=186 nM, [NADPH]=100 μM. Results presented as specific activity of SCoR2 (μM substrate consumed/min/mg protein). Assay performed in triplicate. (L) Summary model showing SCoR2-mediated regulation of carbohydrate metabolism, including PPP and polyol compounds, to generate NADPH and phosphocreatine in the mouse heart. Green arrows indicate pathway upregulation in the absence of SCoR2, and red arrows indicate downregulation. Blue boxes indicate metabolic pathways, tan boxes indicate metabolites, and pink boxes indicate metabolites of particular significance. Thick black arrows indicate directions of SCoR2-regulated metabolic changes. This panel was created using Biorender.com. Statistical significance in (A) determined by Student’s t-test; (B–H, J) determined by multiple independent Student’s t-tests performed between genotypes in each condition; (I) determined by two-tailed Mann–Whitney test; (K) determined by two-tailed Mann–Whitney test between SNO-CoA condition and each carbohydrate condition (N = 3–8 independent replicates per condition). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.
Tables
Table 1
Input metabolites to the pentose phosphate pathway (PPP) via xylulose.
Relative quantity, quantified in mean ion abundance ± standard deviation by LC/MS-based untargeted metabolomic platform, as described in the Methods.
| Metabolite | Organ | SCoR2+/+ | SCoR2-/- | ||||
|---|---|---|---|---|---|---|---|
| sham | MI (1 hr) | MI (4 hr) | sham | MI (1 hr) | MI (4 hr) | ||
| Galactaric acid | Heart | 4.88E+04 +/- 2.14E+04 | 7.08E+04 +/- 3.62E+04 | 2.16E+04 +/- 1.64E+04 | 1.09E+05 +/- 2.37E+04 | 1.17E+05 +/- 2.44E+04 | 2.84E+05 +/- 2.13E+05 |
| Galactaric acid | Plasma | 4.90E+05 +/- 1.83E+05 | 3.83E+05 +/- 8.86E+04 | 8.05E+05 +/- 2.16E+05 | 6.14E+06 +/- 1.84E+06 | 5.82E+06 +/- 2.11E+06 | 1.42E+07 +/- 6.43E+06 |
| Galacturonic acid | Heart | 4.70E+04 +/- 4.03E+04 | 5.53E+04 +/- 2.38E+04 | 1.24E+05 +/- 2.36E+04 | 3.71E+05 +/- 1.63E+05 | 3.47E+05 +/- 8.03E+04 | 9.85E+05 +/- 5.72E+05 |
| Galacturonic acid | Plasma | 6.06E+07 +/- 2.18E+07 | 5.32E+07 +/- 8.19E+06 | 1.54E+08 +/- 3.76E+07 | 5.05E+08 +/- 9.52E+07 | 4.42E+08 +/- 7.53E+07 | 8.55E+08 +/- 2.53E+08 |
| Glucaric acid | Heart | 3.08E+05 +/- 1.36E+05 | 3.31E+05 +/- 9.75E+04 | 4.46E+05 +/- 1.74E+05 | 9.58E+06 +/- 4.84E+06 | 9.93E+06 +/- 2.53E+06 | 3.35E+07 +/- 2.68E+07 |
| Glucaric acid | Plasma | 2.13E+06 +/- 6.93E+05 | 2.26E+06 +/- 7.27E+05 | 5.92E+06 +/- 1.28E+06 | 1.81E+08 +/- 3.10E+07 | 1.66E+08 +/- 4.77E+07 | 2.68E+08 +/- 9.96E+07 |
| Xylose | Plasma | 1.36E+06 +/- 1.95E+05 | 1.35E+06 +/- 2.51E+05 | 1.68E+06 +/- 1.61E+05 | 3.37E+06 +/- 7.05E+05 | 2.91E+06 +/- 5.66E+05 | 4.70E+06 +/- 1.35E+06 |
| Lyxose | Plasma | 1.08E+06 +/- 1.69E+05 | 1.10E+06 +/- 1.88E+05 | 1.39E+06 +/- 1.71E+05 | 2.84E+06 +/- 6.11E+05 | 2.41E+06 +/- 4.26E+05 | 4.02E+06 +/- 1.20E+06 |
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (mouse, male and female) | Akr1a1tm1Dgen, mixed 129x C57BL/6 | Deltagen; Zhou et al., 2019 | Referred to as SCoR2-KO | |
| Genetic reagent (E. coli) | OneShot Omnima competent cells | Invitrogen | C8540-03 | |
| Genetic reagent (E. coli) | BL21-CodonPlus competent cells | Agilent | 230240 | |
| Cell line (human) | HEK293 | ATCC | CRL-1573 | |
| Transfected construct (human) | BDH1 cDNA in pDONR223 | DNAsu | Clone: HsCD00352477 | |
| Transfected construct (human) | BDH1 cDNA in pcDNA-DEST40 | This study | Mammalian expression, V5 epitope tag | |
| Transfected construct (human) | BDH1 C115S cDNA in pcDNA-DEST40 | This study | Mammalian expression, C115S mutant, V5 epitope tag | |
| Biological sample (human) | Post-mortem myocardium | Duke Human Heart Repository | ||
| Antibody | Anti-AKR1A1/SCoR2 (rabbit polyclonal) | Proteintech | 15054-1-AP | 10 µg for IP; 1:1000 for blotting |
| Antibody | Anti-PKM2 (rabbit monoclonal) | Cell Signaling | D78A4 | 1:1000 for blotting |
| Antibody | Anti-p97 (mouse monoclonal) | Fitzgerald | 10R-P104A | 1:1000 for blotting |
| Antibody | Anti-BDH1 (rabbit polyclonal) | Proteintech | 15417-1-AP | 1:1000 for blotting |
| Recombinant DNA reagent | AKR1A1 cDNA in pET21b | Anand et al., 2014 | Bacterial expression, CT 6xHis tag for purification | |
| Sequence-based reagent | forward: 5′-CGTCCAGCTCAATGTCTCCAGCAGCGAAGAGGreverse: 5′-CCTCTTCGCTGCTGGAGACATTGAGCTGGACG | This study | PCR primers for human SCoR2 C115S mutagenesis | |
| Sequence-based reagent | forward: 5′-GCAGAGATTCAACAAGTCTCCCCTCmutant reverse: 5′-GGGCCAGCTCATTCCTCCCACTCATwild-type reverse: 5′-AGCTAAGGCTCCGAGCAGTGCTAAC | Zhou et al., 2019 | PCR primers for mouse SCoR2 (Akr1a1) genotyping | |
| Peptide, recombinant protein | SCoR2/AKR1A1-6xHis | Anand et al., 2014 | Bacterial expression, CT 6xHis tag for purification | |
| Commercial assay or kit | QuikChange II site-directed mutagenesis kit | Agilent | 200523 | |
| Commercial assay or kit | Troponin-1 ELISA kit | Kamiya | KT-470 | |
| Commercial assay or kit | LDH activity assay | Sigma | MAK066-1KT | |
| Commercial assay or kit | NADP/NADPH-Glo assay | Promega | G9071 | |
| Commercial assay or kit | Cyclic GMP Complete ELISA kit | Enzo | ADI-900-164 | |
| Chemical compound, drug | Thiopropyl-Sepharose | Seth et al., 2023 | ||
| Software, algorithm | GraphPad Prism statistics software (v9) | https://www.graphpad.com/ | ||
| Other |
Additional files
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Supplementary file 1
SCoR2-dependent cardiac S-nitrosoproteome and SCoR2 interactome.
- https://cdn.elifesciences.org/articles/106601/elife-106601-supp1-v1.xlsx
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Supplementary file 2
SCoR2-dependent cardiac and plasma metabolome.
- https://cdn.elifesciences.org/articles/106601/elife-106601-supp2-v1.xlsx
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Supplementary file 3
Statistical values for all quantitative experimental results.
- https://cdn.elifesciences.org/articles/106601/elife-106601-supp3-v1.xlsx
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Supplementary file 4
Population characteristics of patients from whom heart samples were assessed.
- https://cdn.elifesciences.org/articles/106601/elife-106601-supp4-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/106601/elife-106601-mdarchecklist1-v1.pdf
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The denitrosylase SCoR2 controls cardioprotective metabolic reprogramming
eLife 14:RP106601.
https://doi.org/10.7554/eLife.106601.3
