Knockout of SCoR2 protects from myocardial injury.

(A) Representative myocardial infarct staining after 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-hour 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-hour 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-hour reperfusion). (A-D): N=5 +/+, 6 –/– mice. (E) Serum Troponin-1 concentration in +/+ and –/– mice after I/R injury (4-hour reperfusion); N=3-4 mice/condition. (F) Serum LDH concentration in +/+ and –/– mice after I/R injury (4-hour 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. (H, I) Quantification of TUNEL+ (apoptotic) nuclei in post-MI +/+ vs. –/– myocardium at 4 hr post-reperfusion (N=3 each), with representative images in (I). Red asterisks indicate TUNEL+ nuclei. 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, ns = not significant.

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 (1hr reperfusion) as assessed by ELISA; N=3 mouse hearts per group. (B) SNO-CoA (60μM) added to +/+ mouse heart lysate (1mg/mL) for 10min 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 100μM 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 ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

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 vs 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 (4hr reperfusion). Ascorbate was omitted from the SNORAC assay (-Asc) as a specificity control. (B): Three coordinated screens in +/+ vs. –/– mouse heart tissue, i.e. (1) SNORAC/MS (SCoR2-dependent S-nitrosoproteome 4 h after I/R, elevated >1.2-fold in –/– vs +/+) (Table S1; N=3), (2) SCoR2 co-IP interactome (Table S1; N=4), and (3) untargeted metabolomic screening in heart and plasma (Table S2; 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) i.e., the cardiac SCoR2-dependent S-nitrosoproteome and the cardiac SCoR2 interactome. Image created with BioRender.com/8bscxsc.

SCoR2 regulates S-nitrosylation of BDH1 at Cys115, impacting ketolytic energy availability.

(A-C): 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 Fig. S3A. (D-F) 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 250μM 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 4 independent experiments. (H, I): CHX pulse-chase assay, in which HEK293 cells transfected with V5-tagged BDH1 were treated with 100μM ECNO or vehicle, in addition to 100μg/mL CHX for 6-24hr. 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 (1hr or 4hr 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 Fig. S3G,H. Correlation of SNO-BDH1 (normalized to SNO-p97) vs. 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 vs. 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, *** p ≤ 0.001, **** p ≤ 0.0001, ns = not significant.

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 Fig. S3G,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 (100μM SNO-CoA) or carbohydrates (1mM); [SCoR2] = 186nM, [NADPH] = 100μM. 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. Image created with BioRender.com/mb3bycd. 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.

Input metabolites to the PPP via xylulose.

Relative quantity, quantified in mean ion abundance +/- standard deviation by LC/MS-based untargeted metabolomic platform, as described in the Methods.

Characterization of SCoR2-/- mice at baseline and 24 hours post-MI.

(A-K) 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.

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 (1), SNO synthase enzymes produce S-nitrosothiols (SNOs) from NO (2), S-nitrosylase (aka transnitrosylase) enzymes transfer SNO to target proteins to regulate activity, interactions, localization or stability (3), denitrosylase enzymes (such as SCoR2) remove SNO from substrate proteins (4). Image created with BioRender.com/htrukr3. (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.

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 Fig. 4A-C. (B) SNO-BDH1 assessed by SNORAC in SCoR2+/+ (+/+) and SCoR2-/- (–/–) liver tissue (N=4 each) from mice (1hr reperfusion), quantified in Fig. 4D-F. (C) Representative Western blot, quantified in Fig. 4H,I, denoting a CHX pulse-chase assay, in which HEK293 cells transfected with V5-tagged BDH1 were treated with 100μg/mL CHX to block new protein synthesis, together with 100μM ECNO or vehicle, with samples collected at 6-24hr. 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 black arrow. T. nigroviridis protein product ID is CAG04267. (G,H) 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.