Nitrosative stress drives heart failure with preserved ejection fraction

Abstract

Heart failure with preserved ejection fraction (HFpEF) is a common syndrome with high morbidity and mortality for which there are no evidence-based therapies. Here we report that concomitant metabolic and hypertensive stress in mice-elicited by a combination of high-fat diet and inhibition of constitutive nitric oxide synthase using Nω-nitro-L-arginine methyl ester (L-NAME)-recapitulates the numerous systemic and cardiovascular features of HFpEF in humans. Expression of one of the unfolded protein response effectors, the spliced form of X-box-binding protein 1 (XBP1s), was reduced in the myocardium of our rodent model and in humans with HFpEF. Mechanistically, the decrease in XBP1s resulted from increased activity of inducible nitric oxide synthase (iNOS) and S-nitrosylation of the endonuclease inositol-requiring protein 1α (IRE1α), culminating in defective XBP1 splicing. Pharmacological or genetic suppression of iNOS, or cardiomyocyte-restricted overexpression of XBP1s, each ameliorated the HFpEF phenotype. We report that iNOS-driven dysregulation of the IRE1α-XBP1 pathway is a crucial mechanism of cardiomyocyte dysfunction in HFpEF.

Conflict of interest statement

Competing Interests Declaration. G.G.S., T.G.G., and J.A.H. are co-inventors on a patent application (PCT/US/2017/037019) that was filed in June 2017 (provisional filed in June 2016). The patent relates to the diet used for modeling HFpEF.

Figures

Extended Data Figure 1. Systemic and cardiac phenotype of mice after five or fifteen weeks of different dietary regimens.

a, Body weight (BW) of mice of different experimental groups after five or fifteen weeks of diet (n=10 mice per group for each time point). b, Intraperitoneal glucose tolerance test (ipGTT) after five or fifteen weeks of diet (for five weeks n=10 mice per group; for fifteen weeks n=5 mice per group). c, Bar graphs depicting the area under the curve of the 5- and 15-week time points ipGTT experiment (for five weeks n=10 mice per group; for fifteen weeks n=5 mice per group). d, Systolic blood pressure (SBP) and e, diastolic blood pressure (DBP) of different experimental groups after five or fifteen weeks of treatment (n=10 mice per group for each time point). f, Percent left ventricular ejection fraction (LVEF%), g, LV global longitudinal strain (GLS), h, Ratio between mitral E wave and A wave (E/A), i, Ratio between mitral E wave and E’ wave (E/E’), j, Ratio between wet and dry lung weight (LW), k, Ratio between heart weight and tibia length (HW/TL) and l, Runninng distance during exercise exhaustion test of mice after five weeks of diet (for LVEF%, E/A ratio, E/E’ ratio, HW/TL ratio, running distance and LW wet/LW dry ratio n=10 mice per group. For LV GLS n=5 mice per group). Results are presented as mean±S.E.M. a, c-l One-way ANOVA followed by Sidak’s multiple comparisons test. b, Two-way ANOVA followed by Sidak’s multiple comparisons test. a, c-l Numbers above square brackets show significant P values. b, for five weeks 15’ ***P=0.0003 CHOW vs. HFD, ***P=0.0004 CHOW vs. HFD+L-NAME; 30’ ***P=0.0008 CHOW vs. HFD, **P=0.006 CHOW vs. HFD+L-NAME; 45’ *P=0.010 CHOW vs. HFD, ***P=0.0008 CHOW vs. HFD+L-NAME; 60’ *P=0.049 CHOW vs. HFD, **P=0.0096 CHOW vs. HFD+L-NAME. for fifteen weeks, 15’ **P=0.008 CHOW vs. HFD, ****P<0.0001 CHOW vs. HFD+L-NAME; 30’ **P=0.005 CHOW vs. HFD, ****P<0.0001 CHOW vs. HFD+L-NAME; 45’ **P=0.009 CHOW vs. HFD, ****P<0.0001 CHOW vs. HFD+L-NAME; 60’ *P=0.028 CHOW vs. HFD, **P=0.0020 CHOW vs. HFD+L-NAME.

Extended Data Figure 2. Heart morphology and vascular characterization of mice after five weeks of different dietary regimens.

a, Representative images of hematoxylin & eosin (H&E), wheat germ agglutinin (WGA), Masson’s Trichrome (MT) and lectin staining in transversal sections of left ventricle of mice of different experimental groups. Images are representative of four independently performed experiments with similar results. Scale bars: 500 μm for H&E; Scale bars: 50 μm for WGA, MT and lectin. b, WGA quantification of cardiomyocyte cross sectional area (n=4 mice per group). c, Percentage of fibrosis area of MT-stained transversal sections (n=4 mice per group). d, Myocardial capillary density (n=4 mice per group). e, Aortic pulse wave velocity (PWV) of mice of different experimental groups (n=5 mice per group). f, Representative pulse wave Doppler tracings of coronary flow in CHOW (top panels) and HFD+L-NAME (bottom panels) mice under basal condition (left panels – Isofluorane, Iso 1.5%) and after hyperemic stimulus (right panels – Iso 3%). Images are representative of five independent mice. g, Coronary flow reserve (CFR) quantification (n=5 mice per group). Results are presented as mean±S.E.M. b-d, e, g One-way ANOVA followed by Sidak’s multiple comparisons test. Numbers above square brackets show significant P values.

Extended Data Figure 3. Histological and functional analyses of skeletal muscle in mice after five weeks of different dietary regimens.

a, Representative images of hematoxylin & eosin (H&E), wheat germ agglutinin (WGA), metachromatic ATPase (ATPase), Masson’s Trichrome (MT) and Picrosirius red (PSR) staining of soleus and gastrocnemius/plantaris (G/P) from CHOW and HFD+L-NAME mice. Images are representative of three independently performed experiments with similar results. Scale bars: 50 μm. b, mRNA level of myosin isoforms (MyHC-1, MyHC-2A, MyHC-2X) of soleus and G/P from CHOW and HFD+L-NAME mice (n=5 mice per group). c, Relaxation curve of isolated soleus from CHOW and HFD+L-NAME mice (n=5 mice per CHOW group; n=6 mice per HFD+L-NAME group). In vivo forelimb d, hindlimb e, and grip force measurements of mice of different experimental groups (n=8 mice per CHOW group; n=4 mice per HFD group; n=3 mice per L-NAME group; n=6 mice per HFD+L-NAME group). f, Maximal tetanic stresses in soleus from CHOW and HFD+L-NAME mice (n=5 mice per CHOW group; n=6 mice per HFD+L-NAME group). Results are presented as mean±S.E.M. b, c, f Two-tailed unpaired Student’s t-test (CHOW vs. HFD+L-NAME soleus; CHOW vs. HFD+L-NAME G/P). d, e One-way ANOVA followed by Sidak’s multiple comparisons test. Numbers above square brackets show significant P values.

Extended Data Figure 4. Adult mouse ventricular cardiomyocyte contractility after five weeks of different dietary regimens and functional characterization and IRE1α-Xbp1s axis in ZSF1-Obese rats at twenty weeks of age.

a, Baseline sarcomeric length (n=4 mice and n=35 cells per CHOW group; n=3 mice and n=30 cells per HFD group; n=3 mice and n=30 cells per L-NAME group; n=3 mice and n=31 cells per HFD+L-NAME group). b, Time to peak (n=4 mice and n=36 cells per CHOW group; n=3 mice and n=30 cells per HFD group; n=3 mice and n=30 cells per L-NAME group; n=3 mice and n=31 cells per HFD+L-NAME group). c, Maximum return velocity (n=4 mice and n=36 cells per CHOW group; n=3 mice and n=29 cells per HFD group; n=3 mice and n=30 cells per L-NAME group; n=3 mice and n=31 cells per HFD+L-NAME group). d, Change (Δ) in sarcomeric length related to baseline (n=4 mice and n=36 cells per CHOW group; n=3 mice and n=30 cells per HFD group; n=3 mice and n=30 cells per L-NAME group; n=3 mice and n=31 cells per HFD+L-NAME group). e, Maximum departure velocity (n=4 mice and n=35 cells per CHOW group; n=3 mice and n=30 cells per HFD group; n=3 mice and n=29 cells per L-NAME group; n=3 mice and n=28 cells per HFD+L-NAME group). f, Representative tracings of cardiomyocyte contraction/relaxation during pacing. Each trace depicts one cell representative of the average for each experimental group. g, Body weigth (BW), h, Percent left ventricular ejection fraction (LVEF%), i, Ratio between mitral E wave and A wave (E/A), j, Ratio between mitral E wave and E’ wave (E/E’), k, Ratio between heart weight and tibia length (HW/TL) ratio, and l, Lung weight (LW)/TL ratio of 20-week old Wistar Kyoto (WKY) and ZSF1-Obese rats (for BW, LVEF%, E/A ratio, E/E’ ratio and LW/TL ratio n=5 rats per group; for HW/TL ratio n=5 rats per WKY group and n=4 rats per ZSF1-Obese group). m, Electrophoretic analysis of spliced (s) and unspliced (u) Xbp1 transcript in LV samples from WKY and ZSF1-Obese rats. Tunicamycin-treated neonatal rat ventricular myocytes (TUN) were used as positive control (n=3 rats per group). n, LV mRNA levels of Xbp1s from WKY and ZSF1-Obese rats (n=5 rats per group). o, Immunoblot images of pIRE1α, IRE1α and GAPDH proteins from LV samples of WKY and ZF1-Obese rats (n=5 rats per group). p, Densitometric analysis ratio between pIRE1α and IRE1α protein bands (n=5 rats per group). Results are presented as mean±S.E.M. a-e One-way ANOVA followed by Sidak’s multiple comparisons test. g-l, n, p Two-tailed unpaired Student’s t-test. Numbers above square brackets show significant P values. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 5. Functional characterization and unfolded protein response activation in mice after one week of severe transverse aortic constriction.

a, Experimental design. C57BL/6N male mice were exposed to sham (control) or severe transverse aortic constriction (sTAC) surgery (filled triangle) and evaluated after one week (empty triangle). b, Percent left ventricular ejection fraction (LVEF%), c, Ratio between mitral E wave and A wave (E/A), d, Ratio between mitral E wave and E’ wave (E/E’), e, Ratio between wet and dry lung weight (LW) and f, Ratio between heart weight and tibia length (HW/TL) of sham control and sTAC mice (n=5 mice per group). g, LV Xbp1s mRNA level from sham and sTAC mice (n=4 mice in sham group; n=5 mice in sTAC group). h, Immunoblot images of GRP94, GRP78 and GAPDH proteins in LV samples of sham and sTAC mice. Images are representative of three independently performed experiments with similar results. Arrow indicates the regulated band. i, KDEL (Lys-Asp-Glu-Leu) sequence immunofluorescence staining in LV sections of sham and sTAC mice. Hoechst stains nuclei. Scale bars: 50 μm. Images are representative of three independently performed experiments with similar results. Results are presented as mean±S.E.M. b-g Two-tailed unpaired Student’s t-test. Numbers above square brackets show significant P values. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 6. Functional characterization and IRE1α-Xbp1s axis in mice after transverse aortic constriction and phenotype of cardiomyocyte-restricted Xbp1s transgenic mice.

a, Experimental design. C57BL/6N mice were exposed to sham (control) or transverse aortic constriction (TAC) surgery (filled triangle) and followed up to five weeks (empty triangles). b, Ratio between mitral E wave and A wave (E/A), c, Ratio between mitral E wave and E’ wave (E/E’), d, Percent left ventricular ejection fraction (LVEF%), e, Ratio between heart weight and tibia length (HW/TL), and f, Ratio between lung weight and body weight (LW/BW) of different experimental groups of mice (for E/A ratio, E/E’ ratio and LVEF% n=10 mice per Pre-TAC, TAC 1wk and TAC 3wks groups; n=5 mice per TAC 5wks group; for HW/TL ratio and LW/BW ratio n=10 mice per TAC 1wk and TAC 3wks groups; n=5 mice per sham and TAC 5wks groups). g, LV Xbp1s mRNA levels from sham and TAC 3wks (n=5 mice per group). h, Immunoblot images of pIRE1α, IRE1α and GAPDH proteins from LV samples of sham and TAC 3wks mice (n=3 mice per group). i, Experimental design. Control (CTR) and Xbp1s transgenic mice (TG) were exposed to CHOW or HFD+L-NAME diet (green filled triangle). After five weeks, echocardiographic assessment was performed and doxycycline (Doxy) was removed from the drinking water to induce transgene expression (gray filled triangle). Two weeks after transgene induction (blue filled triangle), mice were subjected to functional analysis and tissue harvesting. j, LVEF% of different experimental cohorts over time (n=5 mice per CHOW CTR and CHOW TG groups; n=7 mice per HFD+L-NAME CTR and HFD+L-NAME TG groups. Each mouse was analyzed at all three time points). k, LV Xbp1s mRNA level in CTR and Xbp1s TG mice fed with CHOW or HFD+L-NAME diet for seven weeks (n=3 mice per group). l, LV mRNA levels of nppa and nppb genes and m, HW/TL at the end of the study (n=5 mice per CHOW CTR and CHOW TG groups; n=7 mice per HFD+L-NAME CTR and HFD+L-NAME TG groups). Results are presented as mean±S.E.M. b-f, One-way ANOVA followed by Sidak’s multiple comparisons test. g Two-tailed unpaired Student’s t-test. j-m Two-way ANOVA followed by Sidak’s multiple comparisons test. Numbers above square brackets show significant P values. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 7. Myocardial nitrosative stress and inflammatory markers in mice after five weeks of different dietary regimens.

a, Top: Cytokines/chemokines antibody array in plasma samples from CHOW and HFD+L-NAME mice with visual estimation of differently abundant cytokines. Each Images are representative of two independently performed experiments with similar results. Bottom: List of cytokines and chemokines represented by the antibody array membrane. b, Left ventricular (LV) TNFα, IL1β and IL6 mRNA levels of CHOW and HFD+L-NAME mice (n=5 mice per group). c, iNOS and GAPDH in LV samples of CHOW and HFD+L-NAME mice. Neonatal rat ventricular myocytes infected with mouse iNOS adenovirus (AdiNOS; 100 multiplicity of infection) were used as positive controls for iNOS bands. Arrow indicates the regulated band (n=3 mice per group). d, LV mRNA levels of eNOS of different experimental groups of mice (n=4 mice per CHOW group; n=6 mice per HFD, L-NAME and HFD+L-NAME groups). e, Immunoblot images of nNOS, eNOS and GAPDH proteins of different experimental groups of mice (n=3 mice per group). f, Immunoblot images of nitrosylated cysteines (Cys-SNO) and GAPDH proteins in LV samples of wild type (WT) and iNOS knock out (iNOS KO) mice after five weeks of CHOW and HFD+L-NAME diet (W/B: without blocking. - Asc: without ascorbate. GSNO: S-Nitrosoglutathione) (n=3 mice per group). g, Densitometric analysis ratio between Cys-SNO/GAPDH proteins (n=3 mice per group). Results are presented as mean±S.E.M. b, Two-tailed unpaired Student’s t-test. d One-way ANOVA followed by Sidak’s multiple comparisons test. g, Two-way ANOVA followed by Sidak’s multiple comparisons test. Numbers above square brackets show significant P values. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 8. iNOS overexpression in cardiomyocytes reduces IRE1α activation and Xbp1s levels without affecting cardiomyocyte viability.

a, Immunoblot images of iNOS and GAPDH proteins of neonatal rat ventricular myocytes infected with increasing multiplicity of infection (MOI) of α-galactosidase adenovirus (AdLacZ) or iNOS adenovirus (AdiNOS) for 24 hours. Images are representative of three independently performed experiments with similar results. b, iNOS mRNA levels in neonatal rat ventricular myocytes transduced with increasing MOI of AdlacZ or AdiNOS for 24 hours (n=4 biologically independent experiments). c, Medium nitrite/nitrate concentration of neonatal rat ventricular myocytes transduced with increasing MOI of AdlacZ or AdiNOS for 24 hours (n=4 biologically independent experiments). d, Lactate dehydrogenase (LDH) release in neonatal rat ventricular myocytes transduced with increasing MOI of AdlacZ or AdiNOS for 24 hours (n=3 biologically independent experiments). e, Immunoblot images of Cys-SNO, iNOS and GAPDH proteins in NRVMs transduced with AdlacZ or AdiNOS for 24 hours (100 MOI). Images are representative of three independently performed experiments with similar results. f, Immunoblot images of pIRE1α, IRE1α, iNOS and GAPDH proteins in neonatal rat ventricular myocytes transduced with increasing MOI of AdlacZ or AdiNOS in the presence or absence of tunicamycin (TUN) for 24 hours. Images are representative of three independently performed experiments with similar results. g, Xbp1s mRNA level of neonatal rat ventricular myocytes transduced with 100 MOI of AdlacZ or AdiNOS in the presence or absence of TUN for 24 hours (n=3 biologically independent experiments). Results are presented as mean±S.E.M. b-d, g One-way ANOVA followed by Sidak’s multiple comparisons test. Numbers above square brackets show significant P values. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 9. Phenotype of iNOS knockout mice after five weeks of different dietary regimens.

a, DNA genotyping of wild type (WT) and iNOS knock out (iNOS KO) mice. This signature was used for genotyping. b, Experimental design. WT and iNOS KO mice were exposed to CHOW or HFD+L-NAME diet for five weeks (filled triangle). Subsequently, mice were subjected to functional analysis and tissue harvesting (empty triangle). c, Percent left ventricular ejection fraction (LVEF%), d, Body weight (BW), e, Systolic blood pressure (SBP), f, Diastolic blood pressure (DBP) and g, Intraperitoneal glucose tolerance test (ipGTT) of different experimental groups of mice (for LVEF% n=10 mice per group; for BW, SBP, DBP and ipGTT n=5 mice per group). h, Bar graphs depicting the area under the curve of the ipGTT experiment (n=5 mice per group). i, Ratio between heart weight and tibia length (HW/TL) of different experimental groups of mice (n=5 mice per group). Results are presented as mean±S.E.M. c-i Two-way ANOVA followed by Sidak’s multiple comparisons test. c-f, h, i Numbers above square brackets show significant P values. g, 15’ ***P=0.0002 CHOW WT vs. HFD+L-NAME WT, ****P<0.0001 CHOW WT vs. HFD+L-NAME iNOS KO; 30’ ***P=0.0002 CHOW WT vs. HFD+L-NAME WT, ****P<0.0001 CHOW WT vs. HFD+L-NAME iNOS KO; 45’ ****P<0.0001 CHOW WT vs. HFD+L-NAME WT, ***P=0.0002 CHOW WT vs. HFD+L-NAME iNOS KO; 60’ ****P<0.0001 CHOW WT vs. HFD+L-NAME WT, ****P<0.0001 CHOW WT vs. HFD+L-NAME iNOS KO; 120’ **P=0.010 CHOW WT vs. HFD+L-NAME WT, **P=0.007 CHOW WT vs. HFD+L-NAME iNOS KO. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 10. Functional characterization and Xbp1s myocardial levels of mice treated with iNOS inhibitor.

a, Experimental design. C57BL/6N mice were exposed to CHOW or HFD+L-NAME diet (brown filled triangle) for five weeks and subsequently injected intraperitoneally (i.p.) with L-N6-(1-iminoethyl)lysine (L-NIL) at the dose of 80 mg/kg body weight or vehicle twice a day for three days (blue filled triangles). After that point, mice were subjected to functional analysis and tissue harvesting (red filled triangle). b, Urinary nitrite/nitrate concentration in HFD+L-NAME mice treated with vehicle or L-NIL (n=5 mice per group). c, Systolic blood pressure (SBP), d, Diastolic blood pressure (DBP), e, Percent left ventricular ejection fraction (LVEF%), f, Ratio between mitral E wave and A wave (E/A), g, Ratio between mitral E wave and E’ wave (E/E’), h, Running distance during exercise exhaustion test and i, LV mRNA levels of Xbp1s of different experimental groups of mice (for nitrite/nitrate level, SBP, DBP, LVEF%, E/A, E/E’ and running distance n=5 mice group; for Xbp1s level n=3 per group). Results are presented as mean±S.E.M. b Two-tailed unpaired Student’s t-test. c-i Two-way ANOVA followed by Sidak’s multiple comparisons test. Numbers above square brackets show significant P values.

Figure 1. Fifteen weeks of HFD+L-NAME dietary regimen in mice recapitulates key alterations of clinical HFpEF.

a, Experimental design. C57BL/6N mice were maintained on different dietary regimens (filled triangle) and followed up to fifteen weeks (empty triangles). b, Representative left ventricular (LV) M-mode echocardiographic tracings. Images are representative of fifteen independent mice. c, Percent LV ejection fraction (LVEF%) (n=15 mice per group). d, LV global longitudinal strain (GLS) (n=10 mice per group). e, Representative pulsed-wave Doppler (top) and tissue Doppler (bottom) tracings. Images are representative of fifteen independent mice. f, Ratio between mitral E wave and E’ wave (E/E’) (n=15 mice per group). g, Ratio between wet and dry lung weight (LW) (n=15 mice per group). h, Ratio between heart weight and tibia length (HW/TL) (n=15 mice per group). i, Running distance during exercise exhaustion test (n=8 mice per group). Results are presented as mean±S.E.M. c, d, f-i One-way ANOVA followed by Sidak’s multiple comparisons test. Numbers above square brackets show significant P values.

Figure 2. IRE1α-Xbp1s signaling pathway is inactive in experimental and human HFpEF and Xbp1s overexpression in cardiomyocytes ameliorates experimental HFpEF

a, Left ventricular (LV) mRNA levels of Xbp1s, Bip, ATF6, ATF4, CHOP in mice of different experimental groups (n=5 mice per group). b, Immunoblot images of PERK, ATF6 and GAPDH proteins in LV samples of CHOW and HFD+L-NAME mice (n=3 mice per group). c, Electrophoretic analysis of spliced (s) and unspliced (u) Xbp1 transcript in LV samples of CHOW and HFD+L-NAME mice. Tunicamycin-treated neonatal rat ventricular myocytes (TUN) were used as positive control. Images are representative of three independently performed experiments with similar results. d, Adult mouse ventricular myocytes (AMVMs) mRNA levels of Xbp1s, Bip, CHOP in CHOW and HFD+L-NAME mice (n=4 mice per CHOW group; n=5 mice per HFD+L-NAME group. AMVMs were isolated from individual mice). e, Immunoblot images of LV pIRE1α, IRE1α and GAPDH proteins of CHOW and HFD+L-NAME mice (n=3 mice per group). f, Densitometric analysis ratio between pIRE1α and IRE1α protein bands (n=3 mice per group). g, mRNA levels of Xbp1s and Xbp1u in human myocardial biopsies from non-failing (CTR), HFpEF and HFrEF subjects (for CTR group n=15 subjects, for HFpEF group n=13 (Xbp1s) and n=14 (Xbp1u) subjects and for HFrEF group n=15). h, Immunoblot images of pIRE1α and IRE1α proteins in human myocardial biopsies from CTR, HFpEF and HFrEF subjects (n=8 subjects per group). i, Densitometric analysis ratio between pIRE1α and IRE1α protein bands (n=8 subjects per group). j, Ratio between mitral E wave and A wave (E/A) and k, Ratio between mitral E wave and E’ wave (E/E’) of control (CTR) and Xbp1s transgenic mice (TG) fed with CHOW or HFD+L-NAME diet over time (n=5 mice per CHOW CTR and CHOW TG groups; n=7 mice per HFD+L-NAME CTR and HFD+L-NAME TG groups. Each mouse was analyzed at all three time points). l, Running distance during exercise exhaustion test and m, Ratio between wet and dry lung weight (LW) at the end of the study (n=5 mice per CHOW CTR and CHOW TG groups; n=7 mice per HFD+L-NAME CTR and HFD+L-NAME TG groups). Results are presented as mean±S.E.M. a, g, i One-way ANOVA followed by Sidak’s multiple comparisons test. d, f, Two-tailed unpaired Student’s t-test. j-m Two-way ANOVA followed by Sidak’s multiple comparisons test. Numbers above square brackets show significant P values. For gel source data, see Supplementary Fig. 1.

Figure 3. iNOS-dependent IRE1α nitrosylation in HFpEF and cardiomyocytes.

a, Left ventricular (LV) mRNA levels of iNOS of different experimental groups of mice (n=4 mice per CHOW group; n=6 mice per HFD, L-NAME and HFD+L-NAME groups b, Adult mouse ventricular myocytes (AMVMs) mRNA levels of iNOS CHOW and HFD+L-NAME mice (n=5 mice per group. AMVMs were isolated from individual mice). c, LV mRNA levels of iNOS from WKY and ZSF1-Obese rats (n=5 rats per group). d, mRNA levels of iNOS in human myocardial biopsies from non-failing (CTR), HFpEF and HFrEF subjects (n=11 subjects per CTR group, n=11 subjects per HFpEF group and n=10 subjects per HFrEF group). e, Immunoblot images of S-nitrosylated IRE1α (SNO-IRE1α), IRE1α and GAPDH proteins in LV samples of wild type (WT) and iNOS knock out (iNOS KO) mice after five weeks of CHOW or HFD+L-NAME diet. Images are representative of four independently performed experiments with similar results. (- Asc: without Ascorbate, + Asc: with Ascorbate GSNO: S-Nitrosoglutathione) f, Densitometric analysis SNO-IRE1α +Ascorbate protein bands in each group (n=4 mice per group). g, Xbp1s mRNA level of neonatal rat ventricular myocytes transduced with increasing multiplicity of infection of of α-galactosidase adenovirus (AdLacZ) or iNOS adenovirus (AdiNOS) for 24 hours (n=4 biologically independent experiments). h, Immunoblot images of SNO-IRE1α and IRE1α proteins in neonatal rat ventricular myocytes transduced with adenovirus for IRE1α wild type (AdIRE1α WT) or AdIRE1α mutated in two target nitrosylation sites (AdIRE1α M1+M2) and AdlacZ or AdiNOS for 24 hours (- Asc: No Ascorbate. GSNO: S-Nitrosoglutathione). Images are representative of three independently performed experiments with similar results. i, Densitometric analysis ratio between SNO-IRE1α and IRE1α protein band intensities (n=3 biologically independent experiments). j, Xbp1s mRNA level of neonatal rat ventricular myocytes transduced with AdIRE1α WT or AdIRE1α M1+M2 and AdlacZ or AdiNOS for 24 hours (n=5 biologically independent experiments). Results are presented as mean±S.E.M. a, d, g, i, j One-way ANOVA followed by Sidak’s multiple comparisons test. b, c Two-tailed unpaired Student’s t-test. f, Two-way ANOVA followed by Sidak’s multiple comparisons test. Numbers above square brackets show significant P values. For gel source data, see Supplementary Fig. 1.

Figure 4. iNOS genetic inhibition ameliorate the HFpEF phenotype and restores IRE1α-Xbp1s signaling pathway in HFpEF.

a, Ratio between mitral E wave and A wave (E/A), b, Ratio between mitral E wave and E’ wave (E/E’), c, Running distance during exercise exhaustion test, and d, Ratio between wet and dry lung weight (LW) of wild type (WT) and iNOS knock out (iNOS KO) mice after five weeks of CHOW or HFD+L-NAME diet (for E/A ratio, E/E’ ratio n=10 mice per group; for running distance and LW wet/LW dry ratio n=5 mice per group). e, Electrophoretic analysis of spliced (s) and unspliced (u) Xbp1 transcript in left ventricular (LV) samples of WT and iNOS KO mice after five weeks of CHOW or HFD+L-NAME diet. Tunicamycin-treated neonatal rat ventricular myocytes (TUN) were used as positive control. Images are representative of three independently performed experiments with similar results. f, LV mRNA levels of Xbp1s (n=5 mice per group) and g, Immunoblot images of pIRE1α, IRE1α and GAPDH proteins in LV samples of WT and iNOS KO mice after five weeks of CHOW or HFD+L-NAME diet (n=4 mice per group). h, Densitometric analysis ratio between pIRE1α and IRE1α protein bands (n=4 mice per group). Results are presented as mean±S.E.M. a-d, f, h Two-way ANOVA followed by Sidak’s multiple comparisons test. Numbers above square brackets show significant P values. For gel source data, see Supplementary Fig. 1.