iNOS aggravates pressure overload-induced cardiac dysfunction via activation of the cytosolic-mtDNA-mediated cGAS-STING pathway

Abstract

Background: Sterile inflammation contributes to the pathogenesis of cardiac dysfunction caused by various conditions including pressure overload in hypertension. Mitochondrial DNA (mtDNA) released from damaged mitochondria has been implicated in cardiac inflammation. However, the upstream mechanisms governing mtDNA release and how mtDNA activates sterile inflammation in pressure-overloaded hearts remain largely unknown. Here, we investigated the role of inducible NO synthase (iNOS) on pressure overload-induced cytosolic accumulation of mtDNA and whether mtDNA activated inflammation through the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway. Methods: To investigate whether the cGAS-STING cascade was involved in sterile inflammation and cardiac dysfunction upon pressure overload, cardiomyocyte-specific STING depletion mice and mice injected with adeno-associated virus-9 (AAV-9) to suppress the cGAS-STING cascade in the heart were subjected to transverse aortic constriction (TAC). iNOS null mice were used to determine the role of iNOS in cGAS-STING pathway activation in pressure-stressed hearts. Results: iNOS knockout abrogated mtDNA release and alleviated cardiac sterile inflammation resulting in improved cardiac function. Conversely, activating the cGAS-STING pathway blunted the protective effects of iNOS knockout. Moreover, iNOS activated the cGAS-STING pathway in isolated myocytes and this was prevented by depleting cytosolic mtDNA. In addition, disruption of the cGAS-STING pathway suppressed inflammatory cytokine transcription and modulated M1/M2 macrophage polarization, and thus mitigated cardiac remodeling and improved heart function. Finally, increased iNOS expression along with cytosolic mtDNA accumulation and cGAS-STING activation were also seen in human hypertensive hearts. Conclusion: Our findings demonstrate that mtDNA is released into the cytosol and triggers sterile inflammation through the cGAS-STING pathway leading to cardiac dysfunction after pressure overload. iNOS controls mtDNA release and subsequent cGAS activation in pressure-stressed hearts.

Keywords: Cardiac dysfunction; Sterile inflammation; cGAS; iNOS; mtDNA.

Figure 1

iNOS contributes to cytosolic mtDNA accumulation and cGAS activation in pressure overload-stressed heart. (A) Representative co-immunostaining of dsDNA and mitochondria (Tom20) showing cytosolic DNA in heart tissues. (B) Cytosolic mtDNA content in the hearts of TAC mice were measured using quantitative-PCR, n = 4. (C) Total mtDNA in the hearts of TAC mice, n = 4. (D-E) iNOS deficiency decreased cGAS-STING expression and IRF-3 phosphorylation in the hearts of TAC mice, n = 6. (F-G) iNOS deficiency reduced the mRNA expression levels of IFN-β and ISG activated by cGAS-STING in TAC hearts, n = 4. Data are presented as the mean ± SEM. Statistical analysis was performed using a 1 or 2-way ANOVA with a Tukey's multiple-comparison post-hoc test comparisons between multiple groups. TAC, transverse aortic constriction; iNOS. inducible NO synthase; mtDNA, mitochondrial DNA; dsDNA, double-stranded DNA; cGAS, cyclic GMP-AMP synthase; STING, stimulator of interferon genes; IFN-β, interferon-β. *, P < 0.05. **, P < 0.01.***, P < 0.001. ****, P < 0.0001.

Figure 2

iNOS-induced mtDNA release activates the cGAS-STING pathway. (A-B) The expression of cGAS and STING in cells transfected with iNOS, n = 6. (C) The ONNO levels were measured to reflect nitrative stress, n = 6. (D) The mRNA expression levels of IFN-β were measured, n = 6. (E) iNOS expression increased the cytosolic mtDNA content in cardiomyocytes, n = 6. (F-H) mtDNA depletion blunted the effects of iNOS on increasing cGAS-STING and IFN-β expression, n = 6. (I-K) mtDNA increased cGAS-STING and IFN-β (N) expression in isolated cardiomyocytes. Data are presented as the mean ± SEM. Statistical analysis was performed using a 1 or 2-way ANOVA with a Tukey's multiple-comparison post-hoc test comparisons between multiple groups. iNOS. inducible NO synthase; SNAP, S-Nitroso-N-acetyl-DL-penicillamine; EtBr, ethidium bromide; IFN-β, interferon-β. *, P < 0.05. **, P < 0.01.***, P < 0.001. ****, P < 0.0001.

Figure 3

iNOS deficiency alleviates pressure overload-induced sterile inflammation and cardiac injury. (A-D) iNOS deficiency decreased the content of M1 macrophages and transcript levels of IL-1β, IL-6 in TAC hearts, n = 6. (E-G) iNOS deficiency improved cardiac function. Representative images of echocardiograms are shown in (E). LV ejection fraction and fractional shortening are shown in (F)and (G) respectively, n = 6. (H-I) Deficiency of iNOS reduced the mRNA expression levels of ANP and BNP induced by TAC, n = 6. (J) Representative hematoxylin-eosin staining of myocardial tissues from WT or iNOS^-/- mice after TAC. Scale bar: 1 mm. The HW/TL ratio are shown in (K), n = 6. (L) Representative WGA staining of midventricular sections to assess hypertrophy of cardiac myocytes. Scale bar: 100 μm. Quantitative analysis of WGA staining is shown in right, n = 6. (M) Representative images of Masson staining of the heart sections. Scale bar: 100 μm. The results of the statistical analyses are shown in right, n = 6. Data are presented as the mean ± SEM. Data were analyzed using a two-way ANOVA with a Tukey's multiple-comparison post-hoc test. TAC, transverse aortic constriction; WT, wildtype; HW/TL, heart weight and tibia length; WGA, wheat germ agglutinin; LV, left ventricular; IL, interleukin; ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide. *, P < 0.05. **, P < 0.01.***, P < 0.001. ****, P < 0.0001.

Figure 4

cGAS-STING pathway activation blunts the protective role of iNOS on TAC heart. (A-C) STING agonist Compound 3 increased IRF-3 phosphorylation and IFN-β expression in the heart of iNOS^-/- mice, suggesting the activation of the cGAS-STING pathway, n = 6. (D) STING agonist Compound 3 aggravated cardiac function in iNOS^-/- TAC mice as shown in the representative echocardiograms. LV ejection fraction and fractional shortening are shown below, n = 6. (E-F) Compound 3 increased the mRNA expression levels of ANP and BNP induced by TAC in iNOS^-/- mice, n = 6. (G) Representative images of Masson staining of the heart sections. Scale bar: 100 μm. Results of statistical analysis shown in right, n = 6. (H) Representative WGA staining to assess hypertrophy of cardiac myocytes. Scale bar: 50 μm. Quantitative analysis of WGA staining was shown in right, n = 6. (I) Representative phalloidine staining of isolated cardiomyocytes. Scale bar: 100 μm. Quantitative analysis was shown in right, n = 6. Data were presented as the mean ± SEM and were analyzed using a two-way ANOVA with a post-hoc Tukey's multiple-comparison test. TAC, transverse aortic constriction; WT, wildtype; WGA, wheat germ agglutinin; ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide. *, P < 0.05. **, P < 0.01.***, P < 0.001. ****, P < 0.0001.

Figure 5

STING knockdown reduces the expression of inflammatory markers and alleviates cardiac injury in TAC heart. (A-B) STING knockdown suppressed the activation of the IRF-3 phosphorylation, n = 6. (C-E) Cardiac-specific cGAS-STING pathway disruption improved cardiac function. Representative echocardiograms are shown in (C). LV ejection fraction and fractional shortening are shown in (D) and (E), n = 6. (F) Representative images of hematoxylin-eosin staining of myocardial tissue from NC or STING knockdown mice. Scale bar: 1 mm. (G) The HW/TL ratio, n = 6. (H-I) Representative images of WGA staining to assess hypertrophy of cardiac myocytes. Scale bar: 50 μm, n = 4. (J-K) Representative images and analysis of Masson staining of heart sections to assess fibrosis. Scale bar: 100 μm, n = 4. Data are presented as the mean ± SEM and were analyzed using a two-way ANOVA with a post-hoc Tukey's multiple-comparison test. TAC, transverse aortic constriction. NC, negative control; KD, knockdown; *, P < 0.05. **, P < 0.01.***, P < 0.001. ****, P < 0.0001.

Figure 6

Cardiomyocyte-specific STING deficiency alleviates cardiac injury in TAC mice. (A-C) Cardiomyocyte-specific STING deficient suppressed the IRF-3 phosphorylation and IFN-β expression in the TAC heart, n = 6. (D) Quantitative PCR was used to measure the transcript levels of IL1β in TAC heart, n = 6. (E-G) Cardiomyocyte-specific STING deficient improved cardiac function. Representative echocardiograms are shown in (E). LV ejection fraction and fractional shortening are shown in (F) and (G), n = 5. (H) Representative images of hematoxylin-eosin staining. Scale bar: 1 mm. (I) The HW/TL ratio, n = 5. (J-K) Representative images and analysis of Masson staining of heart sections to assess fibrosis. Scale bar: 100 μm, n = 5. Data were presented as the mean ± SEM and were analyzed using a two-way ANOVA with a post-hoc Tukey's multiple-comparison test. TAC, transverse aortic constriction. *, P < 0.05. **, P < 0.01.***, P < 0.001. ****, P < 0.0001.

Figure 7

Disruption of the cGAS-STING pathway increases the proportion of M2 macrophages and alleviates cardiac injury in TAC mice. (A) Flow cytometry analysis of macrophages from the hearts of NC, cGAS KD and STING KD mice after TAC. (B-C) Percentage of M1 or M2 macrophages in the TAC heart, n = 4. Data are presented as the mean ± SEM and were analyzed using a one-way ANOVA with a Tukey's multiple-comparison post-hoc test. TAC, transverse aortic constriction. NC, negative control; KD, knockdown; *, P < 0.05. **, P < 0.01.

Figure 8

Relevance of iNOS-mtDNA-cGAS axis in human hypertensive heart. (A) Upper panel: Representative immunohistochemical staining of iNOS. Scale bar: 100 μm. Lower panel: Representative images of co-immunostaining of dsDNA and the mitochondria (Tom20), showing increased cytosolic DNA levels in heart tissue. Scale bar: 10 μm. (B) Results of the statistical analysis of iNOS expression. (C) Results of the statistical analysis of cytosolic mtDNA content. n = 4. (D) Representative images of immunohistochemical staining to assess the expression of cGAS, STING and p-IRF3 in the hypertensive heart. Scale bar: 100 μm. Results of the statistical analysis are shown in right, n = 4. Data are presented as the mean ± SEM and data were analyzed using a Student's t-test and a one-way ANOVA with a Tukey's multiple-comparison post-hoc test. HHD, hypertensive heart disease. cGAS, cyclic GMP-AMP synthase; STING, stimulator of interferon genes; iNOS. inducible NO synthase; mtDNA, mitochondrial DNA; *, P < 0.05. **, P < 0.01.***, P < 0.001. ****, P < 0.0001.

Figure 9

Graphical mechanistic model. The model illustrates that pressure overload induces iNOS expression and then activates the cGAS-STING cascade leading to sterile inflammation and cardiac dysfunction in mice.