Diabetes mellitus aggravates myocardial inflammation and oxidative stress in aortic stenosis: a mechanistic link to HFpEF features

Abstract

Background: Patients diagnosed with both aortic stenosis (AS) and diabetes mellitus (DM) encounter a distinctive set of challenges due to the interplay between these two conditions. This study aimed to investigate the effects of DM on the left ventricle in AS patients, specifically focusing on the inflammatory response, oxidative stress, and their implications for cardiomyocyte function, titin phosphorylation, and the nitric oxide (NO)-soluble guanylyl cyclase (sGC)-cyclic guanosine monophosphate (cGMP)-protein kinase G (PKG) signaling pathway.

Methods and results: Left ventricular myocardial biopsies (in total: n = 28) were obtained from patients with diabetic AS (n = 11) and compared with those from non-diabetic AS patients (n = 17). Enzyme-linked immunosorbent assay (ELISA) demonstrated significantly elevated levels of pro-inflammatory mediators, including high mobility group box protein 1 (HMGB1) and calprotectin, as well as receptors associated with the inflammatory response, such as Toll-like receptor 2 (TLR2), 4 (TLR4), and receptor for advanced glycation endproducts (RAGE). These were correlated with an enhanced NOD-like receptor protein 3 (NLRP3) inflammasome and the release of interleukins (IL) 1, 6, and 18 in diabetic AS patients compared to their non-diabetic counterparts. Additionally, in the diabetic AS cohort, there was an increase in oxidative stress markers (hydrogen peroxide (H₂O₂), 3-nitrotyrosine, lipid peroxidation (LPO), oxidative glutathione (GSSG)/reduced glutathione (GSH) ratio) within the myocardium and mitochondria, accompanied by impaired NO-sGC-cGMP-PKG signaling, decreased titin phosphorylation, and increased passive stiffness (F_passive) of cardiomyocytes relative to non-diabetic AS patients. In vitro anti-inflammatory treatment with an IL-6 inhibitor and antioxidant treatment with GSH effectively normalized the elevated F_passive observed in AS patients with DM to levels comparable to the non-diabetic group. Furthermore, treatment with PKG and the sodium-glucose cotransporter 2 (SGLT2) inhibitor empagliflozin also resulted in a reduction of F_passive in cardiomyocytes from diabetic AS patients, although not to the levels observed in non-diabetic AS patients.

Conclusion: DM exacerbates inflammation and oxidative stress in AS patients, leading to impaired NO-sGC-cGMP-PKG signaling and increased cardiomyocyte F_passive. These conditions are reminiscent of the pathophysiology of heart failure with preserved ejection fraction (HFpEF). These alterations can be ameliorated through anti-inflammatory and antioxidant therapies, indicating potential therapeutic strategies for diabetic patients suffering from AS.

Keywords: Aortic stenosis; Cardiomyocyte Fpassive; Diabetes mellitus; Heart failure with preserved ejection fraction; Inflammation; Oxidative stress; Protein kinase G; Titin.

Fig. 1

Upstream signaling pathways of inflammation and oxidative stress in left ventricular biopsies from patients with aortic stenosis (AS), with (+DM) and without concomitant diabetes (−DM). A Schematic representation of intracellular oxidative and inflammatory signaling pathways. HMGB1: High mobility group box protein 1; NF-κB: nuclear factor kappa B; NLRP3: NOD-like receptor protein 3; RAGE: receptor for advanced glycation end products; TLR: Toll-like receptor. B HMGB1 levels. C Calprotectin levels. D TLR2 levels. E TLR4 levels. F RAGE levels. G NF-κB levels. H. NLRP3 levels. Box and whisker plots (median, 25th to 75th percentiles, minimum, and maximum) are utilized on the left axis to represent selected parameters in each group (n = 10 samples per group). The dashed lines represent the mean values of both groups. P-values are derived from an unpaired t-test; *P < 0.05, **P < 0.01.The right axis shows the difference between the means ± SEM

Fig. 2

Inflammatory markers and cardiomyocyte passive stiffness in left ventricular biopsies from patients with aortic stenosis (AS), with (+DM) and without concomitant diabetes (−DM). A Schematic representation of intracellular inflammatory signaling pathways. F_passive: passive stiffness; HMGB1: high mobility group box protein 1; IL: interleukin; NF-κB: nuclear factor kappa B; NLRP3: NOD-like receptor protein 3; RAGE: receptor for advanced glycation end products; TLR: Toll-like receptor. B–D Levels of IL-1, IL-6, and IL-18. Box and whisker plots (median, 25th to 75th percentiles, minimum, and maximum) are displayed on the left axis to represent selected parameters in each group (n = 10 samples per group). The dashed lines represent the mean values of both groups. P-values are derived from unpaired t-tests; *P < 0.05, ****P < 0.0001. The right axis shows the difference between the means ± SEM. E Stretch protocol; SL: sarcomere length. F Cardiomyocyte F_passive at SL 1.8–2.4 µm in the presence or absence of in vitro treatment with an inhibitor of IL-6 (IL-6_inh). Curves are second-order polynomial fits to the means (± SEM; n = 30–36/5 cardiomyocytes/heart per group), *P < 0.05 AS−DM versus AS+DM, ^‡P < 0.05 AS−DM alone versus AS−DM after IL-6_inh, †P < 0.05 AS+DM alone versus AS+DM after IL-6_inh by one-way ANOVA. P-values were corrected for multiple comparisons by the Tukey method. G. Original recordings

Fig. 3

Oxidative stress markers in left ventricular biopsies from patients with aortic stenosis (AS), with (+DM) and without concomitant diabetes (−DM). A Schematic representation of intracellular oxidative signaling pathways. F_passive: passive stiffness; NF-κB: nuclear factor kappa B; NLRP3: NOD-like receptor protein 3; RAGE: receptor for advanced glycation end products; ROS: reactive oxygen species; TLR: Toll-like receptor. B Quantification of immunostaining of peroxidation (4-HNE, 4-hydroxynonenal). C Whole scan sections of 4-HNE immunostaining (scale bar 1 mm) showing the overall difference in staining. D 4-HNE immunostaining at higher magnitude (scale bar 100 µm). E Measurement of 3-nitrotyrosine (3-NT) levels. F Ratio of myocardial oxidized glutathione (GSSG) over reduced glutathione (GSH). G Measurement of myocardial hydrogen peroxide (H₂O₂) levels. H Measurement of myocardial lipid oxidation (LPO) levels. I Ratio of GSSG to GSH in mitochondria (Mito). J Mitochondrial levels of H₂O₂. K Mitochondrial levels of LPO. Box and whisker plots (median, 25th to 75th percentiles, minimum and maximum) are used on the left axis to represent selected parameters across each group (n = 10 samples per group). The dashed lines represent the mean values of both groups. P-values are from unpaired t-test; *P < 0.05, **P < 0.01. The right axis shows the difference between the means ± SEM

Fig. 4

Cardiomyocyte F_passive before and after antioxidant treatment of left ventricular biopsies from patients with aortic stenosis (AS), with (+DM) and without concomitant diabetes (−DM). A Stretch protocol; SL: sarcomere length. B F_passive at sarcomere length 1.8–2.4 µm in the presence or absence of reduced glutathione (GSH)._−DM+DM C F_passive at SL 1.8–2.4 µm in the presence or absence of the mitochondria-targeted superoxide dismutase mimetic MitoTEMPO. Curves are second-order polynomial fits to the means (± SEM; n = 30–36/5 cardiomyocytes/heart per group), *P < 0.05 AS−DM versus AS+DM, ^‡P < 0.05 AS−DM alone versus AS−DM alone after GSH or MitoTEMPO treatment, †P < 0.05 AS+DM alone versus AS+DM after GSH or MitoTEMPO treatment by one-way ANOVA. P-values were corrected for multiple comparisons by the Tukey method. D. Original recordings

Fig. 5

Myocardial NO/sGC/cGMP/PKG signaling pathway in left ventricular biopsies from patients with aortic stenosis (AS), with (+DM) and without concomitant diabetes (−DM). A Schematic representation of the myocardial nitric oxide (NO)/soluble guanylyl cyclase (sGC)/cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) signaling pathway. F_passive: passive stiffness; GTP: guanosine triphosphate; P: phosphorylation. B NO levels. C Activity of sGC. D Myocardial levels of cGMP. E. Activity of PKG. F. Ratio of phosphorylated N2B and N2BA titin isoforms to total titin. G. Site-specific phosphorylation at Ser4099 of the N2B isoform. Box and whisker plots (indicating median, 25th to 75th percentiles, minimum, and maximum) are utilized on the left axis to represent selected parameters across each group (n = 8–10 samples per group). The dashed lines represent the mean values of both groups. P-values are from unpaired t-test; *P < 0.05. The right axis shows the difference between the means ± SEM. H F_passive at SL 1.8–2.4 µm in the presence or absence of PKG. I F_passive at SL 1.8–2.4 µm in the presence or absence of empagliflozin (EMPA). Curves are second-order polynomial fits to the means (± SEM; n = 30–36/5 cardiomyocytes/heart per group). *P < 0.05 AS−DM versus AS+DM, ^‡P < 0.05 AS−DM versus AS−DM after PKG or EMPA treatment, †P < 0.05 AS+DM versus AS+DM after PKG or EMPA treatment by one-way ANOVA. P-values were corrected for multiple comparisons by the Tukey method

Fig. 6

PKA and CaMKII in left ventricular biopsies from patients with aortic stenosis (AS), with (+DM) and without concomitant diabetes (−DM). A Schematic representation of the myocardial protein kinase A (PKA) and calcium-calmodulin dependent protein kinase II (CaMKII) signaling pathways. AC: adenylyl cyclase; AngII: angiotensin II; β-AR: β-adrenergic receptor; ATP: adenosin triphosphate; Ca²⁺: calcium; cAMP: cyclic adenosine monophosphate; ET-1: endothelin 1; F_passive: passive stiffness; G: G protein; P: phosphorylation; PLC: phospholipase C. B PKA activity. C. Site-specific phosphorylation at Ser4010 of N2B isoform. D. F_passive at SL 1.8–2.4 µm in the presence or absence of PKA. E CaMKII activity. F Expression of CaMKII. G. Site-specific phosphorylation at Ser4062 of N2B isoform. H F_passive at SL 1.8–2.4 µm in the presence or absence of CaMKII. Panels B, C, E, F, G Box and whisker plots (median, 25th to 75th percentiles, minimum and maximum) are used on the left axis to represent selected parameters in each groups (n = 8–10 samples per group). The dashed lines represent the mean values of both groups. P-values are from unpaired t-test; *P < 0.05. The right axis shows the difference between the means ± SEM. Panels D+H. Curves are second-order polynomial fits to the means (± SEM; n = 30–36/5 cardiomyocytes/heart per group). *P < 0.05 AS–DM versus AS+DM, ^‡P < 0.05 AS−DM versus AS−DM after PKA or CaMKII treatment, †P < 0.05 AS+DM versus AS+DM after PKA or CaMKII treatment by one-way ANOVA. P-values were corrected for multiple comparisons by the Tukey method