Sacubitril/Valsartan attenuates progression of diabetic cardiomyopathy through immunomodulation properties: an opportunity to prevent progressive disease

Figures

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  Fig. 1. Plasma neprilysin activity and diastolic function markers in patients with diabetes. A Correlation analysis between plasma NEP activity of patients with diabetes at baseline and change in LA stiffness index (18 months-Baseline; n = 44/60). B, C Plasma neprilysin activity of pre-HFpEF patients with diabetes from the PARABLE trial at baseline (Val n = 25; Sac/Val n = 35) and 3 months after treatment (Val n = 21/25; Sac/Val n = 33/35). D Change LA stiffness index at 18 months from baseline in Valsartan group (n = 20/25) and Sacubitril/Valsartan group (n = 24/35). n = Patients (biological replicates). Correlation using Spearman’s rank-order correlation test (A) and comparison using either Mann–Whitney test (B, C) or Unpaired t test (D). If P values > 0.05 are not shown in graphs. LA = Left atrial; Sac/Val = Sacubitril/valsartan; Val = Valsartan

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  Fig. 2. Measurement of left ventricular function and structure at 24 weeks of study by echocardiography. A–F Mice are evaluated for cardiac function and disease progression using echocardiography. G Representative images of ratio of MV E/A using PW-doppler. H–K Mice are evaluated for cardiac structure. (L) Representative images of left ventricular structure using M-mode. CD n = 12; HFD/STZ n = 11; HFD/STZ + Sac/Val n = 10; HFD/STZ + Val n = 12. n = Number of mice (biological replicates). Comparison using either one-way ANOVA followed by Tukey's multiple comparisons test (A, B, D, E–F, H, J, K) or Kruskal–Wallis test followed by Dunn's multiple comparisons test (C, I). P values for one-way ANOVA/Kruskal–Wallis test < 0.05 for (C–F) and (H–K). If P values > 0.05 are not shown in graphs. BPM = Beat per minute; CD = Control diet; HFD = High-fat diet; STZ = Streptozotocin; BPM = Beat per minute; MV E/A = Ratio of E-wave velocity to A-wave velocity; IVRT = Isovolumic relaxation time; LA = Left atrial; BSA = Body surface area; LVPW = Left ventricular posterior wall. Other abbreviations as in Fig. 1

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  Fig. 3. Histology studies and assessment of pro-fibrotic genes and cardiac remodelling. A Representative photomicrographs of myocardium stained with Picrosirius red for interstitial fibrosis. B Quantification of collagen content from picrosirius red staining (CD n = 12; HFD/STZ n = 11; HFD/STZ + Sac/Val n = 7; HFD/STZ + Val n = 12). C, D Relative mRNA expression of collagen 1a1 and 3a1 (CD n = 12; HFD/STZ n = 11; HFD/STZ + Sac/Val n = 7; HFD/STZ + Val n = 12), respectively. E Representative photomicrographs of cardiomyocyte cross sectional area stained with Haematoxylin and Eosin. F Quantification of cardiomyocyte cross sectional area from Haematoxylin and Eosin (CD n = 12; HFD/STZ n = 11; HFD/STZ + Sac/Val n = 7; HFD/STZ + Val n = 12). n = Number of mice (biological replicates). Comparison using either one-way ANOVA followed by Tukey's multiple comparisons test (B–C, F) or Kruskal–Wallis test followed by Dunn's multiple comparisons test (D). P values for one-way ANOVA/Kruskal–Wallis test < 0.05 for (B–D), and (F). If P values > 0.05 are not shown in graphs. Abbreviations as in Figs. 1 and 2

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  Fig. 4. Measurement of neprilysin activity and correlations among echocardiographic parameters. A Plasma neprilysin activity of control mice (n = 11–12), HFD/STZ mice without NEP inhibitor (n = 23–24), and HFD/STZ + Sac/Val mice (n = 10) at baseline and 24 weeks of study. B Heart neprilysin activity across different groups (CD n = 12; HFD/STZ without NEP inhibitor n = 23; HFD/STZ + Sac/Val n = 7) at 24 weeks of study. C Correlation matrix among echocardiography parameters, neprilysin activity, and diabetic parameters at 24 weeks of study (CD n = 12; HFD/STZ n = 11; HFD + Sac/Val n = 7–10; HFD/STZ + Val n = 12). n = Number of mice (biological replicates). Comparison using Paired t test or Wilcoxon matched-pairs signed rank test (A). Comparison using Kruskal–Wallis test followed by Dunn's multiple comparisons test (B). Correlation using Spearman’s rank-order correlation test (C). If P values > 0.05 are not shown in graphs. NEP = Neprilysin; SV = Stroke volume; CO = Cardiac output; HR = Heart rate; LVAW = Left ventricular anterior wall; IVCT = Isovolumic contraction time; EF = Ejection fraction; FS = Fraction shortening; HbA1c = Haemoglobin a1c. Other abbreviations as in Fig. 1–2. *P <…

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  Fig. 5. Measurement of diabetic parameters. A, B HbA1c and fasting blood glucose levels at 24 weeks of study. C, D Fasting plasma insulin and GLP-1 levels at 24 weeks of study. E Correlation between GLP-1 and fasting plasma insulin (n = 45). F, G Assessment of HOMA-B and QUICKI levels across groups. CD n = 12; HFD/STZ n = 11; HFD/STZ + Sac/Val n = 10; HFD/STZ + Val n = 12. n = Number of mice (biological replicates). Comparison using either one-way ANOVA followed by Tukey's multiple comparisons test (C, F–G) or Kruskal–Wallis test followed by Dunn's multiple comparisons test (A, B, D). Correlation using Spearman’s rank-order correlation test (E). P values for one-way ANOVA/Kruskal–Wallis test < 0.05 for (A–D), and (F–G). If P values > 0.05 are not shown in graphs. GLP-1 = Glucagon-Like peptide-1; HOMA-B = Homeostatic model assessment B-cell function; QUICKI = Quantitative insulin sensitivity check index. Other abbreviations as in Figs. 1 and 2

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  Fig. 6. Single-nuclei RNA Sequencing (snRNA-seq) of DbCM mouse model. A Experimental design of snRNA-seq (control, HFD/STZ, and HFD/STZ + Sac/Val; n = 3 each). Images rendered by Biorender.com. Image usage is covered by BioRender’s Academic License Terms. B t-SNE dimensionality reduction of 11 major cell populations of mouse cardiac tissue. C Top 5 upregulated genes for each cell population where colour indicates strength of expression and size of dot represents percentage of cells expressing the gene. D Relative abundance of each cell population for sample groups (control, HFD/STZ, and HFD/STZ + Sac/Val; n = 3 each). E Relative macrophages abundance in each sample and group (control, HFD/STZ, and HFD/STZ + Sac/Val; n = 3 each). F Pathway analysis comparing between HFD/STZ mice and control mice. G Subpopulations of macrophages/monocytes. (H) Top 5 upregulated genes for Subpopulations of macrophages/monocytes where colour indicates strength of expression and size of dot represents percentage of cells expressing the gene. I, J Differentially expressed genes in TLF+ macrophages were compared to the Ccr2+ macrophages and the MHC-II macrophages. n = Number of mice (biological replicates).…

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  Fig. 7. Abundance of subpopulations of macrophages/monocytes, roles of Irf7 gene in DbCM and cell–cell interaction studies. A Relative abundance of each subpopulation of cell macrophages/monocytes for sample groups (control, HFD/STZ, and HFD/STZ + Sac/Val; n = 3 each). B Relative subpopulation of cell macrophages/monocytes abundance in each sample and group (control, HFD/STZ, and HFD/STZ + Sac/Val; n = 3 each). C Differential gene expression in TLF+ macrophages (HFD/STZ mice vs Control mice; n = 3 each). D Regulon specificity of TLF+ macrophages. E–G Possible interactions between all cell populations across sample groups. H–J Chord diagrams of inferred COLLAGEN signalling pathway networks across sample groups (control, HFD/STZ, and HFD/STZ + Sac/Val; n = 3 each). n = Number of mice (biological replicates). Comparison using one-way ANOVA (A) and Unpaired t test (B). P values for one-way ANOVA test < 0.05 for (A, B). If P values > 0.05 are not shown in graphs. Edge colour is consistent with the signalling source. Segments with large arrows represent signalling targets and inner bars represent signalling sources in which the colours indicate signalling targets. Irf7 = Interferon regulatory…

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  Fig. 8. Studies of M2-like macrophages (TLF+ macrophages) and fibroblasts. A–CIRF7 is induced in M2-like macrophages with high D-glucose condition (n = 3 each for RT-qPCR; n = 6 each for Western blot; biological replicates). D–E Effects of treatments for IRF7 and IL10 on M2-like macrophages in glucose conditions (n = 3 each; biological replicates). F Experimental design for paracrine interaction between M2-like macrophages and HCFs. G–J HCFs are treated with conditioned media from M2-like macrophages experiments with or without treatments (n = 3 each; biological replicates). K–M Western blotting analysis of HCFs treated with conditioned media from M2-like macrophages experiments (n = 3 each; biological replicates). N The siRNA of IRF7 in M2-like macrophages with different concentrations (n = 3 each; biological replicates). O Western blotting analysis for IRF7 of siRNA-IRF7 in M2-like macrophages (n = 3 each; biological replicates). P Expression of IL10 of siRNA-IRF7 in M2-like macrophages (n = 3 each; biological replicates). Q–S HCFs are treated with conditioned media from siRNA of IRF7 in M2-like macrophages experiments (n = 3 each; biological replicates). Comparison using Unpaired t…