Catestatin restores cardiac metabolic flexibility and enhances mitochondrial function

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  Fig. 1. Boolean analysis of RNA-seq data identifies a CST-regulated MyoFibro gene signature. A Schematic illustrating the role of catestatin (CST) in the regulation of blood pressure. B, C Boolean implication analysis used to identify cardiomyocyte- and fibroblast-specific gene sets. D, E Machine-learning analysis of RNA-seq data from CST-KO hearts identifies a 39-gene MyoFibro signature that is upregulated in untreated CST-KO mice and normalized following CST supplementation. F, G Composite MyoFibro score significantly distinguishes disease states (control vs HF) in human heart samples. H Composite MyoFibro score distinguishes disease states across publicly available datasets from human, mouse, and rat. Statistical significance was assessed using a two-sided Welch’s t-test. Predictive performance is reported as the receiver operating characteristic area under the curve (ROC–AUC). C control, CTRL control, CM cardiomyopathy, DCM diabetic cardiomyopathy, HF heart failure, SHR spontaneously hypertensive rat, WKY Wistar Kyoto. Graphical elements were created by the authors using Microsoft PowerPoint (Microsoft Consolidated UCSD Agreement) and NIH BioArt (https://bioart.niaid.nih.gov/). No…

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  Fig. 2. Boolean analysis of RNA-seq data identifies CST-regulated macrophage gene signatures. A Boolean implication analysis was used to identify macrophage-specific gene sets. B, C Machine-learning analysis of RNA-seq data from CST-KO hearts identifies a 34-gene Mac1 signature that is downregulated in untreated CST-KO mice and normalized following CST supplementation. D, E Composite Mac1 score significantly distinguishes disease states (control vs HF) in human heart samples. F The composite Mac1 score does not consistently distinguish disease states across publicly available datasets from human, mouse, and rat. (G, H) Machine-learning analysis identifies a 26-gene Mac2 signature that is upregulated in untreated CST-KO mice and normalized following CST supplementation. I The composite Mac2 score is significantly elevated in untreated CST-KO mice. J Composite Mac2 score significantly and consistently distinguishes disease states (control vs HF) across publicly available datasets from human, mouse, and rat. Statistical significance was assessed using a two-sided Welch’s t-test. Predictive performance is reported as the receiver operating characteristic area under the curve (ROC–AUC).…

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  Fig. 3. CST restores insulin signaling, glucose metabolism, and glycogen storage in the heart. A Representative immunoblots showing insulin-stimulated phosphorylation of AKT in wild-type (WT) and CST-KO hearts. B Densitometric quantification of AKT phosphorylation shown in (A) (n = 3). C Representative immunoblots showing insulin-stimulated phosphorylation of GSK-3β. D Densitometric quantification of GSK-3β phosphorylation shown in (C) (n = 3). E Glucose uptake and utilization in WT and CST-KO hearts in response to CST, including 2-deoxyglucose (2DG) uptake, glucose-6-phosphate (G6P) formation, and G6P utilization (n = 6). F Expression of genes involved in glucose metabolism in WT and CST-KO hearts in response to CST: Slc2a4 (GLUT4, glucose transporter 4), Pfkm (phosphofructokinase, muscle type), and Pdk2 (pyruvate dehydrogenase kinase 2). G Representative immunoblots showing CST regulation of AKT and GSK-3β phosphorylation. H, I Densitometric quantification of AKT (H) and GSK-3β (I) phosphorylation shown in (G) (n = 3–4). J–M Transmission electron micrographs of the subsarcolemmal region showing glycogen granules: saline-treated WT (J), saline-treated CST-KO (K), CST-treated WT (L), and…

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  Fig. 4. CST improves systemic glucose homeostasis, suppresses hepatic glucose output, and enhances insulin sensitivity. A GTT (2 g/kg dextrose, intraperitoneal) showing mean blood glucose curves for saline-treated WT and CST-KO mice and CST-treated WT and CST-KO mice (n = 8 per group). B Individual GTT blood glucose traces from the same cohorts (n = 8). C Quantification of GTT area under the curve (AUC; mg/dL·min) (n = 8). D PTT (2 g/kg sodium pyruvate, intraperitoneal) showing mean blood glucose curves for saline-treated WT and CST-KO mice and CST-treated WT and CST-KO mice (n = 8 per group). E Individual PTT blood glucose traces (n = 8). F Quantification of PTT AUC (mg/dL·min) (n = 8). G ITT (0.5 IU/kg human insulin, intraperitoneal) showing mean blood glucose curves for saline-treated WT and CST-KO mice and CST-treated WT and CST-KO mice (n = 8 per group). H Individual ITT blood glucose traces (n = 8). I Quantification of ITT AUC (mg/dL·min) (n = 8). Data are presented as mean ± SEM. Statistical analyses were performed using one- or two-way ANOVA followed by Tukey’s multiple comparison test. P < 0.05 was considered significant. **P < 0.01; ***P < 0.001; ****P < 0.0001.

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  Fig. 5. CST suppresses fatty acid uptake, oxidation, and lipotoxicity in the heart. A Modulation of insulin-stimulated glucose uptake by FA in WT cardiomyocytes (n = 4–8). B FA uptake in WT and CST-KO hearts in response to CST (n = 6). C FA utilization (β-oxidation) in WT and CST-KO hearts in response to CST (n = 6). D Expression of genes involved in lipid metabolism in WT and CST-KO hearts in response to CST (n = 8): Cd36 (fatty acid translocase, FAT); Slc27a1 (fatty acid transport protein 1, FATP1); Slc27a2 (fatty acid transport protein 2, FATP2); Dgat2 (diacylglycerol O-acyltransferase 2); Sptlc1 (serine palmitoyltransferase long-chain base subunit 1); Cers2 (ceramide synthase 2); and Ppara (peroxisome proliferator-activated receptor-α). E Triglyceride content in WT and CST-KO hearts in response to CST (n = 8). F Ceramide content in WT and CST-KO hearts in response to CST (n = 8). Data are presented as mean ± SEM. Statistical analyses were performed using one- or two-way ANOVA as appropriate, followed by Tukey’s multiple comparison test. P < 0.05 was considered significant. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

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  Fig. 6. CST restores sarcomeric organization, mitochondrial ultrastructure, and mitophagy in the heart. Subsarcolemmal mitochondria (SSM). A, C, E, G Low-magnification electron micrographs showing sarcomeres and mitochondria in saline-treated WT (A), saline-treated CST-KO (C), CST-treated WT (E), and CST-treated CST-KO (G) hearts. B, D, F, H High-magnification images of SSM showing mitochondrial cristae in the corresponding groups. Inter-/intramyofibrillar mitochondria: (IFM): I, K, M, O Low-magnification electron micrographs showing sarcomeres and mitochondria in saline-treated WT (I), saline-treated CST-KO (K), CST-treated WT (M), and CST-treated CST-KO (O) hearts. J, L, N, P High-magnification images of IFM showing mitochondrial cristae in the corresponding groups. Q Morphometric analysis of sarcomere length in the subsarcolemmal region (n = 265–303). R Quantification of SSM cristae surface area (n = 84–85). S Morphometric analysis of sarcomere length in the inter-/intramyofibrillar region (n = 265–303). T Quantification of IFM cristae surface area (n = 84–85). U Expression of mitophagy-associated genes (Pink1, Prkn, and Map1lc3b) in saline- and CST-treated WT and CST-KO hearts (n =…

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  Fig. 7. CST binds mitochondrial F₁-ATP synthase and modulates its structural dynamics. A SDS–PAGE analysis of proteins eluted from biotinylated CST–streptavidin agarose following ligand affinity chromatography of mouse heart homogenate using human CST (hCgA352–372) as bait. Bound proteins were eluted at pH 5.5 and analyzed. B Sequence coverage map of mouse ATP synthase α-subunit identified by trypsin–LC–MS/MS from CST-binding proteins. Peptide confidence is indicated as follows: green, ≥95%; purple, ≥50%; red, lower confidence. C Representative MS/MS spectrum (precursor mass 1286.7) of a peptide derived from the CST-binding protein. D Time-averaged structure of CST shown in cartoon representation, with N- and C-termini and selected residues indicated. E Structure of the F₁-ATP synthase complex, with α-subunits shown in gray, β-subunits in cyan, and the central axle in blue. F Molecular docking of CST to F₁-ATP synthase. α-subunits (chains a–c) are shown in gray and β-subunits (chains d–f) in cyan. ATP and ADP molecules are shown in magenta and blue, respectively. CST (red) binds at the α–β interface corresponding to the lowest-energy complex. The central axle is omitted for clarity. G…

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  Fig. 8. CST localizes to mitochondria and enhances mitochondrial bioenergetics in cardiomyocytes. A–D Fluorescence microscopy of H9c2 cells showing co-localization of endogenous CST with mitochondria: CST (A; AF488), mitochondria (B; MitoTracker™ Deep Red FM), nuclei (C; Hoechst), and merged image (D), in which co-localization appears yellow. Images were acquired using a Keyence microscope. Scale bar, 20 µm. E Representative intensity profiles of red (mitochondria) and green (CST) channels (n = 20 cells). M1 and M2 denote Manders’ coefficients (fractional overlap of red with green and green with red channels, respectively), and P denotes Pearson’s correlation coefficient. F, G Scatter plots showing the distribution of Manders’ coefficients (F) and Pearson’s correlation coefficients (G) across individual cells (n = 20). H Representative immunoblots showing expression of mitochondrial OXPHOS complex proteins. I–M Densitometric quantification of OXPHOS subunits: Complex I (Ndufb8), Complex II (Sdhb), Complex III (Uqcrc2), Complex IV (Mt-co1), and Complex V (Atp5a) (n = 3). (N) Relative mRNA expression of Sirt1 and Pgc1a (n = 8). O Mitochondrial membrane potential (ΔΨm) (n = 8). P…