Targeting the undruggable transcription factor, KLF5, with a peptidomimetic small molecule, NC114, attenuates pressure overload-induced cardiac remodeling and fibrosis

Abstract

Krüppel-like factor 5 (KLF5) is an intrinsically disordered transcription factor involved in cardiac remodeling, cancer, and metabolic diseases. Targeting KLF5 has been a persistent challenge in drug development due to its structural inaccessibility. We investigated cardioprotective effects of NC114, a rationally designed small molecule that mimics a short, hydrophobic α-helical motif in KLF5, thereby disrupting its protein–protein interactions. Adult C57BL/6J male mice underwent transverse aortic constriction (TAC) or sham surgery, followed by administration of NC114 or vehicle. NC114-treated TAC mice exhibited preserved cardiac function, reduced heart weight-to-body weight ratio, and markedly attenuated interstitial fibrosis. Gene expression analysis demonstrated decreased cardiac expression of Klf5, Nppb, Tgfb1, PAI-1, Col1a1, and Fn1. NC114 also suppressed oxidative stress and reduced phosphorylation of PKCδ and expression of HIF-1α during the early phase post-TAC. Metabolomic profiling revealed that NC114 treatment reversed TAC-induced accumulation of organic and amino acids. NC114, a novel peptidomimetic molecule, targets the undruggable transcription factor KLF5 to attenuate cardiac hypertrophy, fibrosis, and metabolic dysregulation in pressure overload-induced heart failure. This study highlights the potential of KLF5 inhibition as a therapeutic strategy in cardiovascular disease.

Supplementary Information: The online version contains supplementary material available at 10.1038/s41598-025-32155-y.

Keywords: Fibrosis; Hypertrophy; KLF5; Metabolic dysregulation; Oxidative stress; Pressure overload.

Fig. 1

NC114 improves cardiac function and survival in TAC mice. (a) Schematic of the experimental design: NC114 was administered at 50 mg/kg/day for 10 days following transverse aortic constriction (TAC). Echocardiography was performed before TAC, and at 1 week and 4 weeks post-TAC. Hearts were collected for analysis, 1 week and 4 weeks post-TAC. (b) Comparison of ejection fraction (EF, %) with various doses of NC114 treatment (n = 3 per group). (c) Heart weight to body weight (HW/BW) ratios of WT, sham, vehicle, and NC114-treated TAC mice at different doses of NC114 (n = 3 per group). (d) Ejection fraction (EF, %) was measured in the left ventricle before TAC, and at 1 week and 4 weeks post-TAC (n = 10 per group). (e) Long-term survival curves showing significantly improved survival rates in NC114-treated TAC mice compared to untreated TAC mice (n = 3 per group; NC114, 50 mg/kg/day for 10 days). (f and g) Left ventricular internal dimensions at end-diastole (LVIDd) and end-systole (LVIDs) measured 4 weeks post-TAC, indicating improved systolic function in NC114-treated TAC mice (n = 10 per group). (h) Representative M-mode echocardiographic images of the left ventricle (LV), 4 weeks post-TAC. Vertical scale bar: 1 mm; horizontal: 100 ms. (i and j) Quantitative real-time PCR analysis of brain natriuretic peptide (Nppb) and β-myosin heavy chain (β-MHC) expression, demonstrating induction after TAC and subsequent reduction with NC114 treatment (n = 10 per group). GAPDH was used as the reference gene. Statistical analyses were performed using one-way and two-way ANOVA followed by Tukey’s multiple comparisons test. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05 vs. vehicle group.

Fig. 2

NC114 attenuates cardiac hypertrophy and reduces pressure overload-induced cardiac fibrosis. (a) Representative images of heart size from WT, sham, vehicle-treated TAC, and NC114-treated TAC mice at 4 weeks post-TAC. (b) Representative cross-sections of cardiomyocytes stained with hematoxylin and eosin (H&E). Scale bars: 2 mm (left) and 200 µm (right). (c) MT staining (MT) of cardiac tissues, highlighting fibrotic areas. Scale bars: 2 mm (left) and 200 µm (right). (d) Heart weight to body weight (HW/BW) ratios among indicated groups (n = 10 per group). (e) Quantification of fibrotic areas in indicated groups (n = 10 per group). (f to i) Quantitative real-time PCR analysis of connective tissue growth factor (Ctgf), Fibronectin (Fn1), Collagen Type I Alpha 1 Chain (Col1a1), and plasminogen activator inhibitor-1 (PAI-1) mRNA levels in left ventricles (LVs) of indicated groups (n = 10 per group). GAPDH was used as the reference gene. Statistical analyses were performed using one-way ANOVA followed by Tukey’s multiple comparisons test. ****P < 0.0001, ***P < 0.001 vs. vehicle group.

Fig. 3

NC114 reduces macrophage infiltration and inflammatory mediators in TAC-induced cardiac stress. (a) Immunohistochemical analysis of CD68 in heart sections to detect macrophage infiltration. Red arrows indicate CD68-positive macrophages. Scale bar: 200 µm. (b) Quantification of macrophage infiltration (CD68-positive cells) in cardiac tissues using ImageJ (version 1.53e) (n = 10 per group). (c and d) Flow cytometry analysis of macrophage populations in heart tissues, specifically CD45⁺CD11b⁺F4/80⁺Ly6C⁺CCR2⁺ and CD45⁺CD11b⁺F4/80⁺Ly6C⁺CCR2⁻ subtypes, 4 weeks post-TAC (n = 6 per group). (e to h) Quantitative real-time PCR analysis of mRNA expression levels of inflammatory mediators, including interleukin 10 (Il10), transforming growth factor-β (Tgfb1), tumor necrosis factor-α (Tnfα), and chemokine ligand 2 (Ccl2) in the heart, 4 weeks post-TAC (n = 10 per group). GAPDH was used as the reference gene. Statistical analyses were performed using one-way ANOVA followed by Tukey’s multiple comparisons test. ****P < 0.0001, ***P < 0.001, **P < 0.01 vs. vehicle group.

Fig. 4

NC114 preserves the cardiac transcriptomic profile upon pressure overload by TAC. (a) Commonly upregulated and downregulated genes in 1-week and 4-week NC114-treated TAC mice, compared to vehicle-treated TAC mice. Commonly upregulated and downregulated genes at both time points are displayed below their respective diagrams. Note: Genes localized to the extracellular region (GOCC:0005576) and mitochondria (GOCC:0005739), based on subcellular compartment annotations, are highlighted in orange (27 genes) and green (5 genes), respectively, in the gene name column. Left: Venn diagram illustrating overlap of upregulated differentially expressed genes (DEGs) between 1-week (blue) and 4-week (red) NC114-treated mice compared to Vehicle-treated controls. Right: Venn diagram illustrating overlap of downregulated DEGs between 1-week (blue) and 4-week (red) NC114-treated mice compared to Vehicle-treated controls. (b) Quantitative real-time PCR analysis of mRNA expression Klf5, Nox4, and PKCδ in heart tissues, 1 week and 4 weeks post-TAC (n = 7–10 per group). GAPDH was used as the reference gene. (c) Western blot analysis of protein expression levels, including KLF5, pPKCδ (S645), and PKCδ in heart tissues from various groups, 1 week and 4 weeks post-TAC (n = 5-7 per group). GAPDH was used as a loading control. Representative blots are shown in Supplementary Figs.19, 21, 22 and 27. Data for 1 week are shown in blue. Statistical analyses were performed using one-way ANOVA followed by Tukey’s multiple comparisons test. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05 vs. vehicle group.

Fig. 5

Substrate metabolism and metabolic flexibility in NC114-treated TAC mice (1 and 4 weeks post-TAC). Metabolic profiling of heart tissues from WT, sham, vehicle-treated TAC, and NC114-treated TAC mice was performed to analyze glycolysis pathway metabolites, methionine and cysteine pathway intermediates, and branched-chain amino acids (BCAAs) (n = 12–14 per group for 1-week TAC mice group, n = 10 per group for 4-week TAC mice). Data were normalized to an internal control, with units indicated as the area ratio where applicable. (a) Glucose-6-phosphate (G6P), pyruvate, and lactate levels were significantly lower in NC114-treated TAC mice compared to vehicle-treated TAC mice. (b) NC114 effectively lowered increased levels of methionine and cysteine that were observed in TAC mice. (c) The oxidative stress index, calculated from the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) (GSH/GSSG), showed improvement in NC114-treated TAC mice. Data for 1 week are shown in blue. Statistical analyses were performed using one-way ANOVA followed by Tukey’s multiple comparisons test. ns = not significant; ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05 vs. vehicle group.

Fig. 6

Levels of essential amino acids in NC114-treated TAC mice (1 and 4 weeks post-TAC). (a) Levels of essential amino acids, including leucine, isoleucine, valine (branched-chain amino acids, BCAAs) (n = 12–14 per group for 1-week TAC mice group, n = 10 per group for 4-week TAC mice). (b) Levels of multiple amino acids, including alanine, aspartate, lysine, phenylalanine, serine, tryptophan, tyrosine, and glycine were analyzed in heart tissues of all groups (n = 12–14 per group for 1-week TAC mice group, n = 10 per group for 4-week TAC mice). Data for 1 week are shown in blue. Statistical analyses were performed using one-way ANOVA followed by Tukey’s multiple comparisons test. ns = not significant; ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05 vs. vehicle group.

Fig. 7

Mechanistic framework of NC114 efficacy in TAC–induced HF. Schematic representation showing how NC114 protects against HF from pressure overload. TAC upregulates KLF5 expression, which promotes fibrosis, oxidative stress, metabolic reprogramming, and production of abundant cytokines. These proinflammatory cytokines activate multiple molecular signaling pathways, leading to cardiomyocyte hypertrophy, apoptosis and fibroblast activation, ultimately resulting in cardiac remodeling and HF. However, NC114 tends to suppress these effects, highlighting the importance of early intervention in preventing maladaptive remodeling and HF progression. NC114 inhibits KLF5, leading to a reduction in Igfbp7, Col1a1, and cytokines such as Tgfb, Ctgf, and Tnfα, which decreases fibrosis. This, in turn, increases PPAR-α, glycine, and Bax levels, indicating a metabolic shift toward improved metabolism, compensatory metabolic adaptation, and additional compensatory mechanisms. Consequently, this shift results in a reduction of oxidative stress and apoptotic markers (Nox4, Bcl2), ultimately decreasing ROS production and PKCδ, reducing inflammation and oxidative damage, and providing heart protection.