Targeting Inflammation by Pioglitazone and its R-Enantiomer Mitigates Pathological Myocardial Remodeling in Murine Hypertrophic Cardiomyopathy

Figures

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  Figure 1. Pio and R-pio Reduce Hypertrophy in α-MHC719/+ (A) Representative transthoracic echocardiographic images in parasternal long-axis view showing left ventricular wall morphology across treatment groups. Orange arrows indicate the maximum left ventricular wall thickness. Scale bar = 1 mm. (B) Quantification of maximal left ventricular wall thickness in end-diastole (mean ± SD). Untreated α-MHC719/+ mice showed significant hypertrophy compared with untreated WT controls 1.07 ± 0.19 mm vs 0.67 ± 0.08 mm; P < 0.001). Treatment with pio reduced wall thickness to 0.76 ± 0.15 mm (29% reduction vs α-MHC719/+; P < 0.001), while R-pio further reduced it to 0.72 ± 0.18 mm (33% reduction vs α-MHC719/+; P < 0.001), reaching near-WT levels. Statistical comparisons between treatment groups and the untreated α-MHC719/+ control were performed using Dunnett's test for multiple comparisons following 1-way analysis of variance. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Data information: WT: untreated WT (n = 18); α-MHC719/+: untreated α-MHC719/+ (n = 16), α-MHC719/+ +pio: α-MHC719/+ treated with pioglitazone (n = 7); α-MHC719/+ +R-pio: α-MHC719/+ treated with R-pioglitazone (n = 9).

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  Figure 2. Attenuation of Fibrosis by pio and R-pio in α-MHC719/+ Mice (A) Representative Masson’s trichrome-stained transverse sections of the left ventricle. Viable myocardium appears red and collagen-rich fibrotic tissue blue (indicated by arrows). Scale bar = 50 μm. (B) Quantification of fibrotic area (percentage of total left ventricular area) for each group (median and 25th-75th percentiles). Untreated α-MHC719/+ mice exhibited a significant increase in fibrosis compared with WT controls (median fibrosis: 2.65% [Q1-Q3: 0.34%-4.82%] vs 0.00% [Q1-Q3: 0.00%-0.01%]; P < 0.001). Preventive treatment with pio reduced fibrosis to 0.94% [Q1-Q3: 0.00%-1.38%] (65% reduction vs α-MHC719/+) and with R-pio to near-baseline levels 0.08% [Q1-Q3: 0.00%-0.03%]; (>95% reduction vs α-MHC719). Statistical comparisons between groups were performed using the Mann-Whitney U test. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Data information: WT: untreated WT (n = 21); α-MHC719/+: untreated α-MHC719/+ (n = 21); α-MHC719/++pio: α-MHC719/+ treated with pioglitazone (n = 7); α-MHC719/++R-pio: α-MHC719/+ treated with R-pioglitazone (n = 9).

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  Figure 3. Transcriptional Profiling Reveals Metabolic Defects and Inflammatory Signaling in α-MHC719/+ Mice, Largely Ameliorated by Treatment With Pio and R-Pio (A) Dot plots of the top enriched Gene Ontology terms for Biological Processes (BP) and Molecular Functions (MF). Untreated α-MHC719/+ myocardium exhibits a marked suppression of fatty acid metabolic processes (blue circles) and up-regulation of structural remodeling pathways (red circles). Treatment with pio or R-pio largely normalizes these signatures. Circle size reflects enrichment significance (–log10 p). (B to E) Heatmaps illustrating normalized expression (Z-scores) of representative differentially expressed genes compared with untreated WT myocardium. (B) Fatty acid and glucose metabolism: Genes governing fatty acid β-oxidation are down-regulated in untreated α-MHC719/+ hearts and restored by treatment, while glucose metabolism genes show a compensatory up-regulation that is modulated by therapy. (C) Mitochondrial bioenergetics: Key components of the TCA cycle and mitochondrial electron transport chain (Complex I–IV) are suppressed in the disease group and upregulated following treatment. (D) Stress and immune activation:…

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  Figure 4. Impact of Treatment With Pio and R-pio on the Proteomic Profiling in α-MHC719/+ Hearts (A) Gene Ontology enrichment of untreated α-MHC719/+ vs WT shows suppressed mitochondrial metabolism and activated inflammatory/proliferative pathways, largely normalized by treatment. Normalized enrichment scores (NES) indicate pathway direction and strength.(B) Heatmap of the top 10 up- and down-regulated proteins in untreated α-MHC719/+ vs WT. Protein abundance relative to the untreated WT controls is expressed as log2 fold change (log2 FC). Untreated α-MHC719/+ vs WT controls show increased extracellular matrix/hypertrophy proteins and decreased mitochondrial/redox proteins, largely normalized by treatment. (C) Log2 FC of MPC1 is significantly reduced in α-MHC719/+ vs WT (adj. P <0.03), but restored to WT levels by both treatments (ns). P values were adjusted for multiple testing using the Benjamini-Hochberg method. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ns = not significant. Data information: WT: untreated WT, α-MHC719/+: untreated α-MHC719/+; α-MHC719/++ pio: α-MHC719/+ treated with pioglitazone; α-MHC719/++ R-pio: α-MHC719/+ treated with R-pioglitazone.

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  Figure 5. Attenuation of Myocardial Macrophage Infiltration and Systemic Inflammatory Signaling (A to D) Immunohistochemical validation of macrophage infiltration in left ventricular tissue. (A and B) Representative staining (A) and quantification (B) of IBA1-positive macrophages (mean ± SD). Scale bar = 50 μm. Untreated α-MHC719/+ hearts show a significant accumulation of IBA1+ cells compared with WT. Treatment with both pio and R-pio significantly reverses this infiltration. (C and D) Representative staining (C) and quantification (D) of CD68-positive macrophages (mean ± SD). Pan-macrophage numbers are elevated in disease. Pio and R-pio treatment significantly reduces CD68+ cell counts. (E) Systemic inflammatory profiling (NULISA) of plasma samples. The heatmap displays the top 10 up- and down-regulated proteins in untreated α-MHC719/+ vs WT. Scatter plots represent individual biological replicates (n = 4 per group); horizontal lines indicate mean ± SD. Statistical comparisons between treatment groups and the untreated α-MHC719/+ control were performed using Dunnett's test for multiple comparisons following 1-way analysis of variance. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Data…