Long-term mitapivat treatment improves inflammatory pro-fibrotic cardiomyopathy in a murine model of β-thalassemia. Free

Hypertrophic cardiomyopathy (HC) is a severe invalidating complication of ß–thalassemia (ß–thal), contributing to reduced patients' life expectancy. Recently we showed that 12-months old male ß-thal mice develop HC independently from heart iron accumulation, and that oxidation is an early and central event in cardiac fibrosis. Mitapivat, a novel pharmacologic activator of both pyruvate kinases (PK) R and M2, has been previously shown to improve anemia in both mouse models and humans with ß–thal. To assess the effects of mitapivat on the development of ß–thal related cardiomyopathy, we first studied 8- or 12- months old male Hbb^3th+mice compared to wild-type animals. In hearts from Hbb^3th+ mice, we observed up-regulation of PKM2 and co-immunoprecipitation with over-activated STAT3 when compared to hearts from wild-type mice. Of note, functional crosstalk between monomer/dimers of PKM2 and STAT3 activity has been previously reported in different models of cardiomyopathy (Federti E et al. Haematologica 108: 1335, 2023). PKM2-mediated activation of STAT3 modulates pro-inflammatory (e.g NLRP3 inflammasome) and pro-fibrotic pathways. The heart metabolomic, lipidomic, proteomic, and cysteine redox proteome profiling of Hbb^3th+ mice revealed extensive metabolic and oxidative remodeling compared to wild-type controls. Specifically, metabolomic analyses showed significant depletion of acyl-carnitine pools, elevation in sphingosine 1-phosphate and higher levels of sulfur-containing amino acids and carboxylic acids, indicative of impaired mitochondrial function and redox imbalance. Lipidomics revealed a shift toward accumulation of diacyl- and triacyl-glycerols, and decreased levels of phosphatidylcholines (PC) and phosphtadylethanolamines (PE). Proteomic profiling further highlighted significant upregulation of stress response proteins and fibrotic markers in Hbb^3th+ hearts, including elevation of myosin heavy chains (Myh6 and 7) and ubiquitin/proteasomal systems for protein degradation. Redox proteomics revealed extensive cysteine oxidation, di-oxidation, and formation of dehydroalanine residues—particularly in mitochondrial and contractile proteins—pointing to severe oxidative post-translational modifications as a hallmark of ß-thal related cardiomyopathy. We then treated Hbb^3th+mice with mitapivat (1200 ppm w/w was added to the Mod LabDiet 5P00 standard rodent diet) from the age of 2 -months up to either 8- or 12-months of age (corresponding respectively to 6 and 8 months of treatment) compared to vehicle treated animals. Mitapivat treated 8- and 12-month-old Hbb^3th+mice displayed mean Hb increase of 1.5 g/dL and 1.6 g/dL, respectively, when compared to vehicle treated Hbb^3th+ animals. Metabolomic analyses in mitapivat-treated Hbb^3th+ mice showed normalization of glycolytic and TCA cycle intermediates consistent with PK activation, including elevation in pyruvate and downstream lactate and citrate levels, and an increase in myocardial ATP content. Lipidomic profiles were characterized by repletion of PC pools, suggesting mitigation of lipid peroxidation. Proteomic and redox-cysteomic data showed decreased abundance of pro-inflammatory and pro-fibrotic proteins, and reduced oxidative modifications of critical cysteine residues, supporting the role of mitapivat in improving redox homeostasis and in myocardial preservation. Hearts from Mitapivat treated 8- months old Hbb^3th+mice showed reduced activation of STAT3 as well as decreased amount of active STAT3 co-immunoprecipitated with PKM2 when compared to vehicle treated mice. This was associated with down-regulation of heart NLRP3 expression. We also observed decreased activation of NF-kB with down-regulation of endothelin-1 (ET-1) and vascular cell adhesion molecule-1 (VCAM-1) expression, supporting a reduction in heart inflammatory vasculopathy. The mitapivat treated 12- months old Hbb^3th+mice displayed decreased heart perivascular sclerosis, an early step of heart interstitial fibrosis, and a reduction in degradation of the sarcoplasmic reticulum calcium ATPase cardiac isoform-2 (SERCA2A), a transport system important for myocardial performance, when compared to vehicle treated Hbb^3th+mice. Taken together our data indicate that long-term mitapivat treatment protects against ß–thal related cardiomyopathy by synergistic effects on anemia and on heart metabolomic profile, preventing the activation of pro-inflammatory and pro-fibrotic pathways.