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. 2023 Sep 1;108(9):2535-2541.
doi: 10.3324/haematol.2022.282614.

Mitapivat, a pyruvate kinase activator, improves transfusion burden and reduces iron overload in β-thalassemic mice

Alessandro Mattè  1 , Penelope A Kosinski  2 , Enrica Federti  1 , Lenny Dang  2 , Antonio Recchiuti  3 , Roberta Russo  4 , Angela Siciliano  1 , Veronica Riccardi  1 , Anne Janin  5 , Matteo Mucci  3 , Christophe Leboeuf  5 , Achille Iolascon  4 , Carlo Brugnara  6 , Lucia De Franceschi  7
Affiliations

Mitapivat, a pyruvate kinase activator, improves transfusion burden and reduces iron overload in β-thalassemic mice

Alessandro Mattè et al. Haematologica. .
No abstract available

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Figures

Figure 1.
Figure 1.
Mitapivat reduces transfusion burden in β-thalassemia mice exposed to chronic transfusion with associated repro-gramming of splenic macrophage phenotype. (A) Experimental study design to assess the effects of mitapivat on hematologic phenotype of β-thalassemia (β-thal) mice exposed to chronic transfusion. (B) Hemoglobin (Hb) changes over time in transfused (Tr.) β-thal (Hbbth3/+) mice treated with either vehicle or mitapivat (50 mg/kg twice daily [BID]) shown as single animals (n=3 vehicle-treated mice; n=4 mitapivat-treated mice). Grey dotted line shows the transfusion threshold (10.5 g/dL). (C) Transfusion time intervals in β-thal (Hbbth3/+) mice treated with either vehicle or mitapivat (50 mg/kg BID). Data are presented as means ± standard error of the mean (SEM) (n=3 vehicle-treated mice; n=4 mitapivat-treated mice); #P<0.05 compared to vehicle-treated transfused β-thal mice. (D) Iron staining (Perl’s Prussian blue is a semi-quantitative method to assess organ iron accumulation) in spleen from Hbbth3/+ mice treated with either vehicle or transfusion plus vehicle or transfusion plus mitapivat. One representative image from 3 with similar results. Left panel: quantification of iron staining in spleen. Data are mean ± SEM (n=3). *P<0.05 compared with vehicle Hbbth3/+ mice and #P<0.05 compared with vehicle-treated transfused Hbbth3/+ mice. (E) Flow cytometric analysis (CD44+Ter119+ and cell size markers, see also the Online Supplementary Figure S2) of bone marrow and spleen from Hbbth3/+ mice exposed to either vehicle or to chronic transfusion with and without mitapivat treatment (see also Matte et al.). Data are mean ± SEM (n=3-4). *P<0.05 compared with vehicle Hbbth3/+ mice and #P<0.05 compared with vehicle-treated transfused Hbbth3/+ mice. (F) Maturation index as ratio between pop II (Baso E.) and pop IV (Ortho E.) in bone marrow and spleen from Hbbth3/+ mice treated with either vehicle or exposed to chronic transfusion with or without mitapivat, analyzed by flow cytometry. Data are mean ± SEM (n=3-4). (G) Flow cytometric quantification of M1 (CD80) and M2 (CD206) expression on spleen macrophage cell surface from wild-type (WT) or Hbbth3/+ mice exposed to either vehicle or mitapivat or to chronic transfusion with and without mitapivat treatment. Spleen macrophages (MΦ) were isolated with the GentleMACS cell dissociator (Miltenyi Biotech, Germany). MΦ were identified and gated as CD45+/F4/80+ cells. Anti-CD45 PE-Cy5.5, F4/80 PE, CD206 PerCP-Cy5.5 and CD80 were from BioLegend, USA. Data are mean ± SEM (n=3-4). MFI: mean fluorescence intensity; RBC: red blood cells.
Figure 2.
Figure 2.
Mitapivat-treated transfused β-thalassemia mice show reduced liver iron accumulation and improved iron homeostasis. (A) Left and central panels: iron staining (Perl’s Prussian blue is a semi-quantitative method to assess organ iron accumulation) in liver from wild-type (WT) and Hbbth3/+ mice treated with either vehicle or transfusion (Tr.) or transfusion plus mitapivat. One representative image from 5 with similar results. Right panel: quantification of iron staining in liver. Data are mean ± standard error of the mean (SEM) (n=5). °P<0.05 compared to WT, *P<0.05 compared with vehicle Hbbth3/+ mice and #P<0.05 compared with vehicle-treated transfused (Tr.) Hbbth3/+ mice. (B) Liver mRNA expression normalized over liver iron concentration (LIC) as determined using the bathophenanthroline method. Data are presented as means ± SEM (n=3). #P<0.05 compared with vehicle-treated transfused Hbbth3/+ mice. (C) Transferrin saturation in Hbbth3/+ mice treated with either vehicle or transfusion or transfusion plus mitapivat. Transferrin saturation was calculated as the ratio between serum iron and total iron binding capacity, using the Total Iron Binding Capacity Kit (Randox Laboratories, UK) and 50 mL of serum, according to the manufacturer’s instructions. Data are presented as means ± SEM (n=3). *P<0.05 compared with vehicle Hbbth3/+ mice and #P<0.05 compared with vehicle-treated transfused Hbbth3/+ mice. RBC: red blood cells.
Figure 3.
Figure 3.
In transfused ^-thalassemia mice, mitapivat reduces kidney iron accumulation and downregulates profibrotic kidney miRNA let-7 expression. (A) Upper panels: iron staining (Perl’s Prussian blue is a semi-quantitative method to assess organ iron accumulation) in kidney from wild-type (WT) and Hbbth3/+ mice treated with either vehicle or transfusion (Tr.) or transfusion plus mitapivat. One representative image from 3-6 with similar results. Lower panels: quantification of iron staining in kidney. Data are mean ± standard error of the mean (SEM) (n=3-6). *P<0.05 compared with vehicle Hbbth3/+ mice and #P<0.05 compared with vehicle-treated transfused (Tr.) Hbbth3/+ mice. (B) Relative expression of miRNA let-7b and -7d in kidneys from WT or Hbbth3/+ mice exposed to either vehicle or mitapivat or to chronic transfusion with and without mitapivat treatment. Small RNA was isolated from frozen kidneys using a silica spin column-based Quick-RNA kit (Zymo Research), quantified with a UV NanoPhotometer (Implen), and reverse transcribed with the qScript microRNA cDNA Synthesis for RT-PCR (QuantaBio). For real time polymerase chain reaction (RT-PCR) analysis of let-7b and let-7d miRNA, 3 ng of cDNA were used as a template in reaction mixtures (10 mL final volume) including a PowerUp SYBR Green Master Mix (5 mL, Applied Biosystems), miRNA-specific forward and universal reverse primers (1 mL each, miRCURY assays, Qiagen), and PCR-grade water. The expression of the indicated mRNA was quantitated by the comparative ΔCt method. RNU6-1 was used as control for normalization. Data are mean ± SEM (n=3-4). *P<0.05. **P<0.01. ***P<0.001. (C ) Phospho-tyrosine immunoprecipitation of kidneys from WT or Hbbth3/+ mice exposed to either vehicle or mitapivat or to chronic transfusion with and without mitapivat treatment, using anti-phospho-tyrosine specific antibodies (IP: PY, clone PY99 from SCBT, Santa Cruz, CA and clone 4G10 from Merck KGaA, Darmstadt, Germany), revealed with anti-TGF-β receptor (Rec) specific antibody. GAPDH in whole-cell lysate (WCL) is used as loading control. One representative gel from 4 others with similar results is presented. Blots were developed using the Luminata Forte Chemiluminescent HRP Substrate from Merck Millipore (Armstadt, Germany), and images were acquired with the Alliance Q9 Advanced imaging system (Uvitec, UK). Densitometric analysis of immunoblots is shown on the right. Data are mean ± SEM (n=4). °P<0.05 compared to WT; *P<0.05 compared with vehicle Hbbth3/+ mice, #P<0.05 compared with vehicle-treated transfused Hbbth3/+ mice. RBC: red blood cells.
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References

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