SIRT7 upregulation protects against TKI-induced cardiotoxicity by inhibiting MCU-mediated mitochondrial calcium overload

Figures

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  FIGURE 1. TKIs induce AC16 cell injury and cytosolic Ca2+ overload. (A–C) IC50 curves for AC16 cells treated with Snt, Tra, and Ima for 72 h. (D,E) At IC10, IC30, IC50, and IC70, TKIs cause a concentration-dependent increase in NT-proBNP and cTnI in AC16 cells (n = 3, compared with the control group, ** P < 0.01). (F) A representative image of TKI-induced cytosolic Ca2+ overload at IC10, IC30, IC50, and IC70, as detected by live-cell imaging (scale: 400 μm). (G) TKI-induced cytosolic Ca2+ overload in AC16 cells (n = 3, compared with the control group, ** P < 0.01). cTnI, cardiac troponin I; Ima, imatinib; NT-proBNP, N-terminal pro-brain natriuretic peptide; Snt, sunitinib; Tra, trametinib; TKIs, tyrosine kinase inhibitors.

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  FIGURE 2. TKIs impair mitochondrial function and morphology in AC16 cells. (A) Effect of TKIs on mitochondrial Ca2+ content of AC16 cells detected by live-cell imaging; scale: 200 μm. (B) Effect of TKIs on Δφm of AC16 cells detected by live-cell imaging; scale: 200 μm. (C) TKI-induced changes in mitochondrial ATP content. (D) Δφm induced by TKIs, expressed as green/red fluorescence intensity ratio (n = 3, compared with the control group, ** P < 0.01). (E) Transmission electron micrograph of AC16 cells showing the effect of TKIs on mitochondrial morphology; the arrow represents the endoplasmic reticulum and M represents mitochondria; scale bar: 2.5 Kx, 50 μm; 20 Kx, 500 nm. (F) MERC formation was quantified. TKIs significantly increased MERC% in AC16 cells (n = 10, compared with the control group, ** P < 0.01). ATP, adenosine triphosphate; M, mitochondria; MERC, mitochondrial–endoplasmic reticulum coupling; TKIs, tyrosine kinase inhibitors.

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  FIGURE 3. Effects of TKIs on protein expression changes in AC16 cells. (A,B) Venn diagram of TKI-induced downregulation and upregulation of proteins in AC16 cells. (C–E) Volcano plots showing TKI-induced changes in AC16 protein expression. (F) Heatmap of TKI-induced AC16 protein expression changes. Each column in the figure represents a sample. Each line represents a protein. TKIs, tyrosine kinase inhibitors.

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  FIGURE 4. SIRT7 overexpression protects against TKI-induced AC16 cell injury and cytosolic Ca2+ overload. (A) SIRT7 protein expression was detected using Western blotting. (B) SIRT7 overexpression (n = 3, compared with the control group, ** P < 0.01). (C) SIRT7 upregulation protects against TKI-induced cell viability decline (n = 4, compared with the control group, ** P < 0.01; compared with TKI treatment alone, ## P < 0.01). (D,E) SIRT7 upregulation protects against TKI-induced elevation of the myocardial injury markers NT-proBNP and cTnI (n = 3, compared with the control group, ** P < 0.01; compared with TKI treatment alone, # P < 0.05, ## P < 0.01). (F) SIRT7 overexpression protects against TKI-induced cytosolic calcium overload; scale: 200 μm. (G) SIRT7 overexpression attenuates TKI-induced cytosolic calcium overload (n = 6, compared with the control group, ** P < 0.01; compared with the TKI treatment group, ## P < 0.01). cTnI, cardiac troponin I; NT-proBNP, N-terminal pro-brain natriuretic peptide; SIRT7, sirtuin 7; TKIs, tyrosine kinase inhibitors.

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  FIGURE 5. SIRT7 upregulation rescues TKI-induced declines in ATP production and Δφm and preserves endoplasmic reticulum–mitochondria functional coupling in AC16 cells. (A) SIRT7 upregulation mitigates TKI-induced mitochondrial Ca2+ overload. Compared with the control group, the three tested TKIs caused AC16 mitochondrial Ca2+ overload, which was reversed by SIRT7 upregulation; scale: 200 μm. (B) Effect of SIRT7 upregulation on Δφm of AC16 cells (scale: 200 μm). (C) SIRT7 upregulation induced changes in mitochondrial ATP content. (D) Δφm induced by SIRT7 upregulation, expressed as green/red fluorescence intensity ratio (n = 3, compared with the control group, ** P < 0.01; compared with TKI treatment, ## P < 0.01). (E) Transmission electron micrograph of AC16 cells showing the effect of SIRT7 upregulation on mitochondrial morphology, in which the arrow represents the endoplasmic reticulum and M represents mitochondria; scale bar: 2.5 Kx, 50 μm; 20 Kx, 500 nm. (F) MERC formation was quantified (n = 10, compared with the control group, ** P < 0.01; compared with TKI-treated alone, ## P < 0.01). ATP, adenosine triphosphate; M, mitochondria; MERC, mitochondrial–endoplasmic reticulum coupling;…

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  FIGURE 6. TKIs activate the MCU, leading to mitochondrial Ca2+ overload in AC16 cells. (A) The tested TKIs significantly reduced SIRT7 expression. (B) TKIs significantly enhanced MCU expression (n = 3, compared with the control group, *P < 0.05, **P < 0.01; compared with the Snt group, ## P < 0.01). (C) Green fluorescence intensity represents the MCU activity. Before TKI administration, preincubation of RU360 at 5 μM for 2 h effectively inhibited MCU functional enhancement; scale, 200 μm. (D,E) TKIs increased AC16 MCU protein expression (n = 3, compared with the control group, **P < 0.01). MCU, mitochondrial calcium uniporter; SIRT7, sirtuin 7; Snt, sunitinib; TKIs, tyrosine kinase inhibitors.

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  FIGURE 7. SIRT7 Upregulation in AC16 cells counteracts TKI-induced MCU activation and mitochondrial Ca2+ overload. (A) Green fluorescence intensity represents the MCU activity. SIRT7 upregulation effectively inhibits MCU functional enhancement before TKI administration; scale: 200 μm. (B,C) SIRT7 upregulation counteracts the increase in MCU protein expression (n = 3, compared with the control group, **P < 0.01; compared with TKI treatment, ## P < 0.01). MCU, mitochondrial calcium uniporter; SIRT7, sirtuin 7; TKIs, tyrosine kinase inhibitors.

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  FIGURE 8. SIRT7 upregulation rescues TKI-induced declines in hiPSC-CMs. (A–F) TKIs inhibited the mean beating frequency at 72 h, and SIRT7 pretreatment protected against the impedance amplitude reduction induced by TKIs (n > 4, compared with the control group, ** P < 0.01; compared with the TKI treatment alone, ## P < 0.01). (G) SIRT7 upregulation mitigates TKI-induced mitochondrial Ca2+ overload. Compared with the control group, the three TKIs caused mitochondrial Ca2+ overload in hiPSC-CMs, which was reversed by SIRT7 upregulation; scale: 200 μm. hiPSC-CMs, human induced pluripotent stem cell–derived cardiomyocytes; SIRT7, sirtuin 7; TKIs, tyrosine kinase inhibitors.

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  FIGURE 9. Treatment with TKIs enhanced MCU activity and suppressed SIRT7 expression in hiPSC-CMs. Green fluorescence intensity represents the MCU activity. Red fluorescence intensity represents the SIRT7 activity. TKIs significantly reduced SIRT7 expression and increased MCU expression; scale: 20 μm. MCU, mitochondrial calcium uniporter. MCU, mitochondrial calcium uniporter; SIRT7, sirtuin 7; TKIs, tyrosine kinase inhibitors.

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  FIGURE 10. SIRT7 alleviates TKI-Induced cardiotoxicity via inhibition of MCU-Dependent mitochondrial Ca2+ overload.