Cardiomyocyte-specific knockout of AARS2 leads to cardiac dysfunction and fibrosis in mice.

(A–B) Western blot and qRT-PCR analysis showing reduced expression of AARS2 proteins (A) or mRNA (B) of 3-day MI hearts compared with sham hearts (n = 3). (C) Construction diagram of α-MHC-MerCreMer (upper) and AARS2fl/fl mice (lower). (D) Western blots showing reduced AARS2 proteins in AARS2cKO hearts compared with AARS2fl/fl hearts (n = 3). (E) Schematic timelines of tamoxifen treatment and echocardiography (ECHO). (F) Representative M-mode tracings of ECHO in control and cKO hearts before and after tamoxifen treatment. (G) Ejection fraction (EF) and fractional shortening (FS) of AARS2fl/fl and AARS2cKO hearts at different time points after tamoxifen induction (n = 10–11). (H) Cardiac output of AARS2fl/fl and AARS2cKO mice at 28 days after tamoxifen induction (n = 8–10). (I) Left ventricular end-diastolic diameter (LVEDD) and left ventricular end-systolic diameter (LVESD) in AARS2fl/fl and AARS2cKO hearts at 28 days after tamoxifen induction (n = 8–10). (J) Survival percentage of AARS2fl/fl and AARS2cKO mice at 28 days after tamoxifen treatment (n = 8–10). (K) WGA immunofluorescence showing cardiomyocyte hypertrophy on heart slices of AARS2cKO group compared with AARS2fl/fl group at 28 days after tamoxifen induction (scale, 100 μm, n = 5). (L) Masson staining and quantitative analysis showing increased cardiac fibrosis in AARS2cKO hearts compared with AARS2fl/fl control hearts at 28 days after tamoxifen induction (scale bar, 1 mm; n = 8). Mean ± s.e.m.

Cardiomyocyte-specific cKO of AARS2 results in cardiomyocyte apoptosis and energy metabolism deficiency.

(A–B) Immunofluorescence staining showing increased numbers of cTnT+ TUNEL+ cardiomyocytes (A) and Western blot showing reduced anti-apoptotic protein Bcl-2 and increased pro-apoptotic protein BAX (B) in AARS2cKO hearts compared with AARS2fl/fl control hearts at 28 days post tamoxifen treatment (scale, 200 μm; n = 6 for panel A; n = 3 for panel B). (C) Seahorse analysis showing reduced oxygen consumption rate (OCR) of cardiac mitochondria in AARS2cKO hearts compared with AARS2fl/fl control hearts at 28 days after tamoxifen induction (n = 4). (D) Seahorse analysis showing reduced extracellular acidification rate (ECAR) of adult mouse cardiomyocytes in AARS2cKO hearts compared with AARS2fl/fl control hearts at 28 days after tamoxifen induction (n = 4). (E–F) Seahorse analysis showing reduced OCR (E) and ECAR (F) of NRCMs in AARS2 siRNA group compared with control group at 3 days after transfection (n = 4). Mean ± s.e.m.

Cardiomyocyte-specific AARS2 overexpression improves cardiac function and decreases cardiac fibrosis in mice post-MI.

(A) Schematic diagram of α-MHC-MerCreMer, and CAG-AARS2 mice that is driven by the CAG promoter. (B) Western blots showing transgenic overexpression of AARS2 proteins in the hearts of AARS2Tg/+ compared with AARS2WT control mice (n = 3). (C) Experimental protocols for Tamoxifen induction for 5 days, and then recovery for 7 days before ECHO and MI. (D) Representative M-mode of ECHO in control and AARS2Tg/+ mouse hearts at 3 days or 28 days post-MI. (E) EF and FS of the AARS2WT and AARS2Tg/+ mouse hearts were measured at different time points before and after MI (n = 10–11). (F) The cardiac output of AARS2WT and AARS2Tg/+ mice was measured before MI and 28 days after MI (n = 10–11). (G) LVEDD and LVESD of AARS2WT and AARS2Tg/+ mice before MI and 28 days after MI (n = 10–11). (H) Masson’s staining showing decreased fibrotic area in the hearts of AARS2Tg/+ compared with AARS2WT mice at 28 days after MI (scale bar, 1 mm, n = 10–11). Mean ± s.e.m.

Overexpression of AARS2 attenuates cardiomyocyte apoptosis.

(A) Immunofluorescence staining showing reduced cTnT+/TUNEL+ cardiomyocytes in AARS2Tg/+ hearts compared with AARS2WT control hearts at 7 days after MI (scale bar, 200 μm, n = 6). (B) Western blots showing increased anti-apoptotic protein Bcl-2 and decreased pro-apoptotic protein BAX in AARS2Tg/+ compared with AARS2WT control hearts at 7 days after MI (n = 3). (C) The serum level of LDH decreased in AARS2Tg/+ hearts compared with AARS2WT control hearts at 28 days after MI (n = 10). (D) Immunofluorescence staining and quantitative analysis showing reduced MitoSOX in AARS2OE NRCMs after 12 h of hypoxia followed by 1 h of reoxygenation (H/R, scale bar, 20 μm; n = 4). (E) Western blots showing increased Bcl-2 and decreased BAX in NRCMs overexpressing AARS2 (AARS2OE) compared with control NRCMs after 12 h of hypoxia followed by 1 h of reoxygenation (n = 3). (F) The level of LDH decreased in AARS2OE NRCMs after 12 h of hypoxia followed by 1 h of reoxygenation (n = 6). Mean ± s.e.m.

Cardiomyocyte overexpression of AARS2 regulates cardiac metabolism.

(A) Mass spectrometry showing increased lactate and pyruvate but reduced acetyl-CoA in AARS2Tg/+ hearts compared with AARS2WT hearts after 7 days of MI (n = 5). (B) Mass spectrometry showing increased lactate and pyruvate but decreased acetyl-CoA in NRCMs overexpressing AARS2 (AARS2OE) for 3 days (n = 9). (C) Seahorse analysis showing OCR of cardiac mitochondria in AARS2WT and AARS2Tg/+ mice at 28 days after tamoxifen induction (n = 3). (D) Seahorse analysis showing ECAR and quantitative analysis of adult mouse cardiomyocytes in AARS2WT and AARS2Tg/+ mice at 28 days after tamoxifen induction (n = 4). (E–F) Seahorse analysis showing OCR (E) and ECAR (F) of NRCMs in Mock control and AARS2OE groups at 3 days after transfection (n = 4). Mean ± s.e.m.

Overexpression of AARS2 increases the protein level of glycolytic PKM2 via enhancing PKM2 translation.

(A) Ribosome RNA-Seq showing elevated translation of signaling pathways of glycolysis in the AARS2OE NRCMs compared to the Mock NRCMs. (B) Western Blots showing the level of AARS2, PDK4 and LDHA proteins in the hearts of AARS2WT control and AARS2Tg/+ transgenic mice (n = 3). (C) Western Blots showing the level of AARS2 and PKM1 proteins in the hearts of AARS2WT control and AARS2Tg/+ transgenic mice (n = 3). (D–F) Western Blots showing the level of AARS2 and PKM2 proteins in the hearts of AARS2WT control and AARS2Tg/+ transgenic mice (n = 3) (D), in Mock control and AARS2OE NRCMs (E) (n = 3), and in the hearts of AARS2fl/fl and AARS2cKO mice (F) (n = 3). (G–H) Western Blots by non-denatured gels (G) and statistics (H) showing the amounts of PKM2 monomers, dimers and tetramers in the hearts of AARS2WT control and AARS2Tg/+ transgenic mice, and in the hearts of AARS2fl/fl and AARS2cKO mice (n = 4). (I) Mass spectrometry analysis measuring the amounts of alanine (Ala) from homogenates of heart tissues (n = 6) and NRCM lysates (n = 6). (J) Ratio of quantitative results of PKM2 dimers and tetramers in the hearts of AARS2WT control and AARS2Tg/+ transgenic mice of panel H (n = 4). (K) Co-immunoprecipitation reveals no evident interactions between PKM2 and AARS2 in NRCMs. (L–M) qRT-PCR showing the comparative level of PKM2 mRNA in the hearts of control sibling and AARS2Tg/+ transgenic hearts; control sibling and AARS2cKO hearts (L); and in control, AARS2OE, and AARS2siRNA NRCMs (M) (n = 3). FC, Fold changes; Mean ± s.e.m.

PKM2 activator TEPP-46 improves cardiomyopathy in AARS2 cKO mice.

(A) Experimental scheme and time points for tamoxifen induction, ECHO and TEPP-46 administration. (B) EF and FS of AARS2cKO mouse hearts at different time points after administration of control solvent and TEPP-46 (n = 7–8). (C) Representative M-mode of ECHO in different groups of 4-week mice. (D) Cardiac outputs of AARS2cKO mouse hearts at different time points after administration of control solvent and TEPP-46 (n = 7–8). (E) Left ventricular end-diastolic diameter (LVEDD) and left ventricular end-systolic diameter (LVESD) at 28 days of AARS2cKO mice after administration of control solvent and TEPP-46 (n = 7–8). (F) Masson staining showing cardiac fibrosis of AARS2cKO mouse hearts at 28 days after administration of either control solvent or TEPP-46 (scale bar, 1 mm; n = 7–8). (G) Measurements of LDH release in NRCMs from the control group and TEPP-46 group (20 μM) after AARS2 siRNA or control siRNA treatment for 72 h (n = 5). (H) Quantitative analysis of MitoSOX immunofluorescence in NRCMs from the control group and TEPP-46 group (20 μM) after AARS2 siRNA or control siRNA treatment for 72 h (scale bar, 20 μm; n = 5). Mean ± s.e.m.

Genotyping of cardiomyocyte-specific AARS2 knockout mice.

(A) Genotyping of AARS2fl/fl and AARS2cKO mice (n = 3; Mut, mutant). (B) Western Blot showing normal expression of AARS2 proteins in the liver, lung, and skeletal muscle of AARS2fl/fl and AARS2cKO mice (n = 3).

Evaluating AARS2 siRNA for knockout efficiency of AARS2 proteins in NRCMs.

(A) Western blots showing knockdown efficiency of 3 different AARS2 siRNAs in NRCMs, respectively (n = 3). (B) Western blots confirming knockdown efficiency of AARS2 siRNA#3 in NRCMs (n = 3). Ctrl, control; si, small interfering; mean ± s.e.m.

Cardiomyocyte-specific overexpression of AARS2 in the heart but not in the liver, lung, and skeletal muscle.

(A) Genotyping of wild type (WT), α-MHC-MerCreMer; AARS2WT (AARS2WT) control, and α-MHC-MerCreMer; AARS2Tg/+ (AARS2Tg/+) mice (n = 3). (B) Immunofluorescence staining showing ectopic AARS2 proteins in cardiomyocytes of AARS2Tg/+ transgenic mice compared with AARS2WT control mice (scale bar, 50 μm). (C) Western blots showing comparable expression of AARS2 proteins in the liver, lung, and skeletal muscle of AARS2WT control and AARS2Tg/+ transgenic mice (n = 3). Mean ± s.e.m.

Overexpression of AARS2 in cardiomyocytes has no apparent effect on cardiomyocyte proliferation, hypertrophy, and angiogenesis after MI.

(A–B) At 7 days after MI, immunofluorescence staining showing comparable cTnT+/Ki67+ (A) and cTnT+/pH3+ (B) cardiomyocytes in the heart sections of AARS2WT and AARS2Tg/+ groups (scale, 200 μm; n = 5). (C–D) Immunofluorescence staining showing comparable numbers of cTnT+/Ki67+ (C) and cTnT+/pH3+ (D) cardiomyocytes at 48 h after infection of NRCMs with Mock lentivirus or AARS2 lentivirus (scale, 50 μm; n = 3). (E) At 7 days after MI, immunofluorescence staining showing comparable CD31+/α-SMA+ coronary vessels in the heart sections of AARS2WT and AARS2Tg/+ groups (scale, 200 μm; n = 5). (F) WGA staining showing no evident cardiomyocyte hypertrophy in heart slices of AARS2WT and AARS2Tg/+ groups at 28 days after MI (scale, 100 μm; n = 6). Mean ± s.e.m.

Overexpression of AARS2 increases the translation level of some cellular proteins.

(A–D) Ribosome RNA-Seq showing elevated translation of signaling pathways of genes encoded by mitochondria(A), lipoprotein metabolic process (B), cellular response to hypoxia (C), and sodium ion transport (D) in the AARS2OE NRCMs compared to the Mock NRCMs. FC, fold changes.