De-succinylation-induced accumulation of TRMT10C in the nucleus plays a detrimental role in coronary microembolization via its m1A modification function

Abstract

Coronary microembolization (CME) refers to embolism in the coronary microcirculation. This study showed a reduction in succinyl transferase (CPT1A) and the succinylation substrate (succinyl-CoA) in cardiomyocytes in CME models, suppressing the succinylation of the mitochondrially localized protein TRMT10C. Suppression of succinylation promotes KPNA4 recognition of two nuclear localization signals (NLSs), KAKR and KKK(X)₁₀KVKK, in TRMT10C, which induces the transport of TRMT10C from the cytoplasm to the nucleus rather than to the mitochondria. Nuclear TRMT10C induces YTHDF2-mediated decay of TAFAZZIN and NLRX1 through m1A modifications. The reduction in TAFAZZIN and NLRX1 is associated with multiple detrimental effects, such as inflammation mediated by NF-κB and NLRP3, reactive oxygen species (ROS) production, and suppression of mitophagy. TRMT10C knockdown suppressed the accumulation of TRMT10C in the nucleus. It restored NLRX1 and TAFAZZIN protein levels in cardiomyocytes under hypoxia. However, the deficiency of TRMT10C in the mitochondria did not improve-or even worsened-with TRMT10C knockdown. Inducing TRMT10C succinylation via CPT1A overexpression led to the redistribution of TRMT10C to the mitochondria rather than the nucleus, which is likely a better approach for improving cardiomyocyte function under hypoxia than direct TRMT10C knockdown. This study reveals a novel pathological mechanism underlying CME and suggests potential therapeutic targets for this disease.

Keywords: TRMT10C; coronary microembolization; inflammation; m1A modification; mitophagy; nuclear localization; succinylation.

Figure 1

Succinylation modification of proteins is suppressed in CME models in vivo and in vitro. A. A rat CME model was established by injecting microspheres to occlude microvasculature. The black arrow indicates the microspheres in the heart tissues. Hematoxylin and eosin (HE) staining of heart tissues from both the sham and CME rat models. The bar in the figures represents 120 μm. B. Pan-succinylation levels in protein in both sham and CME groups were detected by western blotting with the Succ(K) antibody. C. The levels of Succinyl-CoA in the heart tissues from the sham and CME groups were detected using the detection kit. ***p<0.001 vs. Sham (n = 6). D. The protein levels of SIRT5, CPT1A, KAT2A, and SIRT7 in heart tissues from the sham and CME groups were detected by western blotting. **p<0.01 and ***p<0.001 vs. Sham (n = 6). Sha: Sham. E. Pan-succinylation levels of the protein were examined by western blotting in cardiomyocytes AC16 and H9c2 under hypoxic conditions for 48 hours. F. The levels of succinyl-CoA were examined in cardiomyocytes AC16 and H9c2 under hypoxic conditions. *p<0.05, **p<0.01 and ***p<0.001 vs. 0 hour (n = 3). G. The protein levels of SIRT5, CPT1A, KAT2A, and SIRT7 were examined in cardiomyocytes AC16 and H9c2 under hypoxia by western blot assay. H. Total protein and succinylated proteins were analyzed in AC16 cells via proteomic experiments. The numbers of proteins exhibiting downregulated and upregulated expression and succinylation are presented in a bar chart. I. Intersection analysis was conducted for proteins with downregulated and upregulated expression and succinylation. J. The distribution of proteins with changed succinylation in each cell fraction. K. Gene Ontology (GO) enrichment analysis of the biological processes associated with proteins exhibiting altered succinylation was performed. L. Cluster analysis of the biological processes related to proteins with altered succinylation is presented.

Figure 2

Succinylation modification affects the distribution of TRMT10C in the mitochondrion and nucleus. A. A rat model of CME was established by injecting microspheres to occlude the microvasculature. Immunohistochemical analysis was performed to assess the abundance and localization of TRMT10C protein in the heart tissues from both the sham and CME groups. The scale bar in the figures at 200x magnification represents 120 μm, while the scale bar at 400x magnification represents 50 μm. B. Western blot analysis was conducted to evaluate TRMT10C levels in each cell fraction from the sham and CME groups. ***p < 0.001 vs. Sham (n = 6). C. Expression vectors for Flag-TRMT10C, His-CPT1A, and Myc-SIRT5 were transfected into AC16 and H9c2 cells under hypoxic conditions. Co-immunoprecipitation (Co-IP) analysis was performed by pulling down the Flag-labeled TRMT10C protein, followed by examination of CPT1A, SIRT5, and succinylated proteins using western blotting. D. CPT1A expression vectors and control vectors were transfected into AC16 and H9c2 cells prior to hypoxic exposure. Co-IP analysis was performed to assess the succinylated proteins associated with TRMT10C. E. CPT1A expression vectors were transfected into AC16 and H9c2 cells before hypoxic exposure. An immunofluorescence (IF) assay was conducted to determine the localization of TRMT10C protein within the cells. TOM20 protein fluorescence was used to label the mitochondria, while DAPI staining was employed to visualize the nuclei. The scale bar in the figures represents 5 μm. F. Western blot analysis was performed to examine TRMT10C levels in the mitochondria, cytoplasm (excluding mitochondria), and nucleus of AC16 and H9c2 cells. Hypo: Hypoxia; T10C: TRMT10C; OE: Overexpression.

Figure 3

TRMT10C and CPT1A regulate proliferation, apoptosis, inflammatory response, and ROS levels in cardiomyocytes under hypoxia. A. siRNA targeting TRMT10C and expression vectors for CPT1A were transfected into AC16 and H9c2 cells, with some cells exposed to hypoxia. Cell viability was assessed using the CCK-8 assay. Abbreviations: T10C: TRMT10C; Hypo: Hypoxia; CPT: CPT1A; KD: Knockdown; OE: Overexpression. B. Cell proliferation was evaluated using EdU staining. Red fluorescence indicates EdU-labeled nuclei, while blue fluorescence corresponds to DAPI-stained nuclei. The cell proliferation rate is expressed as the percentage of EdU-labeled nuclei relative to DAPI-stained nuclei. The scale bar in the figures represents 10 μm. C. Cardiomyocytes were trypsinized, incubated with Annexin V-FITC and propidium iodide (PI), and analyzed by flow cytometry using FlowJo software. D. Levels of IL-1β and TNF-α in the culture medium were measured using ELISA detection kits. E. DCFH-DA reacts with reactive oxygen species (ROS) to form the fluorescent compound dichlorofluorescein. ROS levels in the cells were assessed using flow cytometry with the fluorescence probe DCFH-DA. For all studies, *p < 0.05, **p < 0.01, and ***p < 0.001 (n = 3).

Figure 4

Succinylation affects the binding of TRMT10C to the nuclear localization signal protein KPNA4. A. This study established FLAG-tagged constructs of human and rat wild-type (WT) TRMT10C proteins, along with three mutants: human mutant 1 (K173Q, M1), human and rat mutant 2 (K325Q, M2), and the human double mutant (K173Q/K325Q, M3). Individual constructs were transfected into AC16 and H9c2 cells, and proteins were isolated by immunoprecipitation using anti-FLAG antibodies. The succinylation levels of the wild-type and mutant proteins were examined by western blotting. B. Immunofluorescence was performed in AC16 cells after transfection with the WT, M1, M2, and M3 constructs to detect the distribution of TRMT10C protein in various cell fractions. The scale bar in the figures represents 5 μm. C. Western blotting was conducted to analyze the levels of wild-type and mutant TRMT10C proteins in each cell fraction. Abbreviations: Mito: mitochondria; Nucl: nucleus; Cyto: cytoplasm. D. A schematic diagram illustrates two potential nuclear localization signals (NLS) in the human TRMT10C protein. The first NLS (NLS1) is located near the succinylation site at K173, while the second NLS (NLS2) is adjacent to another succinylation site at K325. E. AC16 and H9c2 cells were transfected with wild-type and mutant constructs prior to hypoxic exposure. CPT1A was overexpressed to restore succinylation in hypoxic cells. Co-immunoprecipitation was performed using anti-FLAG antibodies to pull down TRMT10C and its binding partners. Abbreviations: Hypo: hypoxia; OE: overexpression; CPT: CPT1A. F. Protein structure modeling demonstrates the binding of KPNA4 to TRMT10C. G. Wild-type TRMT10C proteins, as well as human M3 and rat M2 mutants, were transfected into AC16 and H9c2 cells with KPNA4 knockdown. Western blotting was conducted to detect proteins in each cell fraction. Abbreviations: Ctrl: control; KD: knockdown; Mito: mitochondria; Nucl: nucleus; Cyto: cytoplasm.

Figure 5

TRMT10C suppresses TAFAZZIN and NLRX1 expression via m1A modification. A. High-throughput sequencing was performed to evaluate changes in gene expression following TRMT10C knockdown under both normoxic and hypoxic conditions. Abbreviations: Ctrl: control; KD: TRMT10C knockdown. B. Gene Ontology (GO) enrichment analysis was conducted on the biological processes related to differentially expressed genes (DEGs). C. M1A-IP sequencing was performed to examine changes in mRNA m1A levels after TRMT10C knockdown under hypoxic conditions. D. According to M1A-IP sequencing, the distribution of m1A modifications across different regions of mRNA was analyzed. E. GO enrichment analysis was conducted for biological processes associated with mRNAs exhibiting changes in m1A modifications. F. Intersection analysis was performed between DEGs and mRNAs with altered m1A modifications. G. M1A-IP sequencing revealed a reduction in m1A levels in the mRNA from the first exon of TAFAZZIN following TRMT10C knockdown. A similar reduction in m1A levels was observed in the mRNA from the seventh exon of NLRX1 after TRMT10C knockdown. Abbreviations: Hypo: hypoxia; Ctrl: control; KD: TRMT10C knockdown. H. A dot blot assay was conducted to detect overall m1A levels in cells after exposure to hypoxia and TRMT10C knockdown. The gray value of the dot blot was used to evaluate m1A levels. The same membrane was stained with 0.02% methylene blue (MB) as a loading control. I. An M1A-IP-PCR assay was performed to examine m1A levels in both TAFAZZIN and NLRX1 mRNA in cells following hypoxic exposure and TRMT10C knockdown. ***p < 0.001 vs. Control (n = 3); ###p < 0.001 vs. Hypoxia group (n = 3). J and K. PCR and western blot assays were conducted to assess the mRNA and protein levels of TAFAZZIN and NLRX1 in cells. ***p < 0.001 vs. Control (n = 3); ###p < 0.001 vs. Hypoxia group (n = 3).

Figure 6

m1A modification attenuates the stability of TAFAZZIN and NLRX1 mRNA. A. To assess the effect of m1A modification on mRNA stability, Actinomycin D was added to the cells to inhibit the synthesis of new mRNA. PCR was performed to evaluate mRNA levels at different time points. Abbreviations: Hypo: hypoxia; Ctrl: control; KD: TRMT10C knockdown. ***p < 0.001 vs. Control (n = 3). B. A RNA immunoprecipitation (RIP) assay was conducted in cardiomyocytes following hypoxic exposure and TRMT10C knockdown. Antibodies targeting YTHDF1, YTHDF2, and YTHDF3 were used to precipitate protein-mRNA complexes. Subsequently, PCR was performed to assess mRNA levels. ***p < 0.001 vs. Control (n = 3); ##p < 0.01 and ###p < 0.001 vs. Hypoxia group (n = 3). C. TRMT10C and YTHDF2 were either overexpressed or knocked down in cardiomyocytes under hypoxic conditions. PCR was conducted to detect the mRNA levels of TAFAZZIN and NLRX1. Abbreviations: T10C: TRMT10C; YT2: YTHDF2. ***p < 0.001 vs. Control (n = 3); #p < 0.05 and ###p < 0.001 vs. Hypoxia group (n = 3); &&&p < 0.001 vs. Hypoxia + TRMT10C overexpression group (n = 3). D. Western blotting was performed to measure the protein levels of TAFAZZIN and NLRX1. E. The schematic diagram illustrates the m1A modification sites in TAFAZZIN and NLRX1 mRNAs. F. In the luciferase reporter assay, we constructed a wild-type (WT) vector containing the cDNA of the first exon of TAFAZZIN and the seventh exon of NLRX1. Additionally, three mutants (MT) of TAFAZZIN were generated by substituting adenine with guanine: MT1 (A₃₀₅ to G), MT2 (A₄₁₃ to G), and MT1/2 (both A₃₀₅ to G and A₄₁₃ to G). Three mutants of NLRX1 were also constructed: MT1 (A₂₄₂₃ to G), MT2 (A₂₅₆₁ to G), and MT3 (both A₂₄₂₃ to G and A₂₅₆₁ to G). Relative luciferase activity was evaluated following various cell treatments. *p < 0.05, **p < 0.01, and ***p < 0.001 (n = 3).

Figure 7

TRMT10C influences cell proliferation, apoptosis, inflammatory response, and ROS levels in cardiomyocytes under hypoxia by regulating TAFAZZIN and NLRX1. A. AC16 and H9c2 cells were transfected with expression vectors for TAFAZZIN and NLRX1 to prevent their reduction under hypoxic conditions. Additionally, TRMT10C was knocked down either alone or in combination with TAFAZZIN and NLRX1. Cell viability was assessed using the CCK-8 assay. Abbreviations: T10C: TRMT10C; TAFA: TAFAZZIN; KD: Knockdown; OE: Overexpression. B. Cell proliferation was evaluated using EdU staining. Red fluorescence indicates EdU-labeled nuclei, while blue fluorescence corresponds to DAPI-stained nuclei. The cell proliferation rate is expressed as the percentage of EdU-labeled nuclei relative to DAPI-stained nuclei. The scale bar in the figures represents 10 μm. C. Cardiomyocytes were trypsinized, incubated with Annexin V-FITC and propidium iodide (PI), and analyzed by flow cytometry using FlowJo software. D. Levels of IL-1β and TNF-α in the culture medium were measured using ELISA detection kits. E. Reactive oxygen species (ROS) levels in the cells were assessed using flow cytometry with the fluorescent probe DCFH-DA. For all studies, ***p < 0.001 vs. Control (n = 3); #p < 0.05, ##p < 0.01, and ###p < 0.001 vs. Hypoxia group (n = 3); &p < 0.05, &&p < 0.01, and &&&p < 0.001 vs. Hypoxia + TRMT10C knockdown group (n = 3).

Figure 8

TAFAZZIN and NLRX1 regulate mitophagy in cardiomyocytes under hypoxia. AC16 and H9c2 cells were transfected with expression vectors for TAFAZZIN and NLRX1 to prevent their reduction under hypoxic conditions. Additionally, TRMT10C was knocked down in AC16 and H9c2 cells under these conditions. A. Co-immunoprecipitation (Co-IP) and western blot assays were performed to evaluate the interaction between PINK1 and Parkin, as well as their protein levels in AC16 and H9c2 cells. B. The protein levels of LC3B in the mitochondrial fraction were assessed by western blotting, with COV IV protein levels serving as the loading control. C. The overall protein levels of LC3B and p62 in the cells were detected by western blotting. D. A lysosomal inhibitor, bafilomycin A1 (BafA1, 20 nM), was applied to block the autophagy/mitophagy flux. Following this treatment, the overall protein levels of LC3B and p62 in the cells were analyzed by western blotting. E. Immunofluorescence using the MitoMark probe and anti-LC3 antibody was conducted to observe the interaction between mitochondria (green fluorescence) and autophagosomes (red fluorescence). The scale bar in the figures represents 10 μm. Abbreviations: T10C: TRMT10C; TAFA: TAFAZZIN; KD: Knockdown; OE: Overexpression.

Figure 9

TAFAZZIN and NLRX1 regulate inflammatory signaling in cardiomyocytes under hypoxia. A. AC16 and H9c2 cells were transfected with expression vectors for TAFAZZIN and NLRX1 to prevent their reduction under hypoxic conditions. Additionally, TRMT10C was knocked down in AC16 and H9c2 cells under these conditions. Immunofluorescence analysis was performed to observe the distribution of p65 in the cytoplasm and nucleus. The scale bar in the figures represents 10 μm. B. Following similar cell treatments, western blotting was conducted to detect NF-κB (p65) in the cell nucleus, as well as p-p65, p65, NLRP3, ASC, and Caspase-1 in the cells. Co-immunoprecipitation (Co-IP) assays were performed to assess the interaction between NLRP3 and ASC.

Figure 10

Knockout (KO) of TRMT10C attenuates cardiac damage in a mouse model of CME. A. The flowchart illustrates the process of TRMT10C knockout in C57BL/6 mice. B. A PCR assay was conducted to detect the mRNA levels of TRMT10C, TAFAZZIN, and NLRX1 in the sham and CME models of TRMT10C-Ctrl and TRMT10C-KO mice. C. Western blotting was performed to assess the protein levels of TRMT10C, TAFAZZIN, and NLRX1 in the sham and CME models of TRMT10C-Ctrl and TRMT10C-KO mice. D. Immunohistochemistry was conducted to detect TRMT10C, TAFAZZIN, and NLRX1 proteins in the sham and CME models of TRMT10C-Ctrl and TRMT10C-KO mice. The scale bar in the 200X images represents 120 μm, while the scale bar in the 400X images represents 50 μm. E. Echocardiography was used to measure the left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) to evaluate heart function. **p < 0.01 and ***p < 0.001 (n = 6). F. A TUNEL assay was conducted to assess apoptosis in heart tissues. The scale bar in the figures represents 50 μm. **p < 0.01 and ***p < 0.001 (n = 6).

Figure 11

Mechanistic diagram illustrating the detrimental effects of TRMT10C in the nucleus under hypoxic conditions via m1A modification. CPT1A is a succinyltransferase responsible for protein succinylation. The reduction in CPT1A and succinyl-CoA levels inhibits the succinylation of TRMT10C in cardiomyocytes during CME. This suppression of TRMT10C succinylation enhances its binding to KPNA4, facilitating the transport of TRMT10C from the cytoplasm to the nucleus instead of to the mitochondria. In the nucleus, TRMT10C promotes the decay of TAFAZZIN and NLRX1 through m1A modification. The reduction of TAFAZZIN and NLRX1 is associated with impaired mitophagy, which may lead to increased apoptosis and reactive oxygen species (ROS) production. Additionally, the decrease in TAFAZZIN and NLRX1 facilitates the activation of inflammatory signaling pathways, such as NF-κB and NLRP3. The reduced levels of NLRX1 promote the interaction between ASC and NLRP3, thereby activating the NLRP3 signaling pathway and inducing the production of inflammatory factors.