Fig 2: Proteasome inhibition did not change expression levels of LONP1 but caused a reduction of UCP2 and VDAC1 protein in neuronal cells. A, B: Representative Western blot images of LONP1, UCP2 and VDAC1 after 100 nM BTZ and CFZ treatment for 3 and 24 h in human neuronal cells. C-E: Quantification of LONP1, UCP2 and VDAC1 after 100 nM BTZ and CFZ treatment for 3 and 24 h in human neuronal cells. Data represent means ± SEM, n = 3–4, *p < 0.05 versus control group.

Fig 3: Loss of LonP1 in cardiomyocytes leads to metabolic reprogramming through enhancing glycogenesis and amino acid metabolism. (A) Western blot analysis demonstrating LonP1 stable knockdown in H9c2 cells. (B) LonP1 stable knockdown increases intracellular ROS (H2O2) production in H9c2 cells, as measured using ROS assay kit (DCFH-DA). Data are plotted as percentages of increase in the MFI and are shown as means ± SEM (n = 3, ***P < 0.001). (C) Mitochondrial superoxide levels of control and LonP1 knockdown stable H9c2 cells were detected by MitoSOX staining and analyzed by flow cytometry. Data are plotted as percentages of alteration of the MFI and are shown as means ± SEM (n = 3, ***P < 0.001). (D) LonP1 stable knockdown reduces the mitochondrial membrane potential of H9c2 cells. Cells were stained with JC-1 and analyzed by flow cytometry. The ratio of fluorescence intensities Ex/Em = 490/590 and 490/530 nm (FL590/FL530) were recorded to show the mitochondrial membrane potential level of each sample. Data are presented as means ± SEM (n = 3, ***P < 0.001). (E and F) The intact cellular OCR of H9c2 cells in the indicated conditions were measured in real time using the Seahorse XF-96 Extracellular Flux Analyzer. Basal OCRs were measured at 3 time points, followed by sequential injection of the ATP synthase inhibitor oligomycin (1 μM), the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (1 μM), the complex I inhibitor rotenone (1 μM), and the complex III inhibitor antimycin A (1 μM). Data are represented as means ± SEM (***P < 0.001). (G) Heatmap showing protein abundance changes in some proteins involved in gluconeogenesis, aspartate and glutamate metabolism, and the biosynthesis of amino acids obtained via high-throughput quantitative proteomics of enriched heart tissues. The relative abundance changes in WT and cLKO mice are expressed using the protein ratio in relation to each control at 10 and 20 weeks of age. (H and I) Western blot and quantification of representative protein levels of ATF4 and PCK2 in WT and cLKO hearts at 4 and 10 weeks, using GAPDH as the loading control. Data are represented as means ± SEM (n = 3, ***P < 0.001). (J) qRT-PCR analysis the transcripts of Suclg2, Pck2, Gls, Phgdh, and Psat1 in WT and cLKO hearts at 10 weeks. Data are represented as means ± SEM (n = 3, *P < 0.05, **P < 0.01, and ***P < 0.001).

Fig 4: Cardiomyocyte-specific deletion of LonP1 causes pathological heart remodeling and impaired heart function. (A) Kaplan–Meier survival curves in WT and cLKO mice (n = 20). (B) Heart weight-to-body weight ratios in WT and cLKO mice of 2, 4, 6, 8, 10, 15, 20, 40, and 60 weeks of age (n = 6, *P < 0.05). (C) Heart morphologies of 10-, 20-, and 60-week-old WT and cLKO mice. (D) Hematoxylin and eosin staining of WT and cLKO hearts of 10-, 20-, and 60-week-old cLKO and WT mice. (E) Representative images showing Sirius Red staining of the cardiac tissues from 10-, 20-, and 60-week-old cLKO mice and their littermate controls (left). Scale bars, 100 μm. Quantitative analysis of myocardial fibrosis in 10-, 20-, and 60-week-old WT and cLKO mice (right). Data are represented as means ± SEM (n = 3, ***P < 0.001). (F to J) Representative images of transthoracic M-mode echocardiographic tracings and echocardiographic parameters of 10-, 20-, and 60-week-old WT and cLKO mice. IVS:D, interventricular septal thickness; LVID:D, LV internal diameter in diastole; EF, ejection fraction; FS, LV fraction shortening. Data are represented as means ± SEM (n = 8 to 10, *P < 0.05 and **P < 0.01). See also Fig. S5.

Fig 5: LonP1 deletion impairs MAMs. (A) Western blot analysis of the LonP1 protein level in homogenate (WCL), cytosolic (Cyto), mitochondria (Mito), ER, and MAM fractions in 4-week-old LonP1-deficient heart. VDAC1 was used as a mitochondrial marker, PDI was used as an ER marker, and IP3R3 was used as a marker of MAM. (B) Subcellular localization of ER and mitochondria in control and LonP1 knockdown H9c2 cells. Representative images show colocalization of mitochondria (MitoTracker Red) and the ER marker protein PDI (green). Scale bar, 5 μm. The amount of ER colocalizing with mitochondria is represented by Pearson’s correlation coefficient. Data are represented as means ± SEM (n = 3, ***P < 0.001). (C) Representative TEM images indicating the ER and mitochondrial contacts in the hearts of 4-week-old cLKO mice and their littermate controls. Arrows indicate the contacts between ER and mitochondria. Scale bars, 50 nm. See also Fig. S1. IF, immunofluorescence.