LonP1 Links UPR mt and UPR ER to Regulate Heart Function

Protein quality control is pivotal to cellular homeostasis and integrity of cardiomyocytes for maintenance of normal heart function. The unfolded protein response (UPR) is an adaptive process to modulate protein quality control in the endoplasmic reticulum (ER) and mitochondria, and is accordingly termed UPR ER and UPR mt , respectively. Lon protease (LonP1) is a highly conserved mitochondrial protease to modulate UPR mt , which is involved in regulating metabolism, mitophagy, and stress response. However, whether LonP1 regulates UPR ER remains elusive. To investigate the regulation of protein quality control in cardiomyocytes, we generated cardiac-specic LonP1 deletion mice. Our ndings show that LonP1 deciency caused impaired mitochondrial respiratory function and fragmentation. Surprisingly, both UPR ER and UPR mt is substantially induced in LonP1-deletion heart suggesting of LonP1 as a novel regulator of UPR ER ; however, the activation of UPR ER occurs earlier than UPR mt in response to LonP1 deletion. Consequently, cardiac-specic LonP1 deciency causes aberrant metabolic reprogramming of cardiomyocytes, pathological heart remodeling, as well as impeded heart function. We uncovered the novel function of LonP1 as an UPR mt mediator, and reciprocal orchestration of UPR mt and UPR ER and mitochondrial dynamics regulated by LonP1 in the cardiomyocytes that is critical to maintain heart function, which offers exciting new insights into the potential therapeutic strategy for heart failure. mainly involved in the degradation of misfolded and aggregated abnormal proteins within the matrix. Here, we show that LonP1 ablation causes many compensatory responses in cLKO mice, including activation of the UPR ER . Strikingly, the UPR ER is activated prior to the UPR mt , suggesting that LonP1 expression is a ne-tuning sensor of the UPR ER and UPR mt . Upregulation of both the UPR ER and UPR mt in cardiomyocytes of LonP1-deletion mice results in well-tolerating mice with no major changes of longevity under unstressed conditions. In addition to the activation of both the UPR ER and UPR mt in the hearts of cLKO mice, FGF21, a key regulator of glucose and lipid metabolism and energy balance, is also increased dramatically to compensate for the deletion of LonP1 expression. The increased expression of FGF21 further ensures cLKO mice survival under unstressed conditions. protein and mRNA expressions are stimulated by LonP1 ablation, indicating a synergistic effect to strengthen PCK2 activity in LonP1-decient cardiomyocytes. Moreover, we found that ATF4 was upregulated, indicating that PCK2 could be transcriptionally regulated by ATF4 through regulating a putative ATF/CRE composite site containing the PCK2 promoter, which caused transcriptional upregulation of PCK2 Our results argue that ATF4-induced PCK2 expression could promote LonP1-deletion cardiomyocytes survival in energy- and intermediate metabolites-limited environments. These results suggest that LonP1-deletion cardiomyocytes may activate ATF4/PCK2-mediated metabolic reprogramming to orchestrate energy and intermediate metabolite homeostasis, which contributes to myocardial cell survival.


Introduction
Mitochondria are important intra-cellular organelles present in most eukaryotic cells, which play a crucial role in ATP production through oxidative phosphorylation (OXPHOS) as well as in determining cell survival or death (1,2). The OXPHOS dysfunction causes a group of multi-systemic diseases which are often progressive or fatal disorders with defects in the cellular ATP supply (3). Given that the heart is highly dependent on aerobic metabolism, it is particularly susceptible to mitochondrial dysfunction.
Energy depletion has been highlighted as an important contributor to the pathology of heart failure (4). There is accumulating evidence indicating that increased oxidative stress is involved in cardiac hypertrophy and dysfunction (5), and reducing of oxidative stress prevents left ventricular remodeling and dysfunction (5). The ATP-dependent Lon protease (LonP1) belongs to the AAA + family of proteins (ATPase associated with various cellular activities), which exhibits a quality-control protease in selectively eliminating certain abnormal proteins and cellular stress responses in mitochondria (6)(7)(8).
LonP1 was upregulated in hypoxia-induced cardiomyocytes and downregulation of LonP1 reduced hypoxia-induced cardiomyocyte apoptosis through decreasing reactive oxygen species (ROS) production (14). In addition, overexpression of LonP1 enhanced ROS generation and induced apoptosis of cardiomyocytes under normoxic conditions (14). Despite its high conservation throughout evolution, the role of LonP1 in vivo in physiological condition and the pathogenesis of human diseases remains largely unknown. Recent studies reported that LonP1 protein and/or mRNA levels are increased in a variety of tumors (15)(16)(17)(18)(19) and LonP1 controls tumor bioenergetics by reprogramming mitochondrial OXPHOS complexes (20). Furthermore, a link between LonP1 and human diseases has been reported in several genetic diseases (21-27). Heart failure is closely related to mitochondrial dysfunction; however, the underlying molecular mechanism remains poorly understood. In a mouse model with pressure overload heart failure, the protein carboxylation, tyrosine nitration and cysteine oxidation of LonP1 increased and the protease activity markedly reduced in the isolated mitochondria, which led to mitochondrial respiration de ciency and left ventricular contractile dysfunction (28). By using haploinsu ciency of LonP1 (Lonp1 +/− ) and cardiac-speci c overexpression of LonP1 (LonTg) mice model, Venkatesh et al found that upregulation of LonP1 alleviates cardiac injury by preventing oxidative damage of proteins and lipids, preserving mitochondrial redox balance and reprogramming bioenergetics by reducing Complex I content and activity (29). However, the role of LonP1 in the pathogenesis of cardiomyopathy and cardiac dysfunction is still unclear.
The unfolded protein response (UPR) is a protective or an adaptive process to modulate protein quality control in the endoplasmic reticulum (ER) and mitochondria, and is accordingly termed UPR ER and UPR mt , respectively. Both UPR ER and UPR mt cause global alteration of transcription networks to increase ER and mitochondrial localized molecular chaperones and proteases to promote the recovery of organellar protein homeostasis (proteostasis) of cell to maintain their function properly (30-32). However, the UPR mt also promotes a rewiring of cellular metabolism that includes an increase in glycolysis and amino acid catabolism genes with a simultaneous repression of TCA-cycle-and OXPHOS-encoding genes potentially to relieve mitochondrial stress and/or alter cellular metabolism to promote survival (33). Nevertheless, it may have important clinical implications, for example, mitochondrial changes in response to ER-stress conditions have been described for several human diseases, including type II diabetes and Alzheimer's disease (34).
In this study, we developed a mouse model lacking LonP1 in cardiomyocyte to characterize its role in the heart. We demonstrate that both UPR ER and UPR mt are activated in the heart of LonP1 de cient mice.
Surprisingly, UPR ER is induced earlier than UPR mt . Moreover, our results show that cardiac-speci c LonP1 deletion leads to mitochondrial fragmentation and dysfunction. Finally, our data show that LonP1 de ciency in the heart results in aberrant metabolic reprogramming and pathological heart remodeling and eventually progresses to heart failure. Collectively, these ndings identify LonP1 as a novel regulator of cardiomyocyte fate and offers exciting new insights into the potential therapeutic strategy for heart failure.

Mice
All mice used in this study were housed at the Wenzhou Medical University Animal Center in a speci c pathogen free (SPF) facility with individually ventilated cages. The room in the animal facility has controlled constant temperature (20-25°C) and humidity (30-70%), with a 12-h light/dark cycle. Mice before sending to our lab. Cell lines were also routinely tested and con rmed to be mycoplasma free during this study.

Hematoxylin and eosin staining (H&E)
Heart tissues were xed, embedded in para n, and sectioned following standard protocols. The sections were stained with hematoxylin for 15 min and washed in running tap water for 20 min. The sections were then counterstained with eosin from 30 sec to 1 min. Finally, the sections were dehydrated in 95% and absolute ethanol and mounted in Permount (Thermo Fisher Scienti c).

Sirius red staining
Sirius Red staining was performed using a Picro Sirius Red staining kit according to the manufacturer's instructions (Thermo Fisher Scienti c). Brie y, para n-embedded heart sections were depara nized /hydrated, and a series of washes was performed, followed by staining with Picro Sirius Red for 1 h and washing for 1 min in 0.1 N HCl. Slides were dehydrated and mounted in synthetic resin.

Masson Trichrome Stain (Connective Tissue Stain)
Masson trichrome staining was performed using a Trichrome Stain Kit (Abcam, ab150686) according to the manufacturer's instructions. Brie y, para n-embedded heart section was depara nized/hydrated, and a series of washes was performed. Then, the slides were placed in preheated Bouin's uid for 60 min, followed by a 10 min cooling period. Equal parts of Weigert's (A) and Weigert's (B) were mixed, and slides were stained with working Weigert's iron hematoxylin for 5 min, then rinsed slide in running tap water for 2 min. Then, Biebrich Scarlet /Acid Fuchsin solution was applied to the slides for 15 min. The slides were rinsed in distilled water and differentiated in phosphomolybdic/phosphotungstic acid solution for 10-15 min or until collagen was not red. Without rinsing, aniline blue solution was applied to the slides for 5-10 min, and the slides were rinsed in distilled water. Then, acetic acid solution (1%) was applied to the slides for 3-5 min. Finally, the slides were dehydrated very quickly in 2 changes of 95% alcohol, followed by 2 changes of absolute alcohol, then cleared in xylene and mounted in synthetic.

Echocardiography
An experienced researcher blinded to the study performed echocardiographic evaluations to avoid biases. A Vevo 3100 high-resolution micro-ultrasound system (FUJIFILM Visual Sonics Inc.) was used to determine heart function and ventricular dimensions. For this procedure, 1.5% iso urane was used to anesthetize mice, and then the mice were placed on a heating table in a supine position. M-mode and two-dimensional (2D) images were recorded along a short-axis view from the mid-left ventricle at the tips of the papillary muscles. The interventricular septal thickness (IVS) and left ventricle (LV) internal diameter (LVID) were measured at end-diastole and end-systole. The fractional shortening (FS) and ejection fraction (EF) were calculated from the LV dimensions in the 2D short-axis view.

Transmission electron microscopy (TEM)
The heart tissue samples were xed with 2.5% (v/v) glutaraldehyde buffer for 15 min at room temperature, followed by overnight at 4°C. The following day, the samples were treated with 1% osmium tetroxide and 0.1 M cacodylate buffer for 1 h. Samples were stained in 1% uranyl acetate and dehydrated with ethanol. Epoxy-resin-embedded samples were sectioned, and placed on formvar/carbon-coated copper grids. Grids were stained with uranyl acetate and lead nitrate. Then the samples were examined with a JEM1400 transmission electron microscope (JEOL).
Isolation and puri cation of mitochondria

Mitochondrial complexes activity assay
The enzymatic activities of the individual mitochondrial respiratory chain (MRC) complexes I, II, III and IV were determined using the mitochondrial fraction from hearts of Cont and cLKO mice as described (35).

Western blot analysis
Tissue samples were washed 3 times with ice-cold PBS and homogenized using a homogenizer (Kinematica AG) in 1.5 ml tissue RIPA lysis buffer (50 mM Tris-HCl, pH 7.4, 1.0% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl) supplemented with a protease inhibitor cocktail tablet, and PhosSTOP phosphatase inhibitor cocktail tablet. Tissue homogenates were cleared by centrifugation at 18,000 ⋅ g for 25 min at 4°C, and the supernatants were collected in clean microcentrifuge tubes on ice. A similar procedure was used to prepare whole-cell extracts from cells. Brie y, cells were washed with icecold PBS and lysed in RIPA lysis buffer supplemented with protease and phosphatase inhibitors on ice for 20 min, followed by centrifugation at 18,000 ⋅ g for 30 min at 4°C, and the supernatants were collected. Protein concentrations of the tissue homogenates or whole-cell extracts were determined using the Pierce BCA protein assay kit (Thermo Fisher Scienti c).
Tissue or cell extracts equivalent to 20 µg total protein were resolved in 10% SDS-PAGE gels followed by electrophoretic transfer onto a nitrocellulose membrane (Bio-Rad) in Tris-glycine buffer. Blots were blocked at room temperature for 1.5 h in 5% nonfat milk in Tris-buffered saline (TBS)-Tween (TBST) on a shaker, and then incubated with the primary antibodies as indicated in 5% nonfat milk TBST overnight at 4°C. The membrane was washed in TBST at least 3 times for 10 min and then incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse immunoglobulin G at room temperature for 1 h with gentle shaking. Immunoreactive proteins were detected by ECL reagent according to the manufacturer's protocol (Thermo Fisher Scienti c). The optical density of the Western blot signals was quanti ed using the National Institutes of Health (USA) ImageJ software.

RNA preparation and quantitative Real-Time PCR (qRT-PCR)
The total RNA was extracted from heart tissues using TRIzol (Life Technologies) according to the manufacturer's instructions. The total RNA (1 µg) from each ample was used to synthesize rst-strand cDNA by reverse transcription using the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara), according to the manufacturer's instructions. The cDNA produced was subsequently used as a template for qRT-PCR. qRT-PCR analysis, which was performed using a 2 µl cDNA /20 µl reaction volume on a CFX Connect™ real-time PCR detection system (Bio-Rad) using SYBR Green according to the manufacturer's protocol. The primer sequences used for qRT-PCR are listed in Supplemental Table 2. Thermal cycling was performed using the following parameters: 95°C for 10 min, then 45 cycles of denaturation at 95°C for 10 sec and extension at 60°C for 30 sec. The threshold cycle number (CT) was recorded for each reaction. The CT value was normalized to that of GAPDH. Each sample was assayed in triplicate and repeated at least three times.

RNA interference
psi-LVRU6P vectors (carrying a puromycin antibiotic resistance gene) containing control and shRNA oligonucleotides were purchased from Gene Copoeia (Rockville, MD, USA). The two shRNAs sequences are listed in Supplemental Table 3.

Lentivirus production and transduction
Lentiviral production and transduction were conducted according to the manufacturer's instructions (Gene Copoeia). Brie y, lentiviral vectors and LonP1 or control shRNA were packed with the Lenti-Pac™ HIV Expression Packaging Kit using HEK293T cells and incubated overnight, followed by replacement of the old culture medium with fresh DMEM supplemented with 5% heat-inactivated FBS and penicillinstreptomycin. Titer Boost reagent (1/500 volume) was added to the culture medium and incubated at 37°C in a humidi ed incubator with 5% CO 2 . At 48 h post transfection, the supernatants containing lentivirus particles were collected, ltered through 0.45 µm syringe lters (Millipore), and used immediately to infect H9c2 cells. To select stably transfected cells, the old medium was replaced by fresh complete medium containing puromycin (2.0 µg/ml) every 3 days until drug-resistant colonies become visible. The knockdown of LonP1 was validated by qRT-PCR and Western blot analysis. Positive clones with stable knockdown of LonP1 were expanded and maintained in medium supplemented with 2.0 µg/ml puromycin.

Fluorescence detection in cultured cells
For measuring the colocalization of LonP1 with mitochondria, cells were stained with 50 nM MitoTracker Green for 30 min at 37°C and then treated with 4% paraformaldehyde for 5 min, followed by incubation with mouse anti-LonP1 antibody at 4°C overnight. Slides were then incubated with Alexa Fluor 555conjugated donkey anti-rabbit antibodies (A-31572, Invitrogen) at room temperature for 2 h. Cells were then stained with DAPI for 30 min at room temperature. Finally, xed or living cells were visualized by confocal microscopy with a Leica TCS SP8 microscope with a 633 numerical aperture [NA] 1.35 oil objective. To determine mitochondrial morphology, 100 cells were randomly selected for quantitative analysis and visually scored into four classi cations (tubular, short tubular, fragmented, and large spherical).

Cell proliferation assay
Control and LonP1 knockdown H9c2 cells were seeded into a 96-well plate at a density of 2 × 103 cells per well and incubated overnight. The viability of H9c2 cells was determined at 1, 2, 3, 4 and 5 days using an MTT Cell Proliferation and Cytotoxicity Assay Kit according to the manufacturer's protocol.

XF extracellular ux analyzer experiments
The intact cellular OCR of LonP1 stable knockdown and control H9c2 cells was measured using a Seahorse XF-96 Extracellular Flux Analyzer (Seahorse Bioscience) as described previously (40). Results were obtained by performing three independent experiments with 8 replicates of LonP1 stable knockdown and control H9c2 cells. After the assay was completed, the protein concentration was determined by a BCA protein assay kit to normalize the OCR according to the manufacturer's instructions.
Brie y, Lon stable knockdown H9c2 or vector control cells were trypsinized, washed with DMEM, and incubated with DCFH-DA at a nal concentration of 10 µM in DMEM for 30 min at 37°C, then the cells were washed three times with DMEM. Mitochondrial superoxide levels were detected by MitoSOX staining according to the manufacturer's protocol (Thermo Fisher Scienti c). Brie y, control and LonP1 stable knockdown H9c2 cells were incubated with 5 µM MitoSOX Red for 10 min at 37°C and then subjected to ow cytometry analysis. Data are plotted as median uorescence intensities (MFI). The mitochondrial membrane potential was analyzed by JC-1 staining according to the manufacturer's protocol (Beyotime).
Brie y, LonP1 stable knockdown H9c2 or vector control cells were stained with 2.0 µM JC-1 in complete medium and incubated for 20 min at 37°C in the dark. To remove excess JC-1, cells were washed once with prewarmed PBS and pelleted by centrifugation. Cell pellets were resuspended by gently icking the tubes, and 500 µl PBS were added to each tube. Cell samples were analyzed immediately using a BD Accuri TM C6 Plus ow cytometer (BD Biosciences). All experiments were carried out at least 3 times independently, with 3 technical replicates in each experiment.

Statistical analysis
All values are presented as the mean ± SEM, obtained from at least three independent experiments, and two-tailed Student's t-test was used for comparison between two groups. All statistical analyses were performed by GraphPad Prism 8.0 or SPSS 20.0. Statistical signi cance was considered as P < 0.05, and denoted as follows in the gures: *P < 0.05; **P< 0.01; ***P < 0.001; N.S., not signi cant.

Generation of cardiomyocyte-speci c LonP1 knockout mice
To investigate the function of LonP1 in the heart, we generated mice de cient in LonP1 speci cally in the heart by mating Lonp1 LoxP/LoxP mice with cardiomyocyte-speci c-MHC-Cre (αMHC-Cre) mice (Fig. 1A, B). The loss of LonP1 was con rmed at both the transcript and protein levels. Levels of Lonp1 mRNA and protein were extremely low and almost undetectable in the heart of LonP1 conditional knockout mice (cLKO) compared with that in littermate controls (Lonp1 LoxP/LoxP , Cont) after two weeks (Fig. 1C, D).
However, we found that there was no difference in LonP1 expression in the other organs of cLKO and Cont mice (Fig. 1E). Thus, our results indicate that LonP1 was e ciently and speci cally deleted in the cardiomyocytes of cLKO mice.
Cardiomyocyte-speci c deletion of LonP1 causes pathological heart remodeling and impaired heart function To determine the effects of cardiomyocyte-speci c deletion of LonP1 on the heart, we rst monitored survival of in Cont and cLKO mice. As shown in Fig. 2A, cLKO mice were viable but had a dramatically shortened life span. We next examined heart function from an early age up to 60 weeks of age in cLKO and Cont mice. There were no signi cant differences observed in the heart-to-body weight ratio between Cont and cLKO mice before 10 weeks (Fig. 2B). However, the cLKO mice at 20 weeks developed cardiomyopathy characterized by increased heart size with age ( Fig. 2C and Supplementary Fig. 1A, B). Based on H&E staining of Cont and cLKO mice hearts at different ages, we found a slight increase in the left ventricular (LV) wall thickness in 20-week-old cLKO mice and a signi cant increase in the cardiac size and LV dilation in 60-week-old cLKO mice ( Fig. 2D and Supplementary Fig. 1A, B). We observed myocardial brosis, as shown by Sirius Red and Masson's trichrome staining, in the hearts of cLKO mice at 20 and 60 weeks (Fig. 2E).
Serial echocardiography analyses revealed progressive heart dysfunction in cLKO mice, which became apparent at 20 weeks and was characterized by a statistically signi cant increase of the interventricular septal depth (IVS:D) and left ventricular internal dimension (LVID), although the left ventricular ejection fraction (EF) was still normal compared with the Cont mice. However, cLKO mice at 60 weeks exhibited severe LV dilation, accompanied by signi cant EF and FS reduction (Fig. 2F). In conclusion, the cardiomyocyte-speci c deletion of LonP1 leads to dilated cardiomyopathy (DCM) and heart failure, which reveals that LonP1 is indispensable for heart function.

LonP1 deletion leads to mitochondrial fragmentation
Mitochondrial dynamics is critical for mitochondrial function, and the balance between ssion and fusion ensures the health of cardiac myocyte. Cardiomyocyte speci c deletion of fusion proteins Mitofusin 1 and 2 (MFN1 and MFN2), optic atrophy 1 (Opa1), and OPA1-processing peptidases YME1L and OMA1, in adult mice causes mitochondrial fragmentation, and which results in the mice developing dilated cardiomyopathy and heart failure (37)(38)(39)(40)(41). We therefore determined the protein levels of the mitochondrial ssion and fusion related proteins dynamin-related protein 1 (DRP1), MFN1 and MFN2, as well as OPA1 and OMA1 in cLKO mice by immunoblotting.
To validate the effect of LonP1 ablation to the mitochondrial morphology, we knocked down LonP1 stably in H9c2 rat cardiomyocytes. Using confocal microscopy, we observed signi cant mitochondrial fragmentation in shLonP1 H9c2 cells compared with shCont H9c2 cells (Fig. 3A, B). We further con rmed that the loss of LonP1 in cardiomyocytes of 4-and 10-week-old mice caused mitochondrial fragmentation by using TEM (Fig. 3C, D).
Furthermore, we observed a signi cant reduction in L-OPA forms a and b, and S-OPA forms c and e were increased dramatically, whereas S-OPA1 form d remained unchanged in the hearts of 4-and 10-week-old cLKO mice in comparison to control mice. OMA1 is a protease that cleaves OPA1 from L-OPA1 to S-OPA1 (42); however, no changes were observed in OMA1 expression in the hearts of 4-and 10-week-old cLKO mice (Fig. 3E, F). Next, we examined Drp1, MFN1 and MFN2 in the hearts of 4-and 10-week-old cLKO and control mice. We found that there was no difference in protein expression of Drp1, MFN1, and MFN2 in the hearts of 4-week-old cLKO and control mice, whereas the protein expression of Drp1 was statistically signi cantly increased, and MFN1 was statistically signi cantly decreased in the hearts of 10-week-old cLKO mice compared with control, while there were no differences in MFN2 (Fig. 3G, H). Notably, there was no difference in any of these proteins involved in mitochondrial fusion and ssion in the hearts of 2week-old cLKO mice and their littermate controls (Supplementary Fig. 2A,B).
These results together indicate that LonP1 ablation in cardiomyocytes promotes OPA1 processing and Drp1 expression, as well as reduces MFN1 expression to enhance mitochondrial fragmentation.

Loss of LonP1 induces UPR ER prior to UPR mt
To further understand the effects of LonP1 deletion to mitochondria and ER, we subsequently examined the UPR ER and UPR mt at different postnatal stages of mice. To test this, we determined the expression level of UPR ER -related proteins in cLKO mice and controls. We observed that inositol-requiring enzyme 1α (IRE1α), eukaryotic initiation factor 2α subunit (eIF2α), and the phosphorylation of eIF2α (p-eIF2α), activating transcription factors 4 and 6 (ATF4 and ATF6), increased signi cantly in response to LonP1 depletion in 4-and 10-week-old cLKO mice (Fig. 4A and B). In addition, we observed that protein disul de isomerase (PDI) remained unchanged in 4-week-old cLKO and control mice, whereas the PDI protein level was signi cantly increased in 10-and 60-week-old cLKO mice (Fig. 4A, B and Supplementary Fig. 3A). Interestingly, ATF4 protein level remained unchanged, whereas ATF6 expression increased signi cantly in 60-week-old cLKO and control mice (Supplementary Fig. 3A).
We next examined the UPR mt -related proteins in cLKO and control mice. We found that ClpX, Tid1-L/S, HSP60, and the ATPase family gene 3-like 2 (AFG3L2) protease involved in the turnover of unfolded proteins, were signi cantly increased in 10-week-old cLKO mice ( Fig. 4C and D). Surprisingly, the UPR mtrelated proteins remained unchanged in 4-week-old cLKO mice ( Fig. 4C and D). We further observed that ClpX, Tid1-L/S, and AFG3L2 protein levels were dramatically upregulated in 60-week-old cLKO mice, whereas there is no difference in HSP60 expression ( Supplementary Fig. 3B). Notably, we observed that UPR ER -and UPR mt -related protein levels remained unchanged between 2-week-old cLKO mice and their littermate controls ( Supplementary Fig. 4A and B). These results suggest that the cardiomyocyte-speci c loss of LonP1 activates both UPR ER and UPR mt ; however, the activation of UPR ER is more sensitive than that of UPR mt to the loss of LonP1 in cardiomyocytes.
We also evaluated the expression of broblast growth factor 21 (FGF21), a marker of mitochondrial stress (43,44), in 4-,10-, and 60-week-old Cont and cLKO mice, and we observed a strong increase of FGF21 protein levels in the hearts of 4-,10-, and 60-week-old cLKO mice (Fig. 4E, F and Supplementary   Fig. 3C). Like the case for UPR ER -and UPR mt -related proteins, there was no difference in FGF21 protein levels between 2-week-old cLKO mice and controls ( Supplementary Fig. 4C).
Taken together, our results reveal that LonP1 ablation in cardiomyocytes activates both UPR ER and UPR mt ; however, LonP1-deletion induces UPR ER prior to UPR mt . Although LonP1 located predominantly in the mitochondrial matrix, it may play a potential pivotal role in maintaining the homeostasis of the ER and mitochondria. Our ndings also indicate that cytokine FGF21 is, indeed, an early marker of mitochondrial dysfunction, suggesting that FGF21 could be a direct target of UPR mt .

Cardiomyocyte-speci c deletion of LonP1 leads to abnormal mitochondrial morphology and dysfunction
We proceeded to investigate the mitochondrial morphology and respiratory chain function in the mitochondria of the hearts of cLKO mice. Similarly, TEM analysis showed increased fragment mitochondria in the LonP1-deletion hearts, which indicated abnormal mitochondrial dynamics (Fig. 5A).
Moreover, we observed distorted mitochondrial morphology in cLKO mice hearts (Fig. 5A). We further observed extensive electron-dense aggregates within the mitochondria of cLKO mice hearts but not in other cell organelles, and no inclusions were observed in Cont mice (Fig. 5A). In the heart mitochondria of 20-week-old cLKO mice, the aggregates were signi cantly increased. Moreover, we found that some large mitochondria were scattered, with dense deposits of different sizes lling the mitochondrial matrix, and the mitochondrial cristae signi cantly decreased or even disappeared in the hearts of 60-week-old cLKO mice (Fig. 5A).
We next examined the steady-state level of assembled OXPHOS complexes by blue native PAGE (BN-PAGE) analysis and subsequent examination of MRC enzyme activities. We observed a signi cant decrease of the super complex (SC) and complex I, III, and IV levels in the heart tissue of both 10-and 20- week-old cLKO mice (Fig. 5B); however, the complex II and V levels only decreased slightly in the heart tissue of both 10-and 20-week-old cLKO mice (Fig. 5B). The enzymatic activities of complex I, III, and IV decreased dramatically in 10-week-old cLKO mice and which was further decreased in 20-week-old cLKO mice (Fig. 5C).
We also detected the protein expression of several MRC enzyme subunits by Western blotting and carried out quantitative analysis. Consistent with the results of BN-PAGE and MRC enzyme activity analyses, we found that SDHA and ATP5a encoded by the nuclear gene remain unchanged, and the mtDNA encoded subunit COX I and COX II, as well as the nuclear gene-encoded subunit NDUFA9 and COX IV, were signi cantly decreased, with the downregulation in 20-week-old more apparent than that in 10-week-old mice ( Fig. 5D and E).
These results together demonstrate that LonP1 knockout in the myocardium induces the accumulation of protein aggregates, impairing mitochondrial structure and morphology, as well as MRC function.

Loss of LonP1 in cardiomyocytes leads to metabolic reprogramming
Our data showed that LonP1 depletion suppressed H9c2 cell proliferation ( Fig. 6A and B), promoted both intracellular ROS and mitochondrial superoxide generation and caused mitochondrial depolarization ( Fig. 6C-E). These results strongly indicate that LonP1 is essential for mitochondrial homeostasis and that the loss of LonP1 may cause oxidative damage in H9c2 cells. To evaluate the impacts of LonP1 on mitochondrial function, we analyzed OCR in control and LonP1-knockdown H9c2 cells. We detected that silencing LonP1 dramatically decreased the overall OCR. Our results further showed that basal respiration, maximum respiration, and ATP production were markedly decreased ( Fig. 6F and G) in LonP1depleted H9c2 cells.
To further explore the underlying molecular mechanism of the LonP1-driven H9c2 cell phenotype, we then checked the protein levels of mitochondrial phosphoenolpyruvate (PEP) carboxykinase (PEPCK-M or PCK2) and activating transcription factor 4 (ATF4) were increased (Fig. 6H and I). It was well elucidated that PCK2, a key enzyme in glycogenesis, could be transcriptionally regulated by ATF4 through binding to a putative ATF/CRE composite site within the PCK2 promoter functioning as an amino acid response element, which mediates PCK2 transcriptional upregulation (45). Consistently, we found that the mRNA transcripts of SUCLG2, PCK2, GLS, PHGDH and PSAT1 were all upregulated in cLKO hearts (Fig. 6J). Collectively, our data indicate that the cardiomyocyte-speci c deletion of LonP1 promotes metabolic reprogramming to overcome LonP1 de ciency caused mitochondrial dysfunction, excessive oxidative stress, and intermediate metabolites limitations, thus promoting cLKO mouse survival.

Discussion
Mitochondrial proteins are continuously damaged by exogenous and/or endogenous stresses, such as ROS, heat, and toxic compounds, which causes proteins damaged due to misfolding or aggregation.
Mitochondrial protein quality control system can sense the damaged protein which are selectively degraded by different proteolytic systems within mitochondrial, therefore maintains the protein homeostasis. However, the defects in mitochondrial protein quality control system will lead to accumulation of damaged proteins in the mitochondria, thus causing mitochondrial dysfunction and ultimately leading to cellular dysfunction. LonP1 has been emerged as an essential and fundamental component of mitochondrial quality control system and as an important stress sensor in the mitochondrial alterations that could be observed in a number of chronic diseases and cancers (7,17,46,47). A recent in vitro study shows that LonP1 is also required for maturation of a subset of mitochondrial matrix proteins and that LonP1 reduction elicits an integrated stress response (48).
Here, we demonstrate that cardiomyocyte-speci c ablation of LonP1 causes the mice develop DCM and ultimately progresses to heart failure, which indicates the essential roles of LonP1 in myocardial cells.
Our study shows that LonP1 mediates UPR ER orchestrating UPR mt to regulate heart function and remodeling. The deletion of Lon1 in cardiomyocytes of mice causes imbalance in mitochondrial dynamics, and OXPHOS dysfunction, thus leading to DCM and eventually to heart failure. These ndings describe a mechanism by which protein quality control system dysfunction predisposes the myocardium to injury via altered mitochondrial fusion and ssion balance as well as susceptibility to chronic mitochondrial stress.
LonP1 mainly involved in the degradation of misfolded and aggregated abnormal proteins within the matrix. Here, we show that LonP1 ablation causes many compensatory responses in cLKO mice, including activation of the UPR ER . Strikingly, the UPR ER is activated prior to the UPR mt , suggesting that LonP1 expression is a ne-tuning sensor of the UPR ER and UPR mt . Upregulation of both the UPR ER and UPR mt in cardiomyocytes of LonP1-deletion mice results in well-tolerating mice with no major changes of longevity under unstressed conditions. In addition to the activation of both the UPR ER and UPR mt in the hearts of cLKO mice, FGF21, a key regulator of glucose and lipid metabolism and energy balance, is also increased dramatically to compensate for the deletion of LonP1 expression. The increased expression of FGF21 further ensures cLKO mice survival under unstressed conditions. Balanced mitochondrial dynamics are critical for myocardial function (49). Mitochondrial ssion ensures biogenesis and e ciently removes damaged or old mitochondria through mitophagy, whereas mitochondrial fusion facilitates the mixing and exchange of vital metabolites and mtDNA between different mitochondria to prevent the accumulation of mitochondrial damage in cells, thus properly maintaining the mitochondrial functions (50,51). OPA1, located in the inner mitochondrial membrane of mitochondria, plays an important role in regulating mitochondrial fusion and ssion (52). OPA1 is cleaved by mitochondrial processing peptidase to form the mature isoform of OPA1 (L-isoform) (53), which is further processed into two shorter isoforms, S1 and S2 OPA1. L-OPA1 is su cient for mitochondrial fusion; however, this feature is lost after cleavage to the S-isoform (53). LonP1-deletion signi cantly reduced L-OPA1 through the cleavage of OPA1 and increased S-OPA1 levels, promoting mitochondrial ssion. Moreover, Drp1, which is the master regulator of mitochondrial division in most eukaryotic organisms (52), was increased in cardiomyocyte-speci c LonP1-deletion mice. These results suggest that the loss of LonP1 triggers stress-induced OPA1 processing and leads to the accumulation of S-OPA1 forms c and e, causing unbalanced fusion and ssion of mitochondria and impairs mitochondria morphology in cardiomyocytes lacking LonP1.
Mitochondrial homeostasis is essential for normal cell physiological equilibrium, and its disruption contributes to multiple diseases (54). Our results clearly show that LonP1 ablation in myocardial cells promotes gluconeogenesis, as well as aspartate and glutamate metabolism, and amino acids biosynthesis, via upregulating multiple vital enzymes. Importantly, mitochondrial PCK2, a key enzyme of gluconeogenesis, is dramatically transcriptionally upregulated due to LonP1 depletion, suggesting that LonP1 ablation may drive PCK2-mediated metabolic adaptation in cardiomyocytes to support TCA cycle metabolism and glycolytic intermediates for biosynthesis (55). The PCK2 activity depends on mtGTP generated by the SUCLG2 form of SCS in the mitochondrial matrix (56). We show that the SUCLG2 protein and mRNA expressions are stimulated by LonP1 ablation, indicating a synergistic effect to strengthen PCK2 activity in LonP1-de cient cardiomyocytes. Moreover, we found that ATF4 was upregulated, indicating that PCK2 could be transcriptionally regulated by ATF4 through regulating a putative ATF/CRE composite site containing the PCK2 promoter, which caused transcriptional upregulation of PCK2 (45,57). Our results argue that ATF4-induced PCK2 expression could promote LonP1-deletion cardiomyocytes survival in energy-and intermediate metabolites-limited environments. These results suggest that LonP1deletion cardiomyocytes may activate ATF4/PCK2-mediated metabolic reprogramming to orchestrate energy and intermediate metabolite homeostasis, which contributes to myocardial cell survival.
In the past, many puzzling results have been reported concerning ER stress induced by mitochondrial dysfunction/stress. Frequently, researchers reveal the mitochondrial structural or functional defects induced by ER stress without a reasonable explanation. Here, we unveiled that LonP1-mediated orchestration of UPR mt , UPR ER and mitochondrial dynamics, and this nding may provide a clue to this long-standing obscurity (Fig. 7). As reduction of LonP1 levels in cardiac myocytes led to DCM and heart failure, it is possible that increased expression of LonP1 may have a cardioprotective effect against DCM and preventing heart failure. It will also be interesting to examine whether there is a correlation between the cardiac level of LonP1 and the incidence of DCM in humans.  Generation of Cardiomyocyte-Speci c LonP1 Deletion Mice. A. Generation of cardiomyocyte-speci c LonP1-de cient mice. Lonp1LoxP/LoxP mice were crossed with cardiomyocyte-speci c-MHC-Cre (αMHC-Cre) mice for deletion of LonP1 in the cardiomyocytes. B. PCR products were electrophoresed on a 1% agarose gel. Genotype identi cation shows Lonp1LoxP/LoxP alleles located at 573 bp, wild type alleles located at 473 bp, and the α-MHC-cre alleles located in 501 bp. Lonp1LoxP/LoxP with α-MHC-cre refers to cLKO mice. C, D. The loss of LonP1 was con rmed at both the transcript (C) and protein (D) levels. Levels of Lonp1 mRNA and protein were extremely low and almost undetectable in the hearts of LonP1conditional knockout mice (cLKO) compared with littermate controls (Lonp1LoxP/LoxP, Cont) after two weeks. In (C), data are presented as the means ± SD (n = 3 mice/group, ***P < 0.001, statistically signi cant by Student's t test). E. There was no difference in Lonp1 expression in the other organs between cLKO and Cont mice. Cardiomyocyte-Speci c Deletion of LonP1 Causes Pathological Heart Remodeling and Impaired Heart Function. A. Kaplan-Meier survival curves of Cont and cLKO mice (n=20). B. Heart weight-to-body weight ratios in Cont and cLKO mice of 2, 4, 6, 8, 10, 15, 20, 40, and 60 weeks of age (n = 6 mice/group, *P < 0.05, Deletion of LonP1 Induces UPRER and UPRmt. A, B. Western blot analysis (A) and quanti cation (B) of endoplasmic reticulum (ER) stress proteins PDI, IRE1α, p-eIF2α, ATF4 and ATF6 in Cont and cLKO hearts at 4 and 10 weeks of age. GAPDH was used as a loading control. Data shown are means ± SEM (n = 3 mice/group, **P < 0.01, ***P < 0.001, statistically signi cant by Student's t test). C, D. Western blot analysis (C) and quanti cation (D) of LonP1 and the mitochondrial unfolded protein response related proteins ClpX, Tid1-L/S, HSP60, and AFG3L2 in Cont and cLKO hearts at 4 and 10 weeks of age. GAPDH was used as a loading control. Data shown are means ± SEM (n = 3 mice/group, ***P < 0.001, statistically signi cant by Student's t test). E, F. Western blot analysis (E) and quanti cation (F) of the mitochondrial stress marker FGF21 protein level in Cont and cLKO hearts at 4 and 10 weeks of age.
GAPDH was used as a loading control. Data shown are means ± SEM (n = 3 mice/group, ***P < 0.001, statistically signi cant by Student's t test). week-old Cont and cLKO mice. Data shown are means ± SD (n = 3 mice/group, *P < 0.05, **P < 0.01, ***P < 0.001, statistically signi cant by Student's t test). D, E. Western blot analysis (D) and quanti cation by ImageJ (E) of protein levels for representative ETC complex subunits from the heart tissue of 10-and 20- week-old Cont and cLKO mice. GAPDH was used as the loading control. Data shown are means ± SD (n = 3 mice/group, ***P < 0.001, statistically signi cant by Student's t test).

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