Ndufs1 Deficiency Aggravates the Mitochondrial Membrane Potential Dysfunction in Pressure Overload-Induced Myocardial Hypertrophy

Mitochondrial dysfunction has been suggested to be the key factor in the development and progression of cardiac hypertrophy. The onset of mitochondrial dysfunction and the mechanisms underlying the development of cardiac hypertrophy (CH) are incompletely understood. The present study is based on the use of multiple bioinformatics analyses for the organization and analysis of scRNA-seq and microarray datasets from a transverse aortic constriction (TAC) model to examine the potential role of mitochondrial dysfunction in the pathophysiology of CH. The results showed that NADH:ubiquinone oxidoreductase core subunit S1- (Ndufs1-) dependent mitochondrial dysfunction plays a key role in pressure overload-induced CH. Furthermore, in vivo animal studies using a TAC mouse model of CH showed that Ndufs1 expression was significantly downregulated in hypertrophic heart tissue compared to that in normal controls. In an in vitro model of angiotensin II- (Ang II-) induced cardiomyocyte hypertrophy, Ang II treatment significantly downregulated the expression of Ndufs1 in cardiomyocytes. In vitro mechanistic studies showed that Ndufs1 knockdown induced CH; decreased the mitochondrial DNA content, mitochondrial membrane potential (MMP), and mitochondrial mass; and increased the production of mitochondrial reactive oxygen species (ROS) in cardiomyocytes. On the other hand, Ang II treatment upregulated the expression levels of atrial natriuretic peptide, brain natriuretic peptide, and myosin heavy chain beta; decreased the mitochondrial DNA content, MMP, and mitochondrial mass; and increased mitochondrial ROS production in cardiomyocytes. The Ang II-mediated effects were significantly attenuated by overexpression of Ndufs1 in rat cardiomyocytes. In conclusion, our results demonstrate downregulation of Ndufs1 in hypertrophic heart tissue, and the results of mechanistic studies suggest that Ndufs1 deficiency may cause mitochondrial dysfunction in cardiomyocytes, which may be associated with the development and progression of CH.


Introduction
Cardiac hypertrophy (CH) is a pathophysiological response characterized by increased thickness of the ventricular wall, greater myocardial cell volume, and enhanced myocardial contractility during the early stage of overload pressure [1]. Primarily, CH is the compensatory response for preservation of cardiac function; however, persistent CH is often associated with disturbed energy metabolism, deteriorated cardiac function, and interstitial fibrosis, which will eventually progress into heart failure [2,3]. Heart failure caused by CH has been shown to be an independent risk factor for various cardiovascular diseases [4,5]. To date, the pathophysiology underlying the progression of myocardial hypertrophy remains elusive. Thus, determination of potential molecular mechanisms is necessary for identification of novel and effective therapies to attenuate myocardial hypertrophy.
The contraction and relaxation of cardiomyocytes require a sufficient energy supply to meet the workload demand, and mitochondria are important primary organelles for energy production in cardiomyocytes [6][7][8]. Mitochondrial dysfunction was shown to be closely associated with the development of heart failure [9][10][11]. Under pathological conditions of CH, the activities of ATP synthase and mitochondrial oxidative phosphorylation complex are attenuated, which results in reduced production of ATP [12,13]. Moreover, attenuated mitochondrial dynamics, reduced mitochondrial volume, and abnormal mitochondrial morphology were detected in cardiomyocytes in CH [14,15]. Mitochondrial dysfunction was shown to increase the production of reactive oxygen species (ROS) via impaired electron transport chains, which can lead to increased oxidative stress and decreased energy production in cardiomyocytes [9,16,17]. Thus, restoration of impaired mitochondrial functions will provide novel strategies to attenuate the progression of CH. NADH:ubiquinone oxidoreductase core subunit S1 (Ndufs1) is one of the core subunits of mitochondrial complex I that regulates mitochondrial oxidative phosphorylation and ROS production [18][19][20]. However, the detailed role of Ndufs1 in the pathophysiology of CH is largely unknown.
In the present study, we initially demonstrated the deregulation of Ndufs1 in heart tissue of mice with CH by analyzing the GSE95140 scRNA-seq dataset. The expression of Ndusf1 was confirmed in heart tissue in a mouse model of CH. Furthermore, in vitro studies determined the molecular mechanisms of Ndusf1-mediated CH. The present study may provide novel insight into the role of Ndusf1 in the pathophysiology of CH.

Materials and Methods
2.1. Analysis of scRNA-seq and Microarray Datasets. RNA sequencing data for single cardiomyocytes were downloaded from the GSE95140 dataset of the GEO database [21]. This dataset is based on the GPL17021 platform and contains 396 single-cardiomyocyte transcriptomes of mice after transverse aortic constriction (TAC) or sham operation assayed on day 3 (D3), week 1 (W1), week 2 (W2), week 4 (W4), and week 8 (W8). The expression in each cell was detected by using the "DropletUtils" package. Gene expression in the cells was calculated using the "QC-Metrics" function in the "scater" package [22]. Ribosomal genes ≥ 10% and mitochondrial genes ≤ 5% were used for subsequent filtering. After filtering, the expression matrix of each sample was normalized by using the "NormalizeData" function of the "Seurat" package (version 3.0) [23]. The genes with the most pronounced differences between the cells were selected using the "FindVariableFeatures" function of the "Seurat" package.
The "ScaleData" function was used to convert the expression data to linear scale. Then, principal component analysis (PCA) was performed using the "RunPCA" function of the "Seurat" package. Principal components (PCs) with standard deviations > 70% were selected. "RunUMAP" of the "Seurat" package was employed to perform UMAP dimensionality reduction analysis. The "FindAllMarkers" function of the "Seurat" package was used to define the criteria for identification of differentially expressed genes (DEGs) as follows: cell population expression ratio > 0:25, log | fold change ðFCÞ | > 0:25, and p ≤ 0:05.
The differentially expressed genes were validated using the GSE24454 microarray dataset. In this dataset, mice were sacrificed 4 weeks after aortic banding (AB) or sham procedure (sAB) and subsequent debanding, including banding and subsequent debanding (DB3) or sham procedure and subsequent debanding (sDB3); the data were obtained at various time points up to day 3 [24]. Thus, the CEL raw data and corresponding annotation platform file were downloaded and preprocessed by background adjustment, normalization, probe summarization, and log 2 transformation of the expression values using the "Affy" package in R.

Gene Ontology (GO) Term Enrichment
Analysis. GO enrichment analysis was performed using the "clusterProfiler" package in R [25]. Notably, the major GO terms of DE genes in biological processes, molecular functions, cellular components, and pathways were evaluated. The Benjamini-Hochberg method was used to adjust the original p values. The GO terms corresponding to the DE genes were enriched with the threshold of correction p value < 0.05. Additionally, the enrichment analyses of the biological processes of the hub genes were carried out with the ToppGene tool (https://toppgene.cchmc.org/), which is a web-based analytic tool used for functional enrichment analysis of the gene lists [26]. Additionally, the cellular compartmentspecific protein-protein interaction network was constructed by the ComPPI database (https://comppi.linkgroup.hu/) [27].
2.3. Gene Set Enrichment Analysis (GSEA). GSEA was used to assess the Kyoto Encyclopedia of Genes and Genomes (KEGG) maps involved in TCA-induced CH development based on time series analysis [28]. Initially, the Kolmogorov-Smirnov method was used to determine the enrichment score (ES); then, the statistical significance of ES was assessed using the empirical phenotype replacement test procedure. The enrichment score (NES) was derived by normalization of ES for each gene set. The false discovery rate (FDR) of each NES was determined.
2.4. Gene Set Variation Analysis (GSVA). The GSVA package of R was used to analyze the activation of the gene sets by unsupervised and nonparametric scoring calculations [29]. The hub pathway-related scores were calculated by the GSVA method in each cell based on the transcription expression matrix after assigning various groups in the TAC model. Significant differences in GSVA scores between various groups were assessed by one-way ANOVA.

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Oxidative Medicine and Cellular Longevity 2.5. Animals and Surgical Intervention. All animal experiments were approved by the Animal Ethics Committee of Sun Yat-sen University (SYSU-IACUC-2020-000469). Sixteen male C57BL/6 mice (8 weeks old) were purchased from Sun Yat-sen University, and the mice were randomly divided into two groups, including the sham (n = 8) and TAC groups (n = 8). Before operation, the animals were anaesthetized by intraperitoneal injection with 100 mg/kg ketamine + 5 mg/ kg xylazine. After the animals reached general anesthesia, a small incision was made in the second intercostal space at the left upper sternal border to open the chest cavity, and the animals were subjected to respiratory ventilation. After exposure of the aortic arch, TAC was performed by tying a 7-0 nylon suture ligature against a 27-gauge needle between the left common carotid artery and the brachiocephalic artery. Then, the needle was quickly retracted to complete the partial constriction procedure. Sham-operated mice were subjected to the same surgical procedures without transverse aortic constriction. The chest was closed with 5-0 nonabsorbable sutures. Postoperatively, the animals were subcutaneously injected with 1.0 mg/kg buprenorphine to relieve postoperative pain every 12 h for 3 consecutive days. The mice were closely monitored every day for body weight and any signs of labored breathing or postoperative pain.
2.6. Echocardiography. Four weeks after ascending TAC operation, the animals from the sham and TAC groups were subjected to echocardiography examination. Briefly, the mice were anaesthetized by 3% isoflurane using an anesthesia machine. The hair on the left chest was carefully removed, and cardiac geometry was determined from the parasternal long axis view with a probe frequency of 30 MHz using a small animal color ultrasonic diagnostic apparatus (Vevo 2100, VisualSonics, Toronto, Canada). The images of the left ventricular area were captured using M-type echocardiography. The interventricular septum (IVS) thickness and left ventricular posterior wall (LVPW) thickness were measured.

Evaluation of Cardiac Index.
After assessment by echocardiography, the animals were sacrificed by an overdose of 5% isoflurane. The heart was immediately dissected and rinsed with ice-cold saline to remove blood clots. After draining the heart tissue on sterile paper, the whole weight of the heart was measured using a digital balance. The left ventricular weight (LVW) was determined by removing the atrium and right ventricle from the whole heart. The heart mass index (HMI) and left ventricular mass index (LVMI) were calculated as follows: HMI = LVW/body weight; LVMI = LVW/body weight. The length of the medial malleolar distance on the right hindlimb to the tibial plateau edge was defined as the tibia length (TL). The ratios of LVW to TL were used as an index of cardiac hypertrophy.
2.8. Hematoxylin and Eosin (H&E) Staining. After animals were sacrificed by an overdose of 5% isoflurane, a part of the heart tissue was fixed with 4% paraformaldehyde and embedded in paraffin. The paraffin-embedded heart tissue was sectioned into 5 μm sections and stained by hematoxylin and eosin. The stained sections were examined under a light microscope (Nikon, Tokyo, Japan).

Transmission Electron Microscopy (TEM).
The mitochondria in the heart tissue were evaluated by TEM. Briefly, the heart tissue was sectioned into 1 mm 3 pieces, which were fixed with 4% glutaraldehyde and 1% osmic acid. Then, the tissue was dehydrated with acetone, embedded in Epon 821, and cut into 70 nm sections. Then, the sections were double stained with uranyl acetate and lead citrate. The mitochondria were examined using TEM (JEM-1230, Tokyo, Japan). Mitochondrial volume and mitochondrial number were evaluated based on the TEM images. 2.13. Western Blot Assay. Proteins from cardiomyocytes or heart tissue were isolated using RIPA buffer supplemented with proteinase inhibitors (Sigma-Aldrich). The concentrations of the protein samples were measured by the BCA method. Equal amounts of proteins (50 μg) were resolved by gel electrophoresis and transferred to a polyvinylidene difluoride (PVDF) membrane. After blocking with 5% nonfat milk at room temperature for 1 h, the membranes were incubated with primary antibodies against NDUFS1 (1 : 1,000; CST, Danvers, USA), atrial natriuretic peptide (ANP; 1 : 1,000; CST), brain natriuretic peptide (BNP; 1 : 1,000; CST), myosin heavy chain beta (β-MHC; 1 : 1,000; CST), and β-actin (1 : 2,000; CST) at 4°C overnight. Then, the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies (1 : 2,000; CST) for 2 h at room temperature. The immunoreactive bands were analyzed by using a chemiluminescence system (Bio-Rad).

Assessment of Mitochondrial Membrane Potential
(MMP). MMP of cardiomyocytes was evaluated using a JC-1 mitochondria staining kit (Thermo Fisher Scientific). Briefly, cardiomyocytes (5 × 10 3 cells/well) were plated in 96-well plates, treated for 24, and incubated with JC-1 fluorescent dye for 20 min at room temperature in the dark. The fluorescent staining by JC-1 was evaluated by fluorescence microscopy. JC-1 monomers were imaged at excitation and emission wavelengths of 490 nm and 530 nm, respectively; JC-1 aggregates were imaged at excitation and emission wavelengths of 525 nm and 590 nm, respectively.
2.16. Detection of Mitochondrial ROS. The production of mitochondrial ROS was determined by using a MitoSOX fluorescent staining kit (Thermo Fisher Scientific, Waltham, USA) according to the manufacturer's protocol. Confocal laser scanning microscopy was used to capture fluorescent images, which were further analyzed using ImageJ software.
2.17. Flow Cytometry Analysis of ROS-Positive Cells. ROS production in cardiomyocytes was evaluated using the 2′,7′ -dichlorofluorescein diacetate (DCF-DA) staining assay (Thermo Fisher Scientific). Briefly, the cells were incubated with DCF-DA for 30 min at 37°C in the dark, washed, resus-pended in PBS, and maintained on ice for immediate assay by flow cytometry (BD Biosciences). The data were analyzed using FACSDiva software (BD) to calculate the number of ROS-positive cardiomyocytes.

Mitochondrial Mass Analysis Using MitoTracker Red
Staining. MitoTracker Red staining was performed to assess mitochondrial mass. Briefly, cardiomyocytes were incubated with 100 nM MitoTracker Red for 30 min at 37°C. Fluorescence was detected at excitation and emission wavelengths of 490 and 516 nm, respectively, using an ELx-800 microplate reader (BioTek; Winooski, VT, USA).

Statistical
Analysis. The data are presented as the mean ± standard deviation. All data analyses were performed using GraphPad Prism software (version 8; GraphPad Software, La Jolla, USA). Statistical significance of differences between various treatment groups was assessed using unpaired Student's t-test or one-way ANOVA followed by the Bonferroni multiple comparison test. p < 0:05 indicated statistical significance.
As shown in Figure 1(c) and Table S2, GSEA was used to analyze the CH-related KEGG pathways, and the KEGG: oxidative phosphorylation pathway was significantly enriched in 4 Oxidative Medicine and Cellular Longevity Furthermore, the GSVA calculated scores of the KEGG: oxidative phosphorylation (OxP score) pathway at various stages of CH were distinctly different from those in the sham operation group. Specifically, CH-related scores were significantly lower in the TAC W1 and W4 groups compared to those in the sham group (all p value < 0.05; Figure 1(d) and Table S3). Moreover, the patterns of GSVA calculated based on the corresponding OxP scores across various cell clusters showed that the scores were significantly lower in cluster 2 than those in cluster 0 (p value < 0.05; Figure 1(e)).

Identification of Candidate Biomarkers Involved in TAC-
Induced CH. DE genes in cluster 2 are illustrated by a volcano plot (Ndusf1: average LogFC = 2:21, adjusted p value = 1:78 E − 38; Figure 2(a)). These genes included genes with log fold change > 1, which were used for Spearman correlation analysis; the results showed that Ndusf1 was highly correlated with the OxP score (R = 0:74, p value < 0.05; Figure 2(b)). The validation of Ndusf1 using GSE24454 showed that Ndusf1 was significantly downregulated in the heart tissue from aortic banding mice compared to that in sham-operated mice (Figure 2(c)). Additionally, NDUSF1 was identified as a key interactor in the protein-protein  Table S4). The biological process enrichment analysis showed that proteins that potentially interact with Ndusf1 were significantly associated with key mitochondrial functions, such as "mitochondrial electron transport, NADH to ubiquinone" (p value = 2:78E − 4), "mitochondrial respiratory chain complex I assembly" (p value = 1:52E − 3), and "NADH dehydrogenase complex assembly" (p value = 1:15E − 2) (Figure 2(f) and Table S5).
Based on the results of bioinformatics analysis, Ndusf1 was selected for subsequent validation and functional analysis.

Ndufs1 Was Downregulated in the Hypertrophic Mouse
Heart. As shown in Figures 3(a) and 3(b), the results of H&E staining indicated that cardiomyocytes were significantly enlarged in the TAC group compared to those in the sham group (p < 0:05). Examination of the dissected heart tissue indicated that the heart weight, HMI, LVMI, and LV/TL were significantly higher in the TAC group than those in the sham group (Figures 3(c)-3(f); all p < 0:05). The results of echocardiography examination showed that the thickness of IVS and LVPW was significantly higher in the TAC group than that in the sham group (Figures 3(g)-3(i); all p < 0:05). qRT-PCR analysis showed that the mRNA expression levels of ANP, BNP, and β-MHC were significantly higher in heart tissue from the TAC group than those in the sham group (Figures 3(j)-3(l); all p < 0:05). Importantly, the expression levels of Ndusf1 mRNA and protein were significantly reduced in the heart tissue from the TAC group compared to those in the sham group (Figures 3(m) and 3(n); all p < 0:05). TEM examination showed that the mitochondrial volume was significantly increased, and the number of mitochondria was significantly decreased in the heart tissue from the TAC group compared    The sarcomeres were enlarged, and myofibrils were disordered in the TAC group. Comparison with the sham group indicated that mitochondria in TAC-induced CH cardiomyocytes showed mild to moderate swelling, intact adventitia, edema of the matrix, disappearance of partial cristae, and aggregation between myofibrils.
3.4. Effects of Ang II on the Expression Levels of ANP, BNP, β-MHC, and Ndusf1. As shown in Figure 4, Ang II treatment caused a significant increase in the mRNA and protein expression levels of ANP, BNP, and β-MHC in cardiomyocytes compared to those in the control group (Figures 4(a)-4(f); all p < 0:05). On the other hand, the mRNA and protein expression levels of Ndusf1 in cardiomyocytes were significantly reduced by Ang II treatment compared to those in the control group (Figures 4(g) and 4(h); all p < 0:05). These results indicated that Ndusf1 may have certain biological functions in Ang II-stimulated cardiomyocytes.     13 Oxidative Medicine and Cellular Longevity the levels of Ndusf1 mRNA and protein compared with those in the cells transfected with pcDNA3.1 (NC group; Figures 6(a) and 6(b); all p < 0:05). As expected, Ang II treatment increased the cardiomyocyte area compared with that in the control group, and Ndusf1 overexpression attenuated Ang II-induced increase in the cardiomyocyte area ( Figure 6(c); p < 0:05). Consistently, Ang II treatment caused a significant increase in the levels of mRNA and protein of ANP, BNP, and β-MHC (all p < 0:05), and Ndusf1 overexpression partially counteracted the stimulatory effects of Ang II on ANP, BNP, and β-MHC expression in cardiomyocytes (Figures 6(d)-6(i); all p < 0:05). The mitochondrial DNA content of cardiomyocytes was significantly reduced by Ang II, and this effect was attenuated by Ndusf1 overexpression (Figure 6(j); all p < 0:05). Moreover, Ndusf1 overexpression attenuated Ang II-induced elevation in the ROS production levels in cardiomyocytes (Figures 6(k) and 6(l); all p < 0:05). Importantly, Ang II treatment significantly repressed MMP and mitochondrial mass in cardiomyocytes, and the effect was significantly alleviated by overexpression of Ndusf1 (Figures 6(m) and 6(n)). In turn, these results indicate that Ndusf1 overexpression may protect against MMP disorder in Ang II-induced mitochondrial dysfunction.

Discussion
CH is a pathophysiological response characterized by increased thickness of the ventricular wall, greater myocardial cell volume, and enhanced myocardial contractility in the early stage of overload pressure [1,3]. The pathophysiology of CH is complex and involves multiple cellular events, and the mechanisms of the development of CH are not fully understood [1,3,30]. Growing evidence indicates that mitochondrial dysfunction is closely related to the development and progression of CH. In the present study, we explored the GSE95140 datasets by using integrated bioinformatics analysis and demonstrated downregulation of Ndusf1 in heart tissue of mice with CH. The expression of Ndusf1 was   thickness of IVS and LVPW were detected in mice subjected to TAC compared with those in sham-operated mice; these findings are consistent with the results of previous studies [31]. Additionally, the mRNA expression levels of ANP, BNP, and β-MHC were elevated in the heart tissue of the mice with CH. ANP, BNP, and β-MHC are commonly used cardiac hypertrophic biomarkers, and elevated levels of these biomarkers have been demonstrated in heart tissue in clinical studies and animal models [32][33][34][35]. Overall, these results suggest successful establishment of CH in mice subjected to TAC treatment. Further validation by qRT-PCR and western blot showed that the expression levels of Ndusf1 mRNA and protein were downregulated in hypertrophic heart tissue.
The data suggest that Ndusf1 may play an important role in CH.
Mitochondria are considered to be dynamic "energy stations", and these morphological changes are characterized by fragmented disconnection and elongated interconnection of mitochondria, which are regulated by activation of mitochondrial fission and fusion proteases or posttranslational modifications of proteins during the development of CH [1,36]. Ndusf1 (NADH:ubiquinone oxidoreductase core subunit S1) is the largest subunit of complex I; the corresponding gene encodes a 75 kDa subunit of NADH-ubiquinone oxidoreductase [37] and thus has gained particular attention due to its significant role in the activity of mitochondrial oxidative phosphorylation. Mutations or deficiency of this gene can destabilize complex I assembly and result in defects of the activity of the electron transport chain (ETC) and elevated ATP production, which can cause mitochondrial fusion and fission to restore damaged mitochondrial DNA and remove damaged mitochondrial fragments, respectively [38][39][40]. However, the role of Ndusf1 in the pathophysiology of CH has not been reported; however, the importance of Ndusf1 in cardiovascular diseases has been emphasized by several research groups. Qi et al. showed that Akap1 deficiency exacerbates diabetic cardiomyopathy in mice by Ndusf1mediated mitochondrial dysfunction and apoptosis [19]. Ndusf1 was shown to be upregulated in cardiac cells upon cyclic stretch, which may be associated with mitochondrial biogenesis [41]. Sato et al. showed that Ndusf1 is associated with the cardiac response to iron deficiency [42]. Additionally, the cardiac levels of acetylated forms of Ndusf1 are decreased in caloric restriction [43]. Impaired electron transport chains in mitochondria contribute to the development and progression of CH [44,45]. Ndusf1 is one of the key regulators of the electron transport chain; thus, we speculate that Ndusf1 may be a key factor in CH. The results of our in vitro studies indicated that the Ang II-induced increase of the expression of hypertrophic biomarkers was accompanied by downregulation of Ndusf1 in cardiomyocytes. Ang II is widely used to induce a cellular model of CH in cardiomyocytes [46,47]. Schwartz et al. used serial analysis of gene expression to demonstrate that continuous Ang II treatment induces downregulation of Ndusf1 in the mouse heart [48]. The activation of the endogenous renin-angiotensin aldosterone system increases the protein levels of Ndusf1 in urine in hypertensive patients [49]. Additionally, Ang II exacerbates mitochondrial dysfunction and oxidative stress to cause heart failure [50]. Thus, in combination with these findings, our results may imply that Ang II-induced CH in cardiomyocytes may be associated with downregulation of Ndusf1.
To gain additional insight into the mechanism of action of Ndusf1 in Ang II-induced CH, we performed loss-and gain-of-function studies in cardiomyocytes. Our results showed that Ndusf1 knockdown decreased the mitochondrial DNA content and MMP and increased mitochondrial ROS production; in addition, Ndusf1 knockdown increased the expression levels of hypertrophic biomarkers in cardiomyocytes, possibly indicating that Ndusf1 knockdown promoted CH by impairing mitochondrial functions. Downregulation of Ndusf1 inhibits the neuroprotective effects of pyrroloquinoline against rotenone injury in cultured SH-SY5Y cells and cultured midbrain neurons, and rotenone can impair mitochondrial dysfunction [51]. Lopez-Fabuel et al. showed that Ndusf1 knockdown impairs mitochondrial O 2 consumption and increases ROS production in neurons [52]. On the other hand, our results showed that overexpression of Ndusf1 attenuated Ang II-mediated effects in cardiomyocytes. Thus, Ndusf1 may be involved in Ang II-mediated CH by modulating mitochondrial functions in cardiomyocytes.
Balanced regulation of mitochondrial fusion and fission is very delicate and susceptible to the development of CH and heart failure; thus, efficient myocardial therapies that target mitochondrial lesions and dysfunction may be a new strategy to suppress susceptibility to myocardial remodeling and attenuate myocardial fibrosis; however, these possibilities are poorly characterized [1,5,11]. Ndufs1 has been illustrated to play a central role in the regulation of morphological dynamics and oxidative stress and may play an important role in mitochondrial crista remodeling, cytochrome release, and mitochondrial respiration in the development of CH and heart failure. A decrease in the expression of Ndufs1, in turn, contributes to aggravated mitochondrial membrane potential disorder, mitochondrial ROS production, and mitochondrial DNA damage.
In conclusion, our results demonstrated downregulation of Ndusf1 in hypertrophic heart tissue, and the results of the mechanistic studies suggest that Ndusf1 deficiency causes mitochondrial dysfunction in cardiomyocytes, which may be associated with the development and progression of CH.

Limitations
There are several limitations in the present study. First, the expression of Ndusf1 in the mouse cardiac tissue was determined 4 weeks after TAC surgery in mice, which cannot reflect the changes in Ndusf1 during the progression of CH. Thus, future studies may determine the expression of Ndusf1 at various time points to confirm the role of Ndusf1 in the progression of CH. Second, the expression of Ndusf1 was validated only in mouse cardiac tissue and cardiomyocytes, and future studies should determine the expression of Ndusf1 in the clinical samples of hypertrophic hearts. Third, loss-and gain-of-function studies were only performed in vitro, and the functional role of Ndusf1 in vivo