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. 2026 Mar 26;24(3):597–608. doi: 10.9758/cpn.25.1392

Therapeutic Lithium Enhances Klotho Expression and Mitochondrial Size and Branching in Human Cardiac Fibroblasts Involving Nuclear Factor-kappa B Inhibition

Pao-Huan Chen 1,2, Ting-Wei Lee 3,4, Shuen-Hsin Liu 5,6, Tzu-Yu Cheng 7,8,9,✉, Yu-Hsun Kao 5,10,✉, Yi-Jen Chen 5,7,8
PMCID: PMC13429796  PMID: 42528353

Abstract

Objective

Emerging studies suggest that lithium might be cardioprotective. Although studies have suggested that lithium improves mitochondrial dysfunction, promotes antiaging processes, and reduces cardiac fibrosis, whether lithium modulates Klotho, an antiaging protein, and mitochondrial morphology in human cardiac fibroblasts remains unclear. This study investigated the effects of therapeutic lithium on Klotho expression and mitochondrial morphology in human cardiac fibroblasts and underlying mechanisms.

Methods

We conducted microRNA sequencing, pathway enrichment analysis, mitochondrial morphology assessment, real-time reverse-transcription polymerase chain reaction, western blotting, and migration assay upon human cardiac fibroblasts and myocardial tissues of rats subjected to mania model.

Results

Transcriptomic analysis of myocardial tissues from rats subjected to mania model unveiled that the significantly enriched pathways targeted by lithium were related to mitochondrial function. Mitochondrial morphology assessment showed that human cardiac fibroblasts treated with LiCl (1.0 mmol/L) for 48 hours exhibited greater mitochondrial area, perimeter length, and degree and length of branching than did control fibroblasts. Additionally, the messenger RNA and protein expression levels of Klotho were upregulated in human cardiac fibroblasts treated with LiCl (1.0 mmol/L) for 48 hours compared with control fibroblasts, whereas the levels of phosphorylated nuclear factor-kappa B (NF-κB) p65 were downregulated. Furthermore, human cardiac fibroblasts treated with the combination of LiCl (1.0 mmol/L) and Bay 11-7802 (3 μM) exhibited similar migration ability and mitochondrial size and branching relative to LiCl (1.0 mmol/L)-treated fibroblasts.

Conclusion

Lithium at a therapeutic concentration enhances Klotho expression and mitochondrial size and branching in human cardiac fibroblasts probably through NF-κB inhibition.

Keywords: Lithium, Klotho proteins, Mitochondria, MicroRNAs, High-throughput nucleotide sequencing

INTRODUCTION

Cardiac fibrosis plays a critical role in the exacerbation of heart failure [1,2]. A primary mechanism of cardiac fibrogenesis is the senescence of cardiac fibroblasts [3]. Among the regulators of accelerated heart aging, Klotho, an antiaging protein, has drawn increasing attention given that numerous studies have demonstrated that Klotho expression is negatively associated with the development of cardiac fibrosis and heart failure [4-8]. Furthermore, increasing evidence suggests that Klotho exerts antifibrogenic effects through the regulation of genes encoding proteins involved in the renin–angiotensin system, inhibition of fibrosis-related growth factor pathways, control of calcium homeostasis, and modulation of mitochondrial function and morphology [9-12]. Hence, targeting Klotho may mitigate cardiac fibrosis, thus conferring cardioprotective benefits.

Lithium is a first-line medication used to treat bipolar disorder [13]. In addition to its neuroprotective effects [14], lithium has been reported to inhibit cardiac fibrogenesis by blocking calcium entry and downregulating the expression of proteins involved in profibrotic pathways [15,16]. Moreover, lithium improves mitochondrial dysfunction and alleviates oxidative stress [17-19], promotes antiaging processes and longevity [20-23], and reduces the risk of heart failure [24,25]. These findings suggest that lithium exerts antifibrotic effects through its antiaging effects and its protective effects on mitochondria. However, whether lithium at a clinically therapeutic concentration can modulate Klotho expression and mitochondrial morphology in human cardiac fibroblasts remains unclear. Therefore, this study investigated the effects of therapeutic lithium on Klotho expression and mitochondrial morphology in human cardiac fibroblasts and the underlying mechanisms.

METHODS

Animal Housing and Care

Male Wistar rats aged 8 weeks (250−300 g) were used in this study. Before the experiments, rats were housed in a group of four to ensure social stability, and were maintained on a 12:12-hour light–dark cycle. In addition, rats were provided ad libitum access to food and drinking water. Cage cleaning was performed by the staff of the Laboratory Animal Center of Taipei Medical University in accordance with standard procedures. The study protocol was approved by the Institutional Animal Care and Use Committee of Taipei Medical University (Protocol No.: LAC−2021−0461).

Rapid-eye Movement (REM) Sleep Deprivation Rat Model and Lithium Treatment

Rats were subjected to REM sleep deprivation in accordance with our previously described methods [16]. The REM sleep deprivation rat model was adopted in this study because rats with deprivation of REM sleep have been well established to exhibit manic-like behaviors and to develop cardiac fibrosis [26-29]. Briefly, a group of four rats were placed inside a 100-cm-long × 45-cm-wide × 30-cm-high water tank. The water tank included 10 circular platforms that were 6 cm in diameter, 10 cm in height, and placed 8 cm apart in two rows. Each platform was fixed at the bottom of the tank. The water level in the tank was maintained at 2 cm below the platform. During the experiments, the rats could move in the tank by jumping from one to another platform while awake. However, during REM sleep, the rats fell off the platform as a result of muscular atonia, and they awoke upon contact with water. Rats were subjected to REM sleep deprivation for 16 hours per day (16:00−08:00) over 4 weeks. Rats spent the remaining 8 hours (8:00−16:00) in their home cages. Rats receiving lithium treatment were orally administered LiCl (#62476, Sigma-Aldrich) at a dosage of 1 mmol/kg per day for a total of 4 weeks by gastric gavage. In our previous study, this dosage of lithium exerted cardioprotection and antifibrotic effects on the hearts of REM sleep-deprived rats [16].

RNA Isolation and Sequencing

At the age of 12 weeks, rats were anesthetized through the inhalation of 5% isoflurane and were euthanized. Subsequently, their hearts were rapidly dissected and kept in ice-cold saline. To obtain samples for microRNA (miRNA) sequencing, myocardial tissues were immediately collected from the anterior walls of the left ventricles, snap-frozen in liquid nitrogen, and stored at −80°C until analysis. RNA was extracted from myocardial tissue samples using the Trizol Reagent (#15596026, Thermo Fisher Scientific) according to the manufacturer’s instructions. The quality of RNA was assessed using an Agilent Technologies 2100 Bioanalyzer (Agilent Technologies). Next-generation sequencing of miRNA was conducted per our previously described methods [30].

Functional Bioinformatics Analysis

Differentially expressed miRNAs between the hearts of REM sleep-deprived rats with and without lithium treatment were identified using the DESeq2 package in R software (https://www.r-project.org/), with p value < 0.05 as the screening threshold. Functional bioinformatics analysis was performed using the mirPath (v.3.0) tool and TargetScan from the DIANA tools (http://diana.imis.athena-innovation.gr/DianaTools/index). Individually obtained pathways were merged for enrichment analysis using the “pathways union” mode based on the Fisher’s exact test. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used for pathway denomination.

Cell Culture and Treatment

Human cardiac fibroblasts isolated from normal adult heart tissue (#CC-2903, Lonza) were seeded on uncoated culture dishes containing FGM-3 Cardiac Fibroblast Growth Medium-3 BulletKit (Lonza) supplemented with 14.999 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 14.010 mmol/L sodium bicarbonate, and these cells were cultured as monolayers at 37°C with 5% CO2 per our previously described methods [15]. To avoid pseudo-replication, at least two sets of cells from passages 4 to 6 were used for each experiment, and at least three independent experiments were conducted. LiCl (#62476, Sigma-Aldrich) at a therapeutic concentration of 1.0 mmol/L and Bay 11-7802 (#HY-13453, MedChemExpress) at a concentration of 3 μM for 48 hours were applied in the following experiments. The study was confirmed exemption by the Taipei Medical University–Joint Institutional Review Board (Protocol No.: N202312152) because this study only used commercialized cells obtained from a legal vendor and complied with the regulation of the Ministry of Health and Welfare in Taiwan (https://dep.mohw.gov.tw/DOMA/fp-2782-9538-106.html).

Assessment of Mitochondrial Morphology

Mitochondrial morphology was assessed using MitoTracker Green FM dye (#M7514, Invitrogen) and a confocal fluorescence microscopy system (STELLARIS 8 Confocal Spectral Scanning System, Leica Microsystems). In brief, human cardiac fibroblasts were seeded at a density of 1 × 104 cells per well on an 8-well chamber (#80806, ibidi GmbH). Before assessment using confocal fluorescence microscopy, human cardiac fibroblasts were loaded with MitoTracker Green FM dye (500 nM) and Hoechst 33423 (#14533, Sigma-Aldrich; 1 μg/ml) and were incubated for 30 minutes at 37°C in the dark. Fluorescence intensity in four randomly selected fields in each well was measured and quantified using Fiji/ImageJ software (National Institutes of Health).

To characterize mitochondrial morphology, mitochondrial size and shape were analyzed. Mitochondrial size was quantified by measuring the mitochondrial area and perimeter. Mitochondrial shape was evaluated using the aspect ratio (mitochondrial length) and form factor (degree of mitochondrial branching). The aspect ratio was defined as the ratio between the major and minor axes of the ellipse equivalent to a mitochondrion [31]. The form factor was calculated using the following formula: (Pm2) / (4pAm), where Pm is the length of the mitochondrial outline and Am is the area of the mitochondrion. In addition, to assess mitochondrial branch length, branch length per mitochondrion and branch length per branch were computed.

Real-time Reverse-transcription Polymerase Chain Reaction (RT-PCR) Analysis

RT-PCR was performed per our previously described methods [15]. In brief, total RNAs were isolated from human cardiac fibroblasts and reversely transcribed using the SuperScript III reverse transcriptase (Invitrogen). The expression levels of Klotho and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA were analyzed with a quantitative (q)PCR using the ABI PRISM7300 system (Applied Biosystems) and SYBER Green (Applied Biosystems). Relative changes in the transcript levels of Klotho were estimated from the threshold cycle (Ct) value and normalized against the respective Ct value of the GAPDH reference gene in the corresponding sample. The following primers were utilized—Klotho (F: 5’-CCTCCTTTACCTGAAAATCAGCC-3’, R: 5’-CAGGTCGGTAAACTGAGACAGAG-3’); GAPDH (F: 5’-GGACCTGACCTGCCGTCTAG-3’, R: 5’-CCTGCTTCACCACCTTC TTGA-3’).

Western Blot Analysis

Western blotting was performed per our previously described methods [15]. Briefly, proteins were extracted from human cardiac fibroblasts using the Mammalian Protein Extraction Reagent (#78501, Thermo Fisher Scientific). Equal amounts of proteins (30 μg) were resolved through 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Subsequently, the electrophoretic transfer of the separated proteins onto equilibrated polyvinylidene difluoride membranes was conducted. The membranes were blocked with 5% skimmed milk for 1 hour at room temperature. Following this blocking procedure, the membranes were probed with specific antibodies against Klotho 1:3,000 (polyclonal; #LS‑C145689, Lifespan BioScience), total nuclear factor-kappa B (NF-κB) p65 1:2,000 (monoclonal; #8242, Cell Signaling Technology), and phosphorylated NF-κB p65 1:2,000 (monoclonal; #3033, Cell Signaling Technology). Bound antibodies were detected using an enhanced Chemiluminescence detection system (Millipore), and the results were analyzed using AlphaEaseFC software (Alpha Innotech). To confirm equal protein loading, targeted bands were normalized to GAPDH 1:50,000 (monoclonal; #M171-1, MBL).

Migration Assay

Migration assay was conducted per our previously described methods [15]. In brief, before treatment, human cardiac fibroblasts were plated at a density of 105 cells per well into a 6-well culture dish. After human cardiac fibroblasts reached nearly full confluence, cells were incubated in serum-free medium. Wound-healing assays were conducted 8 hours after the cell monolayers were scraped with a P200 pipette tip. Net migration areas were subtracted from those at baseline using ImageJ software. For each condition, the average of four randomly selected regions was calculated.

Statistical Analysis

Quantitative data are presented as the mean ± standard error of the mean. Normality of data was assessed using the Shapiro–Wilk test. Statistical significance in human cardiac fibroblasts exposed to various conditions was determined using a ttest (two-group comparisons) or one-way ANOVA followed by Tukey’s post hoc test (three-group comparisons). The Mann–Whitney Utest (two-group comparisons) or the Kruskal–Wallis test with Dunn post hoc analysis (three-group comparisons) was used for nonnormally distributed data. A two-tailed p value of < 0.05 indicated statistical significance.

RESULTS

Effects of Lithium on miRNA Expression in Hearts of REM Sleep-deprived Rats and Pathway Prediction

To broadly explore whether lithium affects pathways involved in the mitochondrial regulation, we performed hypothesis-free miRNA sequencing on myocardial tissues isolated from REM sleep-deprived rats. Transcriptomic analysis revealed the expression of a total of 642 miRNAs in the hearts of REM sleep-deprived rats, among which 17 miRNAs were differentially expressed between the hearts of REM sleep-deprived rats with and without lithium treatment (Fig. 1A). The KEGG enrichment pathway analysis revealed that many of the most significantly enriched pathways were related to mitochondrial functions, such as metabolism and bioenergetics (Fig. 1B).

Fig. 1.

Fig. 1

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Transcriptomic and functional bioinformatics analyses of hearts from REM sleep-deprived rats with or without lithium treatment. (A) Differentially expressed miRNAs between the hearts of REM sleep-deprived rats with (n = 2) and without lithium treatment (n = 2) were identified using the DESeq2 package in R software (https://www.r-project.org/), with p < 0.05 as the screening threshold. A total of 17 miRNAs were differentially expressed, including 12 miRNAs that were significantly upregulated and 5 miRNAs that were significantly downregulated in the hearts of REM sleep-deprived rats treated with lithium. (B) Functional bioinformatics analysis was performed using the mirPath (v.3.0) tool and TargetScan from DIANA tools (http://diana.imis.athena-innovation.gr/DianaTools/index). KEGG pathway enrichment analysis revealed that the most significantly enriched pathways were mucin type O-glycan biosynthesis, gap junction, extracellular matrix–receptor interaction, endocrine and other factor–regulated calcium reabsorption, fatty acid degradation, arrhythmogenic right ventricular cardiomyopathy, metabolic, and tyrosine metabolism pathways.

REM, rapid-eye movement; miRNA, microRNA; KEGG, Kyoto Encyclopedia of Genes and Genomes; ECM, extracellular matrix.

Effects of Lithium on Mitochondrial Morphology in Human Cardiac Fibroblasts

Because the in vivo study and bioinformatics analysis suggested that lithium potentially affected pathways involved in the mitochondrial functions, we examined whether lithium at the therapeutic concentration of 1.0 mmol/L directly modulated mitochondrial morphology in cardiac fibroblasts (Fig. 2). The results showed that human cardiac fibroblasts treated with 1.0 mmol/L LiCl for 48 hours exhibited a larger mitochondrial area (Fig. 2B) and perimeter (Fig. 2C) than did control cardiac fibroblasts. The findings indicated that human cardiac fibroblasts treated with lithium at the therapeutic concentration exhibited an increased mitochondrial size. Furthermore, we analyzed the parameters of mitochondrial shape in human cardiac fibroblasts with or without lithium treatment. The results revealed that treatment with LiCl at 1.0 mmol/L for 48 hours did not significantly affect the mitochondrial length (aspect ratio) in human cardiac fibroblasts compared with control cardiac fibroblasts (Fig. 2D). By contrast, human cardiac fibroblasts treated with 1.0 mmol/L LiCl for 48 hours exhibited greater mitochondrial branching (form factor) (Fig. 2E), branch length per mitochondrion (Fig. 2F) and branch length per branch (Fig. 2G) relative to control cardiac fibroblasts. The findings suggested that treatment with lithium at the concentration of 1.0 mmol/L increased the degree and length of mitochondrial branching in human cardiac fibroblasts.

Fig. 2.

Fig. 2

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Mitochondrial morphology in human cardiac fibroblasts with or without lithium treatment. (A) Representative images of MitoTracker Green FM (Invitrogen)–stained human cardiac fibroblasts (upper panel) and two-dimensional mitochondrial analysis in Fiji/ImageJ (National Institutes of Health; lower panel). (B, C) Mitochondrial area and perimeter were measured to assess mitochondrial size. Compared with control cardiac fibroblasts (n = 6), human cardiac fibroblasts treated with LiCl at 1.0 mmol/L (n = 6) for 48 hours exhibited increased mitochondrial area and perimeter. (D, E) The aspect ratio (mitochondrial length) and form factor (degree of mitochondrial branching) were analyzed to characterize the mitochondrial shape. Compared with control cardiac fibroblasts (n = 6), LiCl at 1.0 mmol/L (n = 6) for 48 hours did not significantly affect mitochondrial length (aspect ratio) in human cardiac fibroblasts. However, human cardiac fibroblasts treated with LiCl at 1.0 mmol/L (n = 6) for 48 hours exhibited increased mitochondrial branching (form factor). (F, G) Branch length per mitochondrion and branch length per branch were calculated to estimate the mitochondrial branch length. Relative to control cardiac fibroblasts (n = 6), human cardiac fibroblasts treated with LiCl at 1.0 mmol/L (n = 6) for 48 hours exhibited an increase in branch length per mitochondrion and branch length per branch.

*p < 0.05, ***p < 0.001.

Effects of Lithium on Klotho in Human Cardiac Fibroblasts

To evaluate whether lithium at the therapeutic concentration of 1.0 mmol/L modulates Klotho expression, we conducted experiments on human cardiac fibroblasts with or without 1.0 mmol/L LiCl treatment for 48 hours. The messenger (m)RNA expression of Klotho was significantly upregulated in human cardiac fibroblasts compared with control fibroblasts (Fig. 3A). Additionally, treatment with LiCl at the concentration of 1.0 mmol/L upregulated the protein expression of Klotho by 76.3% (Fig. 3B). The findings indicated that treatment with lithium at the concentration of 1.0 mmol/L increased Klotho expression in human cardiac fibroblasts.

Fig. 3.

Fig. 3

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Klotho expression in human cardiac fibroblasts with or without lithium treatment. (A) Compared with control cardiac fibroblasts (n = 6), human cardiac fibroblasts treated with LiCl at 1.0 mmol/L (n = 6) for 48 hours exhibited upregulated mRNA expression of Klotho. (B) Human cardiac fibroblasts treated with LiCl at 1.0 mmol/L (n = 6) for 48 hours exhibited upregulated protein expression of Klotho relative to control cardiac fibroblasts (n = 6).

**p < 0.01.

Effects of Lithium on NF-κB in Human Cardiac Fibroblasts

Because NF-κB is a potential downstream target of Klotho and is involved in the regulation of mitochondrial function and dynamics, we investigated whether lithium at the therapeutic concentration of 1.0 mmol/L modulated the levels of total and phosphorylated NF-κB p65 in human cardiac fibroblasts. LiCl treatment at 1.0 mmol/L for 48 hours led to significant downregulation of the levels of phosphorylated NF-κB p65 in human cardiac fibroblasts compared with control cardiac fibroblasts (Fig. 4). By contrast, no significant differences were noted in the levels of total NF-κB p65 between control and human cardiac fibroblasts treated with 1.0 mmol/L LiCl. The findings suggested that lithium at the therapeutic concentration of 1.0 mmol/L inhibited the activity of NF-κB in human cardiac fibroblasts.

Fig. 4.

Fig. 4

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Expression of nuclear factor-kappa B (NF-κB) in human cardiac fibroblasts with or without lithium treatment. Compared with control cardiac fibroblasts (n = 6), human cardiac fibroblasts treated with LiCl at 1.0 mmol/L (n = 6) for 48 hours exhibited decreased levels of phosphorylated (p-)NF-κB p65. However, LiCl at 1.0 mmol/L (n = 6) exerted no significant effect on the levels of total NF-κB p65 in human cardiac fibroblasts.

*p < 0.05.

NF-κB Inhibition on the Effects of Lithium in Human Cardiac Fibroblasts

To examine whether NF-κB inhibition blocked the effects of lithium on mitochondrial size and branching, human cardiac fibroblasts with or without lithium treatment were incubated with Bay 11-7802 (NF-κB inhibitor). The results showed that combined treatment with 3 μM Bay 11-7802 and 1.0 mmol/L lithium for 48 hours increased the mitochondrial area, mitochondrial perimeter, mitochondrial branching (form factor), branch length per mitochondrion, and branch length per branch in human cardiac fibroblasts to a similar extent as those observed in 1.0 mmol/L lithium–treated fibroblasts (Fig. 5). Furthermore, we examined whether NF-κB inhibition blocked the effects of lithium on the migration ability (i.e., profibrotic activity) of human cardiac fibroblasts. The results demonstrated that lithium treatment at 1.0 mmol/L for 48 hours reduced the migration ability of human cardiac fibroblasts compared with control fibroblasts (Fig. 6). In addition, treatment with the combination of 3 μM Bay 11-7802 and 1.0 mmol/L lithium for 48 hours inhibited the migration ability of human cardiac fibroblasts to a similar extent as that observed in 1.0 mmol/L lithium–treated fibroblasts. The findings suggested that treatment with lithium (1.0 mmol/L) increased the mitochondrial size and branching and decreased the migration ability of human cardiac fibroblasts through NF-κB inhibition.

Fig. 5.

Fig. 5

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Mitochondrial morphology of human cardiac fibroblasts treated with lithium and Bay 11-7802 for 48 hours. (A) Representative images of MitoTracker Green FM (Invitrogen)–stained human cardiac fibroblasts (upper panel) and two-dimensional mitochondrial analysis in Fiji/ImageJ (National Institutes of Health; lower panel). Compared with control cardiac fibroblasts (n = 6), combined treatment with 3 μM Bay 11-7802 and 1.0 mmol/L lithium for 48 hours (n = 6) increased the mitochondrial area (B) and mitochondrial perimeter (C) in human cardiac fibroblasts. No significant between-group differences were observed in the aspect ratio (D). In addition, the combined treatment increased mitochondria form factor (E), branch length per mitochondrion (F), and branch length per branch (G) similar to that observed in lithium–treated fibroblasts (n = 6).

*p < 0.05, ***p < 0.001.

Fig. 6.

Fig. 6

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Migration ability of human cardiac fibroblasts treated with lithium and Bay 11-7802 for 48 hours. (A) Photographs and (B) average data of the migration assay in human cardiac fibroblasts (n = 6). Compared with control cardiac fibroblasts (n = 6), human cardiac fibroblasts treated with LiCl at 1.0 mmol/L (n = 6) for 48 hours exhibited reduced migration ability. In addition, combined LiCl (1.0 mmol/L) and Bay 11-7802 (3 μM)–treated cardiac fibroblasts (n = 6) for 48 hours exhibited comparable migration ability to LiCl (1.0 mmol/L)–treated cardiac fibroblasts.

*p < 0.05, **p < 0.01.

DISCUSSION

To the best of our knowledge, this study is the first to demonstrate that lithium at the therapeutic concentration of 1.0 mmol/L increased the mRNA and protein expression levels of Klotho in human cardiac fibroblasts. Furthermore, lithium at this therapeutic concentration inhibited the migration ability of human cardiac fibroblasts. Evidence has indicated that Klotho exerts antifibrotic effects [9-12] and confers cardioprotective benefits [4-8]. The present finding of the upregulation of Klotho expression by lithium suggests a novel mechanism that mediates the cardioprotective effects of lithium found in previous clinical studies [24,25,32,33].

Impaired mitochondrial quality control contributes to the activation of cardiac fibroblasts and promotes the development of fibrogenesis in the myocardium [34,35]. The machinery of mitochondrial quality control relies on the regulation of mitochondrial dynamics (i.e., fission and fusion cycles) and mitophagy [35,36]. While enhanced mitochondrial fusion facilitates communication between mitochondria and promotes mitochondrial genomic repair, erroneous fission leads to the fragmentation of mitochondria and damage to mitochondrial DNA. In turn, impaired oxidative phosphorylation in the fragmented mitochondria leads to oxidative stress and calcium overload [36,37], resulting in cardiac fibroblast senescence and fibrogenesis [3]. In our previous studies [15,18], we discovered that lithium inhibited mitochondrial oxidative stress and store-operated calcium entry. In the present study, we further uncovered that lithium at the therapeutic concentration of 1.0 mmol/L increased the mitochondrial size and branching in human cardiac fibroblasts, which are the main morphological features characterizing mitochondrial fusion. The findings suggest further research into the actions of lithium on mitochondrial dynamics in addition to the assessment of morphological indices.

Evidence suggests that Klotho rescues mitochondrial damage by modulating signaling pathways regulating mitochondrial function and antioxidants [12,38]. Loss of Klotho expression leads to mitochondrial injury and dysfunction [39,40]. Because studies have suggested that NF-κB is a downstream target of Klotho [41,42] and is involved in the regulation of mitochondrial function and dynamics [43,44], the present study investigated whether NF-κB mediates the beneficial effects of lithium on mitochondrial morphology. The results revealed that lithium at the therapeutic concentration of 1.0 mmol/L inhibited the activity of NF-κB in human cardiac fibroblasts. Furthermore, cardiac fibroblasts treated with the combination of lithium and Bay 11-7802 exhibited similar mitochondrial size and branching relative to lithium-treated cardiac fibroblasts. These findings suggested that therapeutic lithium enhanced mitochondrial size and branching through NF-κB inhibition.

Our bioinformatics analysis predicted that lithium regulates multiple signaling pathways in the myocardium. The findings are similar to those of numerous studies reporting the multiaction targets of lithium in the brain [45,46]; suggesting that the therapeutic effects of lithium are mediated by mechanisms with multiple dimensions ranging from molecular to organ system levels. It is worth noting that in addition to pathways regulating mitochondrial function, pathways associated with the gap junction and extracellular matrix–receptor interaction, which were recognized in our bioinformatics analysis, also contribute to the pathogenesis of cardiac fibrosis [47,48]. Thus, we could not exclude the possibility that lithium inhibited the profibrotic activities of human cardiac fibroblasts through pathways other than Klotho and mitochondrial quality control. Furthermore, human cardiac fibroblasts utilized in this study were isolated from normal adult cardiac tissues. Therefore, future mechanistic studies should conduct experiments on cardiac fibroblasts derived from patients who have specific cardiac diseases.

Epidemiological studies have indicated that patients with bipolar disorder are at high risks of heart failure and cardiac mortality [49,50]. Although the underlying mechanisms are complex and remain unclear, initial evidence suggest accelerated aging in the heart of individuals with bipolar disorder as an underlying mechanism [51,52]. Furthermore, increasing studies have suggested that the pathophysiology of both bipolar disorder and heart failure is related to abnormal mitochondrial morphology and dynamics, neuroimmune dysfunction, and aberrant mitochondrial metabolism and oxidative stress pathways [53,54]. Hence, regarding the clinical implication, this basic research provided laboratory evidence that can be used by future translational studies investigating the potential cardioprotective effects of therapeutic lithium.

This study has several limitations that should be considered when interpreting the findings. First, the miRNA sequencing data were derived from two rats per group. Thus, the findings should be considered exploratory and need to be validated in future studies. Second, mechanistic findings were primarily derived from in vitro experiments, with limited molecular validation in cardiac tissues from lithium-treated animals. Third, conclusions are based on a single lithium concentration and time point, which restricts insight into dose- or time-dependent effects.

As summarized in Figure 7, this experimental study suggests that lithium at the therapeutic concentration of 1.0 mmol/L increases the expression of Klotho (an antiaging protein) and enhances mitochondrial size and branching in human cardiac fibroblasts probably through NF-κB inhibition. Given the fact that patients with bipolar disorder are at high risks of accelerated aging, mitochondrial dysfunction, and heart failure, future studies should validate the mechanisms proposed by this study and include biomarker panels reflecting the biological processes of aging and mitochondrial dysfunction to examine the cardioprotective benefits of lithium in clinical patients.

Fig. 7.

Fig. 7

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Illustration of the proposed mechanisms underlying the effect of therapeutic lithium on Klotho expression and mitochondrial dynamics in human cardiac fibroblasts. Lithium at a therapeutic concentration increased the expression of the Klotho antiaging protein in human cardiac fibroblasts, which in turn inhibited the activity of nuclear factor-kappa B (NF-κB) by downregulating the levels of phosphorylated (p-)NF-κB p65. With the inactivation of NF-κB, mitochondrial dynamics shifted from fission process to fusion process, thereby facilitating mitochondrial repair and protecting human cardiac fibroblasts from senescence and profibrotic phenotypes.

Acknowledgments

The authors appreciate the Laboratory for Precision Medicine at Taipei Medical University Hospital for the technical support in the next-generation sequencing.

Footnotes

Funding

This study was supported by the National Science and Technology Council of Taiwan under grant NSTC 112–2314–B–038–050; Taipei Medical University–Wan Fang Hospital under grants 111TMU–WFH–15, 112TMU–WFH–09, and 113TMU–WFH–19.

Conflicts of Interest

No potential conflict of interest relevant to this article was reported.

Author Contributions

Conceptualization: Pao-Huan Chen, Yu-Hsun Kao, Yi-Jen Chen. Data acquisition: Pao-Huan Chen, Shuen-Hsin Liu. Formal analysis: Pao-Huan Chen, Ting-Wei Lee, Tzu-Yu Cheng. Funding: Pao-Huan Chen, Tzu-Yu Cheng. Supervision: Yu-Hsun Kao, Yi-Jen Chen. Writing—original draft: Pao-Huan Chen. Writing—review & editing: Ting-Wei Lee, Shuen-Hsin Liu, Tzu-Yu Cheng, Yu-Hsun Kao, Yi-Jen Chen.

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