Berberine alleviates myocardial diastolic dysfunction by modulating Drp1-mediated mitochondrial fission and Ca²⁺ homeostasis in a murine model of HFpEF

Abstract

Heart failure with preserved ejection fraction (HFpEF) displays normal or near-normal left ventricular ejection fraction, diastolic dysfunction, cardiac hypertrophy, and poor exercise capacity. Berberine, an isoquinoline alkaloid, possesses cardiovascular benefits. Adult male mice were assigned to chow or high-fat diet with L-NAME (“two-hit” model) for 15 weeks. Diastolic function was assessed using echocardiography and non-invasive Doppler technique. Myocardial morphology, mitochondrial ultrastructure, and cardiomyocyte mechanical properties were evaluated. Proteomics analysis, autophagic flux, and intracellular Ca²⁺ were also assessed in chow and HFpEF mice. The results show exercise intolerance and cardiac diastolic dysfunction in “two-hit”-induced HFpEF model, in which unfavorable geometric changes such as increased cell size, interstitial fibrosis, and mitochondrial swelling occurred in the myocardium. Diastolic dysfunction was indicated by the elevated E value, mitral E/A ratio, and E/e’ ratio, decreased e’ value and maximal velocity of re-lengthening (–dL/dt), and prolonged re-lengthening in HFpEF mice. The effects of these processes were alleviated by berberine. Moreover, berberine ameliorated autophagic flux, alleviated Drp1 mitochondrial localization, mitochondrial Ca²⁺ overload and fragmentation, and promoted intracellular Ca²⁺ reuptake into sarcoplasmic reticulum by regulating phospholamban and SERCA2a. Finally, berberine alleviated diastolic dysfunction in “two-hit” diet-induced HFpEF model possibly because of the promotion of autophagic flux, inhibition of mitochondrial fragmentation, and cytosolic Ca²⁺ overload.

Keywords

HFpEF / berberine / Drp1 / autophagy / Ca ²⁺

1 Introduction

Heart failure is usually classified into heart failure with reduced ejection fraction (HFrEF) and heart failure with preserved EF (HFpEF), with left ventricular EF ≥ 50% being widely considered as the cut-off to distinguish between the two [1–4]. Over the past decades, the prevalence of HFpEF is rapidly escalating mainly because of an aging population and the increased prevalence of comorbidities including obesity, diabetes mellitus, and metabolic syndrome, resulting in high cardiovascular morbidity and mortality [5,6]. HFpEF is a complicated clinical entity with predominant phenotypes of diastolic dysfunction and poor ventricular compliance [7]. The current mainstream therapy for HFpEF involves diuretics for patients with congestion and mineralocorticoid receptor antagonists, along with careful management of comorbidities [5]. Although various studies and clinical trials have been performed to identify possible molecular mechanisms and therapeutic targets for HFpEF, effective drug targets and therapeutic remedies are still lacking [8].

Berberine, an isoquinoline alkaloid from the Chinese herb Coptis chinensis and other plants, is widely used as an antibiotic for intestinal infections and diarrhea [9,10]. Over recent decades, novel biological functions were unveiled for berberine, including anti-inflammation, anti-apoptosis, and anti-oxidative stress, and these findings reveal the protective role of berberine for diseases such as cancer, diabetes mellitus, and Alzheimer’s disease [11]. Moreover, berberine have shown efficacy in various cardiovascular diseases, including myocardial ischemia, hypertension, dyslipidemia, atherosclerosis, arrhythmia, and cardiomyopathies [9,12,13]. Berberine has effectively alleviated pressure overload-induced cardiac injury and heart failure in a transverse aortic contraction (TAC) mouse model [14]. Moreover, berberine ameliorated mitochondrial dysfunction and restored mitophagy in TAC-challenged mice [14]. Given that exercise intolerance governed by mitochondrial dysfunction and oxygen utilization impairment is one of the hallmarks of HFpEF [15], the present study was designed to decipher whether berberine is protective in HFpEF and whether mitochondrial injury and mitochondrial homeostasis are involved in berberine-induced response. A “two-hit” murine model was established to combine both metabolic and mechanical stress to mimic the complicated clinical manifestation of human HFpEF [16]. Berberine was administered to the “two-hit” challenge-induced HFpEF murine model prior to the assessment of cardiac function and mitochondrial dynamics. Our results indicated that berberine ameliorated cardiac diastolic dysfunction in HFpEF through the inhibition of Drp1-mediated mitochondrial fission, indicating its potential for the treatment of HFpEF.

2 Materials and methods

2.1 Experimental animals, HFpEF model, and plasma file

Adult male C57BL/6N mice were randomly assigned into four groups, including a chow or high-fat diet (60% of calories from fat) plus L-NAME (0.5 g/L in drinking water, “two-hit”) group with or without oral treatment of berberine (50 mg/kg daily for 4 weeks) [16]. Mice were anesthetized by a combination of ketamine (80 mg/kg; Pfizer, Berlin, Germany) and xylazine (12 mg/kg; Bayer AG, Leverkusen, Germany). Euthanasia was accomplished by cervical dislocation. Blood glucose levels were measured using a glucometer (Acu-ChekTM, Corydon, IN, USA). The experimental procedures used were approved by the Institutional Animal Use and Care Committee of Zhongshan Hospital Fudan University (Shanghai, China) and Xuhui Central Hospital Fudan University (Shanghai, China) following the NIH Guideline [16].

2.2 Gene ontology (GO) enrichment

The official gene symbols of berberine interaction were obtained from the PubChem database prior to the application of these genes in the DAVID software. The P value was set at ≤ 0.01. Graphics was produced using website [17].

2.3 Echocardiographic assessment

Mice were anesthetized using isoflurane (1.5%–3.0%) before cardiac geometry and function were evaluated using M-mode echocardiography (Vevo 2100, VisualSonics, Toronto, Canada). Fractional shortening (FS) was derived from LV end-diastolic diameter (LVEDD) and end-systolic diameter (LVESD) using the equation of (LVEDD–LVESD)/LVEDD. Ejection fraction (EF) and heart rate (HR) were calculated using Vevo 2100 echocardiography. Peak Doppler blood inflow velocity across mitral valve during early diastole, peak Doppler blood inflow velocity across mitral valve during late diastole, peak tissue Doppler of myocardial relaxation velocity at the mitral valve annulus during early diastole, and early filling deceleration time were also observed. For echocardiogram acquisition in temperature-controlled conditions, isoflurane was reduced to 1.0%–1.5% and was adjusted to maintain a heart rate within the range of 420–550 beats per min [18,19].

2.4 Exercise exhaustion test

After 3 days of acclimatization to treadmill exercise, exhaustion test was performed. First, the mice ran uphill (20°) on the treadmill (Columbus Instruments, Columbus, OH, USA) starting at a warm-up speed of 5 m/min for 4 min before the speed was readjusted to 14 m/min for 2 min. For every 2 mins, speed was increased by 2 m/min until the mice were exhausted (defined as inability to return to run within 10 s of direct contact with an electric-stimulus grid). Running time was measured, and it was used to calculate the running distance [20].

2.5 Blood pressure recordings

Systolic and diastolic blood pressure were measured using a non-invasive procedure in conscious mice by using the tail-cuff method and a CODA instrument (Kent Scientific, Torrington, CT, USA). Mice were placed in individual holders on a platform, and recordings were performed under steady-state conditions. Blood pressure was recorded for at least three measurements per session.

2.6 Proteomics and enrichment analysis

Protein was extracted from mouse hearts and was subjected to NSI source followed by tandem mass tag (TMT)-based mass spectrometry (MS) in Q Exactive TM Plus (Thermo) coupled online to the UPLC. Peptide FDR was adjusted to 1%, and all spectra identified with a score < 20 were discarded. Proteins meeting the criteria of |log₂FC| > 1.2 and P value < 0.05 were identified as differentially changed proteins. Proteins were mapped to relevant biological annotation, and the result of GO and KEGG pathway enrichment analysis was visualized using the “cluster Profiler” package.

2.7 Isolation and culture of adult mouse cardiomyocytes (AMCMs)

AMCMs were isolated from adult mice, and a yield of at least 90% rod-shaped AMCMs was deemed successful [21]. Heart tissues from different groups were utilized, including “two-hit” diet-induced HFpEF and chow diet-mice with or without berberine treatment.

2.8 Histological examination

Following anesthesia by using ketamine (80 mg/kg; Pfizer) and xylazine (12 mg/kg; Bayer AG), mice were sacrificed, and heart samples were immediately placed in 10% neutral-buffered formalin at room temperature for 24 h. Cardiomyocyte cross-sectional areas were calculated using a digital microscope (400×) and the Image J software (version2.3.0, NIH). Masson trichrome staining was used to assess interstitial fibrosis. The percentage of fibrosis was calculated using Adobe Photoshop CS3 (Adobe Systems Inc, San Jose, CA, USA). The fraction of light blue stained area normalized to total area was used as an indicator of myocardial fibrosis. Frozen slices were cut on a cryotome and stained for wheat germ agglutinin (WGA) to measure cross-sectional area of cardiomyocytes [22].

2.9 Detection of reactive oxygen species (ROS)

Myocardial tissue slices were rinsed with PBS and incubated with a 10 µM DCFH-DA fluorescence probe (Beyotime, Shanghai, China, 1:1000) for 20 min at 37 °C. Cells were visualized using a Leica confocal microscope. ROS fluorescence intensity was evaluated using the ImageJ software [22].

2.10 TUNEL

According to the one-step TUNEL apoptosis assay kit (Beyotime S1086) protocol, frozen heart slices were incubated with TUNEL solution for 1 h before fluorescent imaging by using the Leica sp8 confocal fluorescence microscope [23].

2.11 Transmission electron microscopy (TEM)

Small cubic pieces ≤ 1 mm³ from left ventricles were fixed with 2.5% glutaraldehyde in 0.1 M sodium phosphate (pH 7.4) overnight at 4 °C. Following post-fixation in 1% OsO₄, samples were dehydrated through graded alcohols and were embedded in Epon Araldite. Ultrathin sections (50 nm) were prepared using an ultramicrotome (Ultracut E, Leica, Wetzlar, Germany) and stained with uranyl acetate and lead citrate. Specimens were viewed under a Hitachi H-7000 electron microscope (Hitachi High Tech America, Inc., Pleasanton, CA, USA) [22].

2.12 Measurement of cytoplasmic Ca²⁺

Isolated cardiomyocytes were loaded with 1 μmol/L Fluo-4 (Beyotime, S1060) for 20 min. Cardiomyocytes were excited at 494 nm with light from an argon ion laser, and the emission fluorescence (516 nm) was imaged using a laser scanning confocal microscope (Leica sp8). Cytoplasmic Ca²⁺ was calculated based on the fluorescence intensity measurements of the Fluo-4 Ca²⁺ probe [24].

2.13 Measurement of mitochondrial Ca²⁺

The mitochondrial Ca²⁺ dye Rhod-2 (R1245MP, Invitrogen, Thermo Fisher, Shanghai, China) was employed. Cells were incubated with Rhod-2 (5 μmol/L) at 37 °C in the dark for 30 min. Cells were washed thrice to remove excess or non-specific probes in the mitochondria. Cells were visualized under a confocal microscope (Leica sp8). Mitochondrial Ca²⁺ was calculated based on the fluorescence intensity of Rhod-2 [25].

2.14 Mitochondrial isolation and purification

Mitochondria were isolated from AMCMs by using a mitochondrion isolation kit (Abcam, ab110170) per the manufacturer’s instruction. In brief, cells were collected and were homogenized. After centrifugation at 1000× g for 10 min at 4 °C, supernatants were collected, and pellets were resuspended. Supernatants were centrifuged at 12 000× g for 15 min at 4 °C and were resuspend in RIPA buffer. Protein concentration was determined using a BCA protein assay kit (Beyotime) [26].

2.15 Immunofluorescence and confocal imaging of Drp 1 co-localization

Immunofluorescence for Tom20 and Drp1 was performed following the manufacturer’s instruction. Briefly, H9C2 cells were fixed and permeabilized at room temperature. After blocking for 1 h, samples were incubated with rabbit anti-Drp1 and mouse anti-Tomm20 antibodies (1:50) overnight at 4 °C. H9C2 cells were incubated with anti-rabbit Alexa Fluor 488 (1:1000) and anti-mouse Alexa Fluor 546 (1:1000 dilution) secondary antibodies for 1 h in the dark. Immunofluorescence was assessed using a laser confocal microscope equipped with a 630× oil objective (Leica sp8) [26,27].

2.16 ATP level

ATP level was measured using a firefly luciferase-based ATP assay kit (Beyotime, S0026). The assay was operated following the manufacturer’s instructions. Myocardial tissues (20 mg) were lysed in an ATP lysis buffer (200 µL) and were homogenized prior to centrifugation at 12000× g for 5 min at 4 °C. Supernatants were removed for ATP assay. ATP detection reagent (100 μL) and the sample were added into a microwell for 5 min at room temperature, and the contents were mixed in a multifunction microplate reader. ATP level was calculated from the standard curve and was expressed as nmol per mg protein [28].

2.17 Adenoviral transfection

H9C2 cells were transduced with adenoviruses carrying Drp1 (Adv-Drp1) at indicated multiplicities of infection (MOI) for 48 h following the manufacturer’s instruction. For these treatments, cells cultured in serum-free medium with dimethyl sulfoxide (DMSO, final concentration < 0.1%) were employed as controls [29].

2.18 Measurement of mitochondrial membrane potential (ΔΨ)

The ΔΨ value was measured using a ThermoFisher Image-iTTM TMRM kit. Cells were treated with TMRM (50 nmol/L) for 30 min at 37 °C. The samples were visualized, and the fluorescence intensity was recorded using a fluorescence microscope (Leica sp8) [30].

2.19 Berberine and Drp1 molecular docking simulation

The possible structural interplay between berberine and Drp1 was detected by performing molecular docking in AutoDockTools-1.5.7 and Open Babel GUI. First, the molecular structures of Drp1 and ligand molecular berberine were downloaded from the uniprot and cas website. After the removal of water molecules and addition of hydrogen, the ligand and acceptor structures were docked in AutoDock. The docking result was constructed with the lowest binding energy. Finally, molecular docking results were visualized and analyzed using PyMol 2.4.1 [31].

2.20 Cell shortening/re-lengthening

The mechanical properties of adult mouse cardiomyocytes were assessed using an IonOptix soft-edge system (IonOptix, Milton, MA). Cardiomyocytes were field stimulated at 0.5 Hz. Cell shortening and re-lengthening were assessed based on peak shortening (PS), time-to-PS (TPS10, 50, and 90), time-to-10%, -50%, and -90% re-lengthening (TR10, 50, and 90), and the maximal velocity of shortening/re-lengthening (± dL/dt) [21,32].

2.21 Measurement of intracellular and mitochondrial Ca²⁺

Cardiomyocytes were loaded with 1 μmol/L Fura-2 (Beyotime, S1502) for 20 min, and fluorescence intensity was recorded using a dual-excitation fluorescence photomultiplier system (Ionoptix). The qualitative change in Fura-2 fluorescence intensity (FFI) was based on the FFI ratio at the two wavelengths (360/380). Fluorescence decay time (single exponential) was derived as an indicator of intracellular Ca²⁺ clearance [32].

2.22 Western blot analysis

Heart tissues were sonicated in a lysis buffer containing 20 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton, 0.1% sodium dodecyl sulfate, and a protease inhibitor cocktail. Protein samples were incubated with anti-atrial natriuretic peptide (ANP), anti-natriuretic peptide B (BNP), anti-myosin heavy chain β (β-MHC), anti-mitochondrial uncoupling protein 2 (UCP2), anti-BCL-2 Associated X (Bax), anti-microtubule-associated protein 1 light chain 3 (LC3B), anti-protein 62 (p62), anti-autophagy related protein 5 (Atg5), anti-autophagy related protein 7 (Atg7), anti-citrate synthase (CS), anti-pyruvate dehydrogenase (PDH), anti-succinate dehydrogenase (SDH), anti-Na⁺-K⁺-ATPase α1 (ATPase), anti-peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α), anti-sodium/calcium exchanger (NCX), anti-calcium/calmodulin-dependent protein kinase II (CAMKII), anti-sarco(endo)plasmic reticulum Ca²⁺ ATPase 2a (SERCA2a), anti-phospholamban (PLN), anti-voltage-dependent anion-selective channel protein 1 (VDAC1), anti-mitochondrial calcium uniporter protein (MCU), anti-ryanodine receptor 1 (RYR1), anti-inositol 1,4,5-trisphosphate receptor type 1 (IP3R1), anti-dynamin-1-like protein (Drp1), anti-p-Drp1Ser616, anti-p-Drp1Ser637, anti-translocase of the outer mitochondrial membrane 20 (Tom20), anti-vinculin and anti-β-actin (loading control), and anti-VDAC1 (loading control of mitochondrial protein) antibodies. All antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA) or Abcam (Cambridge, UK). After immunoblotting, films were scanned and detected using a Bio-Rad calibrated densitometer, and the band intensity was normalized to respective loading controls (β-actin, VDAC, and Vinculin) [21,22].

2.23 Statistical analysis

Each variable was analyzed using one-way ANOVA and statistical test. P < 0.05 was regarded as the threshold for determining whether the groups significantly differ from each other. Data are expressed as mean ± SEM, and all statistical analyses were performed in GraphPad Prism 8.0.1 software (GraphPad, San Diego, CA, USA).

3 Results

3.1 Berberine treatment was related to mitochondria and oxidative stress signaling pathways

Possible berberine related signaling pathways were examined using the PubChem database and were annotated using the DAVID Bioinformatics Resources database. Enrichment results showed that berberine was closely associated with cell proliferation, oxidative stress, and heart development signaling pathways (Fig.1). Besides, ample genes and proteins in nucleosome, mitochondria, and cytosol were enriched in berberine-related pathways (Fig.1). Berberine-associated genes regulated within pathways are shown in detail in Table S1. Several molecular functions were enriched for berberine including ubiquitin protein ligase, transcription cofactor, and histone deacetylase binding (Fig.1 and 1D).

3.2 Berberine ameliorated cardiac diastolic dysfunction in “two-hit” diet-induced HFpEF mice

Different doses of berberine were tested to verify the safety. Notably, control mice treated with berberine (50 mg/kg/d, p.o.) survived for at least 3 weeks. Thus, this dose was employed as the recommended dosage for the rest of the study (Fig.2). Mice were maintained with high-fat diet (60% of calories from fat, HFD) and L-NAME (0.5 g/L in drinking water) for 15 weeks to establish the “two-hit” animal model for HFpEF. Berberine was intragastrically administered daily for the final 4 weeks (Fig.2). Echocardiographic results showed thickened LV mass, interventricular septum (IVS, d), and posterior wall thickness in diastole (PWT, d) at 15 weeks without affecting left ventricular ejection fraction (LVEF), FS, LVEDD, and LVESD in HFpEF mice compared with chow mice (Fig.2–2K). Non-invasive Doppler technique showed diastolic dysfunction in HFpEF mice, as evidenced by the increased E value, decreased e’ value, and elevated mitral E/A ratio and E/e’ ratio in HFpEF mice. The effect of these processes were reversed by berberine (Fig.2–2P). Besides, berberine did not affect either diastolic or systolic function in mice with a chow diet, and the heart rate remained unchanged among these mice (Fig.2).

3.3 Berberine alleviated exercise intolerance, cardiac hypertrophy, and glucose intolerance in “two-hit” diet-induced HFpEF mice

The “two-hit” diet-induced HFpEF mice exhibited a substantially increase in bodyweight, heart weight-to-tibial length ratio, wet-dry lung weight ratio, and decreased exercise capacity (running distance), and the effect of these processes was attenuated by berberine treatment (Fig.3–3E). Berberine also reversed the increase in systolic blood pressure (SBP) and diastolic blood pressure (DBP) in HFpEF mice (Fig.3 and 3G). Intraperitoneal glucose tolerance test (IPGTT) showed glucose intolerance and insulin resistance in HFpEF mice compared with those in chow group, and the effect of this process was ameliorated by berberine (Fig.3 and 3I). Total cholesterol, total triglyceride, and low-density lipoprotein were also elevated in HFpEF mice, and slight changes in high-density lipoprotein were observed (Fig.3–3M). Besides, berberine did not affect body weight, heart weight-to-tibia ratio, blood pressure, glucose tolerance, and serum cholesterol and lipid levels.

3.4 Berberine alleviated myocardial hypertrophy and interstitial fibrosis in “two-hit” diet-induced HFpEF mice

The current findings revealed that berberine effectively rescued “two-hits” insult-induced interstitial fibrosis (Masson trichrome) and cardiac hypertrophy (H&E and WGA) without eliciting any effect itself (Fig.4–4C). Several biomarkers for cardiac injury were also elevated in HFpEF mice, including anti-atrial natriuretic peptide, anti-natriuretic peptide B, and anti-myosin heavy chain β, and their effects were remarkably attenuated by berberine (Fig.4–4G).

3.5 Berberine alleviated mitochondrial injury and ameliorated autophagic flux in “two-hit” diet-induced HFpEF mice

Pronounced ROS production and apoptosis were observed in HFpEF mice as evidenced by ROS fluorescence and TUNEL assay, and their effect was ameliorated by berberine treatment. Berberine did not affect ROS production or apoptosis in mice administered with chow diet (Fig.5–5D). Transmission electronic microscopy (TEM) was employed to evaluate the ultrastructure of mitochondria, and the results displayed a pronounced increase in circularity index (the minor-to-major axis length ratio), indicating the occurrence of mitochondrial swelling and injury in the cardiomyocytes of HFpEF mice. Notably, the elevated density along with decreased mitochondrial perimeter denoted pronounced mitochondrial fission in mouse heart tissues following HFpEF insult, and its effect was partially inhibited by berberine (Fig.5–5H). The levels of mitochondrial injury marker proteins UCP2 and Bax were also increased in HFpEF mice, and its effect was ameliorated by berberine (Fig.5–5L). Berberine did not affect mitochondrial ultrastructure and dynamics. Moreover, disturbed autophagy flux was observed in HFpEF mice, as indicated by the decreased levels of LC3B, Atg5, and Atg7 along with increased p62, and its effect was partially reversed by berberine (Fig.5–5P). Berberine did not affect autophagy protein markers in chow diet-fed mice. HFpEF group displayed downregulated levels of mitochondria-related genes (CS, SDH, PDH, ATPase, and PGC-1α), and its effects were significantly alleviated by berberine, in which only a slight effect was exerted by berberine in chow mice (Fig.5–5U). Moreover, berberine reversed compromised ATP content in HFpEF group, in which berberine only exerted a slight effect (Fig.5).

3.6 Berberine alleviated mitochondrial fission and Ca²⁺ overload in “two-hit” diet-induced HFpEF mice

The biological processes involved in the pathologies of HFpEF were determined by subjecting the heart tissues from chow and “two-hit” diet-induced HFpEF mice to TMT proteomics analysis. Among the 156 differentially expressed proteins identified, 74 and 82 were upregulated and downregulated, respectively. KEGG enrichment analysis indicated the prominent role for fatty acid degradation, metabolism, and oxidative phosphorylation in HFpEF. Circulating levels of FFA and proinflammatory cytokines were elevated, and these parameters are likely responsible for pathological hypertrophy and diastolic dysfunction. Diastolic dysfunction, which is among the most vital pathological manifestations in HFpEF, was closely related to calcium signaling pathway as enriched in Fig.6. Consistent with the increased mitochondrial fragmentation in TEM images, the analysis of proteomics indicated the elevated fission-related protein Drp1 in HFpEF mice (Fig.6). Fig.6–6E display the crystal structures of Drp1 and the potential targets, in which berberine serves as an adaptor that binds Drp1 with a binding pocket consisting of VAL (A:243) and SER (A:281).

Considering the pronounced mitochondrial injury and fission in HFpEF hearts, mitochondrial Ca²⁺ transfer proteins were evaluated. The results show that MCU was downregulated in HFpEF mice, and its effect was alleviated by berberine. HFpEF was also accompanied with the upregulation of PLN and downregulation of SERCA2a, thus promoting Ca²⁺ re-uptake into sarcoplasmic reticulum. The effect of this process was also ameliorated by berberine. Although NCX, CAMKII, IP3R1, and RYR1 were significantly increased in HFpEF, they were unaffected by berberine in vivo. Berberine did not affect the levels of these mitochondrial Ca²⁺ flux molecules under the chow diet setting (Fig.6–6N).

Mitochondrial fission protein Drp1 localization between mitochondria and cytoplasm was determined by isolating mitochondrial and cytosolic fractions. The results indicated that berberine reversed the elevated Drp1 phosphorylation at S616 site in mitochondrial fraction without affecting Drp1 phosphorylation at S637 in mitochondria and cytoplasm in HFpEF mice (Fig.6–6X). The cytosolic and mitochondrial Ca²⁺ levels in Drp1 transfected H9C2 cells were then evaluated. Considering the lack of a commonly accepted cell model for HFpEF, Drp1 transfection was used to evoke mitochondrial and cytosolic Ca²⁺ overload. The data shown in Fig. S1A–S1F indicate that berberine partially offset cytoplasmic and mitochondrial Ca²⁺ overload and mitochondrial membrane potential collapse in the face of forced Drp1 transfection. In addition, berberine partially suppressed the level of co-localization between Drp1 and mitochondria (Fig. S1G and S1H). These findings favor an obligatory role for Drp1 mitochondrial localization and related Ca²⁺ handling in berberine-elicited beneficial response.

3.7 Berberine alleviated cardiomyocyte mechanical diastolic abnormalities and intracellular Ca²⁺ decay in “two-hit” diet-induced HFpEF mice

A slight difference was observed in resting cell length, peak shortening, maximal velocity of shortening ( dL/dt), and time-to-10%/50%/90% peak shortening (TP10/50/90) between chow and HFpEF groups with or without berberine treatment (Fig.7–7D,7F). However, an increase in time-to-50%/90% re-lengthening (TR50/90, but not 10%) and a decrease in maximal velocity of re-lengthening (–dL/dt) were observed in cardiomyocytes from HFpEF mice, and its effect was reversed by berberine treatment (Fig.7 and 7G). Furthermore, berberine depressed the raised levels of Ca²⁺ in baseline (Fura-2 fluorescent intensity, FFI) and prolonged intracellular Ca²⁺ decay in HFpEF mice without affecting electrically-stimulated rise in intracellular Ca²⁺ (ΔFFI, Fig.7–7K). Berberine did not affect any of the intracellular Ca²⁺ indices tested (Fig.7–7K).

4 Discussion

This study mainly found the cardioprotective role of berberine in the “two-hit” diet-induced HFpEF murine model through the modulation of mitochondrial dynamics and Ca²⁺ homeostasis. Berberine treatment effectively ameliorated HFpEF-evoked anomalies in exercise capacity, insulin sensitivity, cardiac remodeling, and diastolic function. The results of proteomics and KEGG enrichment analyses further indicated the pertinent role for mitophagy and Drp1 involved in the pathological process of HFpEF, which was verified by fragmented mitochondria in heart tissues from HFpEF mice. Cell shortening and re-lengthening results also indicated that berberine alleviated mechanical diastolic abnormalities and prolonged Ca²⁺ decay in cardiomyocytes from HFpEF mice, consistent with the changes in Ca²⁺-related proteins. Besides, berberine remarkably reversed the disturbed autophagic flux in HFpEF mice as indicated by the changes in LC3B and p62 levels. Meanwhile, berberine reversed mitochondria dysfunction. In vitro findings denoted that berberine alleviated forced Drp1 transfection-evoked mitochondrial localization and Ca²⁺ mishandling in berberine-elicited beneficial response. Overall, these findings revealed the protective role of berberine against HFpEF-induced cardiac remodeling and diastolic dysfunction possibly through the inhibition of Drp1-mediated mitochondrial fission and Ca²⁺ overload.

As a growing public health problem, HFpEF has multiple comorbidities including obesity, hypertension, and diabetes [33]. In the present study, a “two-hit” mouse model (high-fat diet along with L-NAME in drinking water) was employed to recapitulate metabolic and hypertensive stress of human HFpEF setting as described [16]. Consistent with other studies, an increase in bodyweight, blood pressure, and insulin resistance was noted in “two-hit” diet-induced HFpEF model. Exercise tolerance (running distance) and cardiac diastolic function were assessed using echocardiography, and the findings show that they were both compromised in HFpEF mice following 15 weeks of “two-hit” challenge without affecting systolic myocardial function. Through the analysis of public database, berberine exhibited a cardioprotective property and displayed enrichment in the perspectives of oxidative stress, cell proliferation, and cardiac development among the other cell signaling pathways. This result supports our current finding that the administration of berberine for 4 weeks effectively ameliorated exercise intolerance and diastolic dysfunction in our “two-hit” HFpEF murine model.

Considering the high energy demand in cardiomyocytes, maintenance of mitochondrial homeostasis is essential for energy supply and cell survival [34]. Mitochondrial quality control involves mitochondrial biogenesis, mitochondrial fusion and fission, and a mitochondria-specific form of autophagy (mitophagy) [35,36]. Fusion and fission occur in adult cardiomyocytes to sustain a network for mitochondrial morphology. Drp1, a crucial mitochondrial fission regulator, is downregulated in cardiomyocytes under normal conditions to mediate the segregation of damaged mitochondria for degradation (mitophagy) [37–39]. The activation of Drp1 along with unchecked mitophagy in pressure overload induced cardiac hypertrophy and heart failure, indicating their essential role in Drp1-mediated mitochondrial fragmentation and injury in cardiac remodeling and heart failure [40–42]. Notably, a Drp1-independent regulatory pathway also exists for mitophagy [34]. Drp1 is also involved in mitochondrial respiration and cell death including apoptosis and necroptosis [42–44]. The activation and S616 phosphorylation of Drp1 have improved mitochondrial fission and ameliorated mitochondrial defect in hypertension-induced HFpEF [45]. In the present study, increased Drp1 level and Drp1 S616 phosphorylation in mitochondrial fraction, Drp1-related mitochondrial fragmentation, and mitophagy were all evident in HFpEF mouse hearts, which are consistent with previous findings on mitochondrial fission and cristae destruction in heart tissues from HFpEF patients with valvular or ischemic heart disease [46].

In the present study, berberine reconciled dysregulated intracellular Ca²⁺ and Ca²⁺ regulating proteins (SERCA2a, phospholamban) in vivo in HFpEF murine model and forced Drp1 transfection-evoked mitochondrial localization and Ca²⁺ mishandling in vitro. A close connection has been suggested between mitochondrial morphology and Ca²⁺ homeostasis based on the fundamental role of Ca²⁺ in energetic metabolism in cardiomyocytes [30]. Drp1-mediated mitochondrial dynamics is essential for biological processes involving Ca²⁺ buffering capacity [47]. Dephosphorylated Drp1 and mitochondrial fission impaired the mitochondrial Ca²⁺ handling and cytosolic Ca²⁺ overload [48]. In the present study, Drp1 level and S616 phosphorylation increased in mitochondrial fraction from the “two-hit” HFpEF murine hearts. Ca²⁺ cycling is essential for maintaining a proper contraction-relaxation, and the Ca²⁺ dysregulation that results from SERCA2a defect leads to the disturbance of Ca²⁺ in heart failure and hypertrophy [49,50]. Ca²⁺ uptake into mitochondrial matrix is fundamental for cell metabolism and function [51]. Ca²⁺ regulates mitochondrial function, ATP production, and cell survival. The disruption of mitochondrial Ca²⁺ cycling is involved in numerous diseases, such as heart failure, diabetes, and neurodegeneration [52]. By using a metabolic risk-related rat model of HFpEF (Zucker/fatty spontaneously hypertensive F1 hybrid ZSF1-lean and ZSF1-obese rats), cardiac mitochondrial and cytosolic Ca²⁺ handling was examined to reveal elevated basal mitochondrial Ca²⁺ in ZSF1-obese rats. Besides, resting cytosolic Ca²⁺ levels and cytosolic Ca²⁺ transient decay time were also higher in ZSF1-obese rats possibly because of the decrease in SERCA2a/phospholamban ratio [53]. This observation supports our findings in cytosolic Ca²⁺ overload and decrease in SERCA2a/phospholamban ratio in the heart tissues from HFpEF mice.

The current study has several limitations. First, murine models of Drp1 knockout or overexpression would provide a better “gain- or loss-of-function” support for a role of mitochondrial fission in modulating Ca²⁺ handling, onset, and development of HFpEF. Next, more sophisticated experimental techniques should be employed for detecting the levels of cytosolic and mitochondrial Ca²⁺ concentrations aside from the level of Ca²⁺ modulation proteins. Considering that HFpEF is a lethal disorder without effective therapies, further study is warranted to elucidate mechanisms and develop potential treatment methods.

The current findings indicate that berberine alleviated exercise intolerance and cardiac diastolic dysfunction in “two-hit” diet-induced HFpEF mice through the inhibition of Drp1-mediated mitochondrial fission and modulation of disturbed cellular Ca²⁺ handling (Fig.8). These findings support that Drp1-dependent mitochondrial fission may serve as a target for management against HFpEF myopathies. Further study is needed to elucidate the mechanism behind HFpEF-induced changes of Drp1 and how berberine offers regulation on Drp1.

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