Dental pulp stem cells ameliorate D-galactose-induced cardiac ageing in rats

Animals

Thirty male Sprague–Dawley rats (8 weeks old, 180–200 g) were purchased from the Theodor Bilharz Research Institute, Imbaba, Egypt, and kept in the animal house of the Faculty of Medicine, Menoufia University, Egypt as previously described by (El-Akabawy et al., 2023). The animals were acclimatized to laboratory conditions for 1 week before the start of the experiment. The rats were kept in standard cages: two were rats kept in each cage, under standard laboratory conditions (22 ± 5 °C, 60 ± 5% humidity, and a 12-h/12-h light/dark cycle). Standard laboratory chow and tap water were provided ad libitum. At the end of the experiment, rats were anaesthetized via an intraperitoneal injection of ketamine (90 mg/kg) and xylazine (15 mg/kg) and were decapitated (El-Akabawy et al., 2023). Rats were euthanized in the event of rapid weight loss or impaired ambulation  via lethal injection of pentobarbital sodium (200 mg/kg). In this study, no rats were euthanized prior to the planned end of the experiment. All experimental procedures involving animals were approved by the Institutional Review Board of Ajman University, UAE [IRB# M-F-A-11-Oct].

DPSC isolation and culture

Dental pulp tissues were obtained from the pulpal cavity of Sprague–Dawley male rat incisors (aged 6–8-week-old) and cultured as previously described (Patel et al., 2009). Briefly, the dental pulp tissues were promptly harvested and enzymatically digested in a solution of 3 mg/mL collagenase type 1 (Sigma-Aldrich, St. Louis, MO, USA) for 1 h at 37 °C. The cells (1 × 10⁶ cells) were cultured in T25 cm flasks (Falcon). The culture media consisted of Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 20% foetal bovine serum (FBS, Gibco), 100 U/mL penicillin, and 100 µg/mL streptomycin. The flasks were incubated at 37 °C in a humidified incubator with 5% CO₂. Media were replaced every three days. The cells were passaged at 80% confluence using 0.05% trypsin–EDTA (Sigma-Aldrich) for 3–5 min. To evaluate cell viability, the cell suspension was mixed with 0.4% Trypan blue (Gibco), and 10 µL of the mixture was loaded in each chamber of a haemocytometer. Counting of the viable and non-viable cells was conducted within 5 min. Cells of passage 4 were evaluated.

Flow cytometry

Cells were resuspended in staining buffer (2% FBS/ phosphate buffered saline (PBS)) and surface-stained with FITC-conjugated mouse anti-rat CD105 (BioLegend, UK), FITCH-conjugated mouse anti-rat CD90 (BD Pharmingen, San Diego, CA, USA), PE-conjugated rabbit anti-rat CD34 (Abcam, Cambridge, UK), or PE-conjugated rabbit anti-rat CD45 (Abcam, Cambridge, UK) at 4 °C for 30 min. Isotype-matched antibodies were used as controls. Cells were analysed using an EPICS XL flow cytometer (Beckman Coulter, Brea, CA, USA) (El-Akabawy et al., 2022).

Experimental design

Rats were randomly divided into three groups: control, D-galactose (D-gal)-treated, and D-gal + DPSCs-treated ( n = 10 in each group). G Power software was used to determine the sample size. Rats (aged 9 weeks) in the D-gal- and D-gal + DPSCs-treated groups were given intraperitoneal injections of d-gal (300 mg/kg, Sigma-Aldrich, St. Louis, MO, USA) daily for 8 weeks. Rats in the D-gal + DPSCs group received intravenous administration into the tail vein of 1 × 10⁶ DPSCs labelled with the membrane-bound fluorescent marker PKH26 (Sigma-Aldrich, St. Louis, MO, USA) once every two weeks (El-Akabawy et al., 2022).

Measurement of body weight and the heart index

Body weights were measured weekly. At the end of the experiment, the rats were anaesthetized via intraperitoneal injection of ketamine (90 mg/ /kg) and xylazine (15 mg/kg) and were decapitated. Hearts were immediately dissected and weighed. Heart indices were calculated as follows: heart tissue weight (mg)/final body weight (g) (El-Akabawy et al., 2022).

Transthoracic echocardiography

All transthoracic echocardiography (TTE) measurements were performed using a linear transducer. A linear-array probe was used at a frequency of 10 MHz and attached to a Mindray M7 premium (Shenzhen Mindray Bio-Medical Electronics Co., Ltd., PR China) ultrasound echocardiography Doppler machine. The rats were anaesthetized by intraperitoneal injection of ketamine hydrochloride (25 mg/kg) and xylazine (5 mg/kg). Anaesthesia was followed by hair removal from the anterior part of the chest and thereafter, rats were kept on a specialized warming table to sustain normothermia (Watson et al., 2019).

Ejection fraction (EF) and fractional shortening (FS), left ventricular internal dimension at end-systole (LVIDs), left ventricular end-systolic volume (ESV), left ventricular posterior wall thickness at end-diastole (LVPWd), left ventricular internal dimension at end-diastole (LVIDd), and left ventricular end-diastolic volume (EDV) were assessed following the American Society of Echocardiography guidelines. This assessment was performed in a blind manner by an independent experienced researcher.

Electrocardiogram recordings

Three touch electrodes of the PowerLab settings (MLA1214, AD Instruments, New South Wales, Australia) were attached to an animal bioamplifier (FE136 Animal Bio Amp, AD instruments, New South Wales, Australia) and fixed to the animals. Lead placement was conducted according to the lead II configuration for determining heart rate (HR) in small laboratory animals (Stohr, 1998).

Electrocardiogram (ECG) recordings were performed as previously described by Harkin, O’Donnell & Kelly (2002). A stretch of the ECG readings was obtained to calibrate the second channel for simultaneous beats per minute (BPM) recordings. The ‘Ratemeter’ (within Chart for Powerlab; Holliston, MA, USA) was utilized simultaneously to measure the HR on channel 2 from the ECG plot in channel 1. HR was adjusted between 0 and 500 BPM. The ratemeter band was adjusted in such a way that the upper boundary remained positioned lower than the R-wave peak, whereas the lower line was higher than the P- and T-waves and any other noise. This facilitated the monitoring of the ECG waves on channel 1 with simultaneous BPM recorded on channel 2. The ECG was recorded at a sampling speed of 500/s and within a voltage range of 500 mV. A high-pass filter was adjusted to 0.3 Hz, and a low-pass filter (50 Hz) was used.

The ECG were digitized and stored using standard PC-based hardware (AD Instruments, Dunedin, New Zealand). PowerLab v.7.3.7 was utilized to illustrate the recording diagrams. The recordings were analysed using LabChart software (AD Instruments, Dunedin, New Zealand). The recorded ECG was used to calculate R-R, QRS, PR, QT, cQT, and ST intervals using the same software. Throughout the experiment, the rats’ body temperature was sustained at 38 °C. The time at the start of the recording was set as 0.0 min. The corrected QT (cQT) was determined using Bazett’s formula [QTc = QT/RR1/2] (Goldenberg, Moss & Zareba, 2006) installed in the software.

Assessment of oxidative stress and antioxidant indices

Malondialdehyde (MDA) and glutathione (GSH) levels, as well as superoxide dismutase (SOD) activity, were measured in the cardiac tissue using a spectrophotometer. Rat cardiac tissues (100 mg) were homogenized in one mL of phosphate-buffered saline (PBS; pH 7.0) to assess the MDA level and determine the degree of lipid peroxidation. After mixing the homogenates with 20% trichloroacetic acid (TCA), the mixtures were centrifuged at 5000 rpm for 15 min. A 5% thiobarbituric acid (TBA) solution was added to the supernatants and boiled for 10 min. The absorbance was obtained at 532 nm and a standard curve was used to quantify the MDA levels. The results are presented as nanomoles (nmol) per milligram (mg) of protein.

Based on the suppression of a nitro blue tetrazolium reduction by O₂ produced by the xanthine/xanthine oxidase system, the superoxide dismutase (SOD) activity was determined, and absorbance was obtained at 550 nm. The findings are represented as units (U) per milligram (mg) of protein. One SOD activity unit was considered as the enzyme concentration required to generate 50% inhibition in one mL reaction solution per mg of tissue protein.

To examine GSH concentrations, cardiac tissue homogenates were incubated with a solution of dithiobis nitrobenzoate (DTNB) for 1 h. The absorbance was obtained at 412 nm. A standard curve was used to determine the GSH level. The results are presented in micromoles (mmol) per mg of protein.

Quantitative reverse-transcription polymerase chain reaction

RNA was isolated from cardiac-tissue homogenates of rats in each group using the RNeasy Purification Reagent (Qiagen, Hilden, Germany). RNA purity was assessed using a spectrophotometer, ensuring a 260/280 nm absorption ratio of 1.8–2.0 for all samples. Subsequently, cDNA synthesis was performed employing Superscript II (Gibco Life Technologies, Waltham, MA, USA). Quantitative PCR (qPCR) was performed on a StepOneTM instrument with software version 3.1 (Applied Biosystems, Foster City, CA, USA). Reaction mixtures contained SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA), gene-specific primer pairs (detailed in Table 1), cDNA, and nuclease-free water. Cycling conditions comprised an initial denaturation step at 95 °C for 10 min, followed by 40 cycles of 15 s at 95 °C and 60 s at 60 °C. Data analysis was conducted using the ABI Prism sequence detection system software, and quantification was performed with the Sequence Detection Software v1.7 (PE Biosystems, Foster City, CA, USA). The comparative cycle threshold method (Livak & Schmittgen, 2001) was used to determine relative expression levels of the target gene, with all values normalized to β-actin mRNA.

Western blot analysis

Western blot analysis was conducted as previously described before by (Al-Serwi, El-Kersh & El-Akabawy, 2021). Using radioimmunoprecipitation buffer (Sigma-Aldrich, St. Louis, MO, USA), proteins were isolated from the cardiac tissues. The homogenates were centrifuged at 12,000 × g at 4 °C for 20 min and the protein level was measured in lysate aliquots using a protein assay kit (Bio-Rad, Hercules, CA, USA). Samples were boiled at 95 °C for 5 min, separated (20 µg/lane) using 7% sodium dodecyl sulphate–polyacrylamide gel electrophoresis, and subsequently transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). Next, the membranes were blocked for 1 h at room temperature (RT) using 5% bovine serum albumin in Tris-buffered saline (TBS), and then incubated for 12 h at 4  °C with primary antibodies specific for anti-sirt1 (Abcam, Cambridge, UK, cat # ab110304), anti-cleaved caspase-3 (Abcam, Cambridge, UK, cat # ab184787), anti-cytochrome c (Abcam, Cambridge, UK, cat # ab133504), anti-Bax (Abcam, Cambridge, UK, cat # ab32503), and anti-Bcl-2 (Abcam, Cambridge, UK, cat ab194583). After rinsing with TBS, the membranes were incubated with secondary horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG antibody (1: 3000, Bio-Rad) for 1 h at RT. Proteins were visualized using enhanced chemiluminescence (ECL Plus; Amersham, Arlington Heights, IL, USA) and measured via densitometry using Molecular Analyst Software (Bio-Rad). The relative expression of each protein band was normalized to that of β-actin.

Histological and immunohistochemical analyses

At the end of the experiment, cardiac tissues were dissected and fixed in 10% formalin and embedded in paraffin wax. For histological assessment, 5-µm left ventricular sections were de-paraffinized, rehydrated using a graded ethanol series (100%, 90%, and 70%), and stained with haematoxylin and eosin (H&E) or Masson’s trichrome.

For immunofluorescence staining, cardiac tissue was fixed at 4  °C for 24 h and cryoprotected in 30% sucrose at 4  °C. Using a cryostat, serial sections (40 µm) were obtained and kept at −20 °C until use. The sections were incubated in 10% blocking solution at RT for 30 min and then incubated at 4 °C overnight with the following primary antibodies; rabbit Anti-Cardiac Troponin T antibody (1:2000, Abcam, Cat. #ab209813), rabbit Anti-Cardiac Troponin I antibody (1:1000, Abcam, Cat. #ab209809), or rabbit anti-connexin-34 (1:1000, Abcam, Cat. #ab259276). Thereafter, sections were rinsed with PBS and incubated with a secondary antibody (1:500, Alexa-488, Cat. #A-11034, Molecular Probes) for 1 h. After washing with PBS, the sections were mounted in Fluoroshield mounting medium containing DAPI (Abcam, Cat. #ab104139).

Quantitative histological assessments

For quantitative evaluation, three H&E- and Masson-stained sections per rat were used. By applying ImageJ software (NIH, Bethesda, MD, USA), the H&E- and Masson-stained sections were examined to determine the cardiomyocyte area and Masson’s-stained area, respectively. A Leica DMLB2/11888111 microscope equipped with a Leica DFC450 camera was used to acquire the images.

Connexin-43 immunofluorescence was measured by randomly capturing five non-overlapping images per slide. A Leica DM5500 B/11888817/12 microscope, fitted with a Leica HI PLAN 10×/0.25 objective and a Leica DFC450C camera, was used to capture the images. Connexin-43-stained spots were manually counted using the plugin/cell counting tool (Rangan & Tesch, 2007) in ImageJ software (National Institutes of Health, Bethesda, MD, USA), and the average per field for each rat was then calculated. This measurement was performed in a blind manner by an independent experienced researcher. For statistical analysis and comparison, ten animals were used per experimental group.

Statistical analysis

Statistical analysis was performed as previously described by El-Akabawy et al. (2022). The data are presented as the mean ± SEM. Normal distributions were assessed using the D’Argostino and Pearson normality tests, and data were analysed using one- or two-way analysis of variance (ANOVA), followed by a post hoc Bonferroni test. P <0.05 was considered statistically significant. Statistical analyses were performed using GraphPad Prism 5.03 software (GraphPad Software, San Diego, CA, USA). No inclusion/exclusion criteria were applied in this study.

Characterization of DPSCs

DPSCs derived from the dental pulp tissue of Sprague–Dawley rats were spindle-shaped after 10 days of culture. Flow cytometry was used to characterize the cells at passage 4. The expression of CD90 and CD105 (mesenchymal cell markers), and CD45 and CD34 (hematopoietic lineage markers) were assessed. Over 90% of the cells were identified as CD90+ and CD105+ and less than 10% were identified as CD45+ and CD34+ (Fig. S1). These findings suggest that most of these cells were MSCs.

DPSC transplantation improves body weight and heart indices

Body weights of the rats in the control, D-gal and transplanted groups did not differ significantly (Fig. 1A). However, the heart index was dramatically increased in aged rats than in control rats, suggesting cardiac hypertrophy. DPSC transplantation dramatically decreased the heart index compared with that of D-gal rats (Fig. 1B), indicating that the transplanted cells attenuated D-gal-induced hypertrophy.

Intravenous injection of DPSCs reduces cardiac dysfunction in D-galactose-induced aged rats

Next, we evaluated the effects of DPSC transplantation on D-galactose (D-gal)-induced cardiac ageing in rats using echocardiography. The results revealed widespread LV systolic and diastolic dysfunction, including reduced EF% and FS%, elevated LVIDs, ESV, and LVPWd in D-gal-treated rats. Compared to D-gal-treated rats, DPSC injection improved EF% (Fig. 2B) and FS% (Fig. 2C) and reduced LVPWd (Fig. 2D), LVIDs (Fig. 2E), ESV (Fig. 2F) measurements. We observed an increase in left ventricular internal dimension at end-diastole (LVIDd) (Fig. 2G) and left ventricular end-diastolic volume (EDV) (Fig. 2H) in aged hearts; however, this difference was not statistically significant. D-gal intraperitoneal injection led to significant changes in the ECG results of treated rats in the form of increased QRS duration and cQT interval, but decreased R amplitude and ST height compared to the control group. Rats in the D-gal + DPSCs group exhibited an improved ECG pattern, as indicated by a significantly decreased QRS duration and cQT interval, and an elevated R amplitude and ST height, compared to the aged group (Table 2).

Effect of DPSC transplantation on the expression of connexin-43

The decreased production of the critical gap junction protein, connexin-43, plays a pivotal role in age-related cardiac dysfunction (Rodriguez-Sinovas et al., 2021). To determine whether DPSC transplantation restored connexin-43 expression in the hearts of aged rats, connexin-43 immunoreactive areas were evaluated. In D-gal-treated rats, immunoreactive connexin-43 areas were sparse, and their expression in cardiac tissue markedly decreased, whereas in the D-gal + DPSCs group, they were markedly upregulated (Figs. 3A–3D).

DPSC systemic transplantation attenuates cardiac histopathological alterations in D-galactose-induced aged rats

To examine the alterations in the myocardial architecture of different groups, left ventricular cardiac tissue slides were stained with H&E. The left ventricular cardiac tissue of D-gal-induced ageing rats exhibited a distorted myocardial structure, characterized by a disorganized arrangement of cardiomyocytes and expanded intercellular space (arrows, Fig. 4A) when compared to the control rats. In contrast, marked improvement was observed in the ageing rats that received DSPSC injection. H&E staining also revealed that the left ventricular cardiomyocyte cross-sectional area was significantly enlarged in D-gal-induced ageing rats, whereas DPSC treatment markedly decreased the cardiomyocyte area in D-gal + DPSC-treated rats (Fig. 4B). Masson’s trichrome staining was used to examine the degree of cardiac fibrosis in the different groups. The collagen-stained area in the interstitial and perivascular areas of the myocardium dramatically increased in D-gal-induced ageing rats. However, DPSC treatment significantly reduced collagen accumulation compared to that in the D-gal group (Figs. 4C and 4D).

DPSCs migrate into and survive in the cardiac tissue with a few transplanted cells differentiated into cardiomyocytes

An immunofluorescence-based examination of the cardiac tissue of D-gal + DPSCs rats was performed to assess the engraftment of injected DPSCs into the heart. Transplanted DPSCs were distinguished from recipient cells via labelling with PKH26, which is a cell membrane-binding dye with red fluorescence. PKH26-labelled cells were distributed in the cardiac tissues of the D-gal + DPSCs rats (Figs. 5B, 5F, 5J, and 5N). Interestingly, a few of the transplanted cells differentiated into cardiomyocytes, as indicated by the colocalization of cardiac troponin T (cTnT) (Figs. 5A–5H). or cardiac troponin I (cTnI) with PKH26-labelled cells (Figs. 5I–5P).

DPSCs upregulate Sirt1 expression and exert an antioxidative effect in D-galactose-induced cardiac ageing in rats

We further investigated whether DPSC transplantation has antioxidant effects. Compared with the control rats, we observed that aged rats exhibited higher concentrations of MDA (Fig. 6A), whereas the SOD (Fig. 6B) and GSH (Fig. 6C) levels were dramatically reduced in aged hearts compared with those in control rats. In D-gal + DPSC-treated rats, MDA concentrations in the cardiac tissue were significantly reduced (Fig. 6A), whereas SOD (Fig. 6B) and GSH (Fig. 6C). levels were higher than those in the aged rats (Figs. 6A–6C). Sirt1 demonstrated its ability to delay ageing and induce cardiac antioxidative effects (Alcendor et al., 2007; Cencioni et al., 2015; Lu et al., 2014; Ministrini et al., 2021). Interestingly, DPSC-treated rats exhibited higher Sirt1 expression than D-gal-treated rats (Fig. 7A).

DPSCs exhibit anti-apoptotic effects in D-galactose-induced cardiac ageing in rats

Cardiac ageing is linked to a marked reduction in Bcl2, an anti-apoptotic marker, and a significant elevation in Bax and cytochrome c, thereby triggering apoptosis (Pollack et al., 2002) Western blot analysis demonstrated that the mitochondria triggered an increase in the pro-apoptotic markers Bax (Fig. 7B), cytochrome c (Fig. 7D), and cleaved caspase-3 (Fig. 7E), whereas the anti-apoptotic marker Bcl-2 (Fig. 7C) was reduced in D-gal-treated rats. However, in D-gal + DPSC-treated rats, the expression of all upregulated apoptotic markers decreased (Figs. 7B, 7D, and 7E), while the expression of Bcl-2 was enhanced (Fig. 7C).

Effects of DPSC administration on senescence-associated markers in D-galactose-induced ageing rats

Senescence-linked β-galactosidase (SA-β-gal) activity is commonly used to recognize cells as senescent. A group of cell cycle regulatory factors, including P21, and P53, are also considered as senescence markers (Shimizu & Minamino, 2019). The expression of senescence-related markers, such as SA- β-gal (Fig. 8A), p53 (Fig. 8B) and p21 (Fig. 8C), was significantly upregulated in aged rats compared to those in control rats. In D-gal + DPSC-treated rats, DPSCs efficiently reduced the expression of all evaluated ageing markers (Figs. 8A–8C).