Abstract
BACKGROUND:
Pathological cardiac hypertrophy is one of the main activators of heart failure. Currently, no drug can completely reverse or inhibit the development of pathological cardiac hypertrophy. To this end, we proposed a silicate ion therapy based on extract derived from calcium silicate (CS) bioceramics for the treatment of angiotensin II (Ang II) induced cardiac hypertrophy.
METHODS:
In this study, the Ang II induced cardiac hypertrophy mouse model was established, and the silicate ion extract was injected to mice intravenously. The cardiac function was evaluated by using a high-resolution Vevo 3100 small animal ultrasound imaging system. Wheat germ Agglutinin, Fluo4-AM staining and immunofluorescent staining was conducted to assess the cardiac hypertrophy, intracellular calcium and angiogenesis of heart tissue, respectively.
RESULTS:
The in vitro results showed that silicate ions could inhibit the cell size of cardiomyocytes, reduce cardiac hypertrophic gene expression, including atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and β-myosin heavy chain (β-MHC), decrease the content of intracellular calcium induced by Ang II. In vivo experiments in mice confirmed that intravenous injection of silicate ions could remarkably inhibit the cardiac hypertrophy and promote the formation of capillaries, further alleviating Ang II-induced cardiac function disorder.
CONCLUSION:
This study demonstrated that the released silicate ions from CS possessed potential value as a novel therapeutic strategy of pathological cardiac hypertrophy, which provided a new insight for clinical trials.
Supplementary Information
The online version contains supplementary material available at 10.1007/s13770-023-00523-2.
Keywords: Cardiac hypertrophy, Silicate ions, Capillaries formation, Cardiac function, Intracellular calcium
Introduction
Pathological cardiac hypertrophy is a maladaptive response of the heart to external stimuli such as hypertension, pressure overload, and myocardial injury [1]. It is characterized by the enlargement of individual cardiomyocytes and the thickening of the ventricular wall, accompanied by multiple pathological changes including cardiac contractile dysfunction, intracellular mitochondrial dysfunction, glucose metabolism disorder, and reduction in the survival rate of cardiomyocytes and so on [2–6]. Moreover, long-term cardiomyocyte enlargement is highly associated with severe cardiovascular diseases such as heart failure, which affects approximately 64.3 million people worldwide and leads to a 5-year mortality rate of 56% [7].
Several treatment strategies including drugs, surgery, interventional therapy, immunotherapy, and gene therapy have been developed for the cardiac hypertrophy, among which drug therapy is the most widely used. For example, β-blockers by targeting the β-adrenergic receptor (β-AR) has been used to treat cardiac hypertrophy, and angiotensin converting inhibitor enzyme has been proven to inhibit cardiac hypertrophy of blood pressure [8–10]. However, those drugs may cause unwanted side effects, and none of them can completely inhibit or reverse the occurrence and development of cardiac hypertrophy in clinical practice [10]. Therefore, it is of great significance to develop new therapy for the treatment of cardiac hypertrophy.
Recently, “ions therapy” based on bioceramics materials has a great potential for tissue regeneration due to its good stability, bioactivity and biocompatibility [11, 12]. Silicate ions derived from silicate bioceramics can inhibit the cardiac hypertrophy after myocardial infarction. In addition, it also showed strong effect on the viability of cardiomyocytes as well as regulate the interactions between cardiomyocytes and endothelial cells, upregulate the expression of connexin43 protein (gap junction protein), regulate the intracellular calcium homeostasis [13], further promote the expression of angiogenesis-related factors (such as vascular endothelial growth factor (VEGF) [14, 15]. Since pathological cardiac hypertrophy is highly correlated with cardiomyocyte apoptosis [16], intracellular calcium homeostasis [17] and sparse capillary density [18], it is reasonable to speculate that silicate ions may have a good therapeutic effect on pathological cardiac hypertrophy. Although the inhibitory effect of silicate ions on cardiac hypertrophy was observed in MI model, how silicate ions regulate cardiac hypertrophy and its mechanism were not completely understood, which promoted us to conduct a more deeply study.
Thereby, in this study, silicate ion extract derived from calcium silicate (CS) bioceramic was obtained prior to use, and then the function of silicate ions was explored on angiotensin II (Ang II)-induced cardiac hypertrophy both in vitro and in vivo. Specifically, the effects of silicate ion extract on cell size of cardiomyocytes and the mRNA expression of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and β-myosin heavy chain (β-MHC) were evaluated in Ang II-injured H9C2 cardiomyocytes. Meantime, the therapeutic effects of silicate ion extract on cardiac hypertrophy were investigated by analyzing cardiac function, cell size of cardiomyocytes, and capillaries formation in the heart of Ang II-induced hypertrophic mouse model.
Material and methods
Preparation of CS extract
The extracted silicate ions were prepared by our previous study as followed [13]. Briefly, CS powders were purchased from Kunshan Chinese Technology New Materials Co., Ltd (Jiangsu, China) and added into serum-free Dulbecco’s modified Eagle’s medium (DMEM) or saline with the weight-to-volume ratio of 1:5 g / mL. The mixed suspension was shaken at a speed of 120 rpm/min at 37 °C for 24 h and centrifuged at a speed of 4000 rpm/min for 5 min, then sterilized using a 0.22 μm filter (Millipore, Burlington, MA, USA) to obtain CS extract. One part in DMEM was diluted with DMEM at a volume ratio of 1/16 for further cell experiments, while the other part in physiological saline solution was obtained for animal experiments.
Effects of CS extract on Ang II-induced cArdiomyocyte hypertrophy in vitro
Cell Culture
The rat cardiomyocyte cell line of H9C2 was purchased from Procell Life Science & Technology Co., Ltd (Wuhan, China) and cultured in high glucose DMEM with 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin at 37 °C in 5% CO2 incubator.
Immunofluorescent staining of cTnI
H9C2 was seeded in 24-well plates with a density of 2 × 103 cells per well for 12 h, then the media were replaced with Ang II (1 μM) containing media for 48 h in the absence/presence of CS extract. The H9C2 cells were fixed with 4% paraformaldehyde (PFA) for 10 min, washed with PBS three times, blocked with goat serum containing 1% Tween 20 for 1 h at room temperature, and then incubated with primary antibody Cardiac Troponin I (cTnI) (15513-1-AP, Proteintech, Rosemont, IL, USA) at 4 °C overnight. The secondary antibody of Cy3-conjugated Affinipure goat anti-rabbit IgG (H + L) (SA00009-2, Proteintech) was added and incubated in the dark at room temperature for another 1 h. Cell nuclear was stained with ready-to-use 4′6-diamidino-2-phenylindole (DAPI, Solarbio, C0065, Beijing, China) for 10 min. The images of H9C2 cells were detected by a Nikon A1 confocal microscopy (Nikon, Tokyo, Japan), and analyzed by the NIS-Elements Viewer software (version 5.21.00). The surface area of cells was calculated using the Image J software (National Institutes of Health, Bethesda, MD, USA).
Quantitative real-time PCR (qRT-PCR) analysis
H9C2 was seeded in 24-well plates with a density of 3 × 104 cells per well for 12 h, then the media were replaced with Ang II (1 μM) containing media for 48 h in the absence/presence of CS extract. Total RNA was extracted from cells by the RNA extraction kit (M3211070, YEASEN), and then RNA concentration was detected with an ultra-micro spectrophotometer (DeNovix, DS-11, Wilmington, DE, USA). Next, mRNA was converted to single-stranded cDNA using Hifair®III 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) (H6201080, YEASEN). The quantitative real-time PCR (qRT-PCR) was conducted following the commercial kit of Hieff® qPCR SYBR® Green Master Mix (No Rox) (H7101190, YEASEN). The relative mRNA expression levels were analyzed by the 2−ΔΔCt method. GAPDH was used as the endogenous control. The cardiac hypertrophic-related genes (ANP, BNP, and β-MHC), and angiogenesis-related genes (VEGF, bFGF, and KDR) in rats or mouse were tested. The gene primers were listed in Table 1.
Table 1.
Primer sequence in this study
| Gene | Sense primer | Antisense primer | |
|---|---|---|---|
| GAPDH | R | 5′-AGTGCCAGCCTCGTCTCATA-3′ | 5′-ACCAGCTTCCCATTCTCAGC-3′ |
| ANP | R | 5′-AGTGCGGTGTCCAACACAGA-3′ | 5′-TCATCTTCTACCGGCATCTTCTC-3′ |
| BNP | R | 5′-GCTTTGGGCAGAAGATAGA-3′ | 5′-CAAGTTTGTGCTGGAAGATAA-3′ |
| β-MHC | R | 5′-CCTCGCAATATCAAGGGAAA-3′ | 5′-TACAGGTGCATCAGCTCCAG-3′ |
| GAPDH | M | 5′-CTTCACCACCATGGAGAAGGC-3′ | 5′-GGCATGGACTGTGGTCATGAG-3′ |
| ANP | M | 5′-ATGGGCTCCTTCTCCATCAC-3′ | 5′-TTATCTTCGGTACCGGAAGCTG-3′ |
| BNP | M | 5′-ATGGATCTCCTGAAGGTGCTGTC-3′ | 5′-CTACAACAACTTCAGTGCGTTAC-3′ |
| β-MHC | M | 5′-ATGTGCCGGACCTTGGAAG-3′ | 5′CCTCGGGTTAGCTGAGAGAGATCA-3′ |
| VEGF | M | 5′-GAGGTCAAGGCTTTTGAAGGC-3′ | 5′-CTGTCCTGGTATTGAGGGTGG-3′ |
| bFGF | M | 5′-GGAGAAGAGCGACCCTCACATCAAG-3′ | 5′-CCAGTTCGTTTCAGTGCCACATACCAA-3′ |
| KDR | M | 5′-ACTGCAGTGATTGCCATGTTCT-3′ | 5′-CAAACAGAAACCCGTGGAGATG-3′ |
ANP, atriopeptin natriuretic peptide; BNP, brain natriuretic peptide (BNP); β-MHC, β-myosin heavy chain; VEGF, vascular endothelial growth factor; bFGF, basic fibroblast growth factor; KDR, vascular endothelial growth factor receptor; R, rat; M, mouse
Intracellular calcium measurements
H9C2 on glass coverslips were cultured and treated with Ang II (1 µM) containing media for 48 h in the absence/presence of CS extract. The cells were loaded with 4 µM Fluo-4AM (Solarbio, F8500) in HBSS (Servicebio, G4203, Wuhan, China) at 37 °C for 20 min followed by 40 min incubation with five fold volume of HBSS (Servicebio, G4210) containing 1% FBS. After that, the cells were washed with HEPES buffer for 3 times. The fluorescence signals were recorded by fluorescence microscopy (Nikon). The mean density rate of each image was calculated using the Image J software (National Institutes of Health).
Effects of CS extract on Ang II-induced cardiac hypertrophy in vivo
Animal and treatment
The animal experiment protocol of this study was approved by the Animal Research and Ethics Committee of Wenzhou Institute of University of Chinese Academy of Sciences (Approval Issue No. WIUCAS22021301). The cardiac hypertrophy mice model was performed according to the previously described protocol [19]. Briefly, 8-week-old male C57/BL6J mice were purchased from the Zhejiang Experimental Animal Center. The mice were fed randomly with free drinking water in a constant temperature (25 ± 2 °C) environment with 12 h daylight and 12 h dark cycles. First, the mice were stabilized in their new environment for 7 days, and then subcutaneously implanted with the Alzet Osmotic Pumps with Ang II. Briefly, Ang II was placed in sustained-release Alzet Osmotic Pumps (Model 2004, DURECT Corporation, Cupertino, CA, USA) at a rate of 1000 ng/kg/min and equilibrated in saline for 42 h at 37 °C. Then, 100 µL silicate ions containing saline or saline control were intravenously injected 7 times every other day. The experimental groups are defined as Ang II + Saline and Ang II + CS. The control group injected with an equal amount of saline without Ang II was designed as Saline control.
Echocardiographic measurements
Mice were anesthetized with isoflurane, and the chest hair was removed. Cardiac function was recorded by M-mode using a high-resolution Vevo 3100 small animal ultrasound imaging system (Visua Isonics, Toronto, Canada) equipped with a 30-MHz transducer. Echocardiographic parameters include left ventricular ejection fraction (LVEF), the long axis fractional shortening (LVFS), the diastolic left ventricular anterior wall (LVAW; d), the systolic left ventricular anterior wall (LVAW; s), the diastolic left ventricular posterior wall (LVPW; d) and the systolic left ventricle posterior wall (LVPW; s) were measured using the Vevo 3100 software. Three consecutive cardiac cycles were measured to calculate those parameters.
Blood Pressure (BP) measurements
The mouse was put into a pouch with its head facing inward, rump wrapped with a cloth, and tail exposed. The pouch was placed into a constant temperature cylinder, and then, the tail of the mouse was inserted into the sensor. Meanwhile, the mouse’s blood pressure was measured when a steady pulse wave appeared. A non-invasive blood pressure monitor (BP-2010A, Softron, Beijing, China) was used to measure the heart rate and blood pressure (BP) including the systolic BP, diastolic BP, and mean BP after 7, 14, and 28 days treatment.
Histological analysis and qRT-PCR
The heart tissues of mice were isolated after 28 days, fixed with 4% PFA overnight, embedded in paraffin, cut into 5 μm serial sections, and stained with hematoxylin and eosin (H&E) for observation of the structure and morphology. Wheat germ Agglutinin (WGA) analysis of cardiomyocytes was performed according to the previous protocol [20]. Briefly, 10 μg/mL WGA (S25064, Shanghai source leaf, Shanghai, China) staining solution was configured and dropwised to the heart tissue sections in the dark and incubated for 30 min at 37 °C. Cell nuclear was stained with ready-to-use DAPI for 10 min. For histo-immunofluorescence analysis, the obtained sections were rehydrated and heated for antigen retrieval in 0.01 M sodium citrate buffer before being blocked with 3% hydrogen peroxide to inhibit the activity of the endogenous enzymes. Then, the sections were blocked with 5% BSA for 1 h at room temperature, followed by incubation with primary antibody Anti-CD31 (GB11063-2, Servicebio) at 4 °C overnight. After that, the secondary antibody Cy3-conjugated Affinipure Goat Anti-Rabbit IgG (H + L) (SA00009-2, Proteintech) was added and incubated in the dark at room temperature for 1 h. Cell nuclei were stained with DAPI for 10 min. The images of sections were detected by fluorescence microscopy (Axio Vert.A1, Carl Zeiss, Oberkochen, Germany). The cell surface area and capillaries number in heart tissue were measured and analyzed by Image J software (National Institutes of Health).
qRT-PCR analysis of the obtained tissues was conducted following the same procedure in Section 2.2. The cardiac hypertrophic-related genes (ANP, BNP, and β-MHC), and angiogenesis-related genes (VEGF, bFGF, and KDR) in mice were examined. The gene primers were listed in Table 1.
Statistical analysis
All data were analyzed using GraphPad Prism7 (Beijing, China) based on the method of one-way variance (ANOVA) with Tukey’s multiple comparison test. The results were expressed as the mean ± SEM, and each test was repeated in at least three independent experiments. A value of p < 0.05 was considered to have a significant difference.
Results
Silicate ions Inhibit Ang II-induced cardiomyocyte hypertrophy in vitro
To investigate the influence of CS extract on cardiomyocyte hypertrophy, we constructed an Ang II-induced cardiomyocyte (H9C2 cell) hypertrophy in vitro model. We first analyzed the cell surface morphology with different concentration of CS treatment in Ang II induced cell model using immunofluorescence staining of anti-cardiac Troponin T I (cTnI). As shown in the Fig. S1, different concentration of CS (from 1/4 to 1/32) could alleviate cardiac hypertrophy, indicating a dose-dependent effect. Among them, the 1/16CS is the optimal concentration, as compared to 1/4 and 1/32. Therefore, we then selected 1/16 for subsequent cell experiments. In details, as shown in Fig. 1A, our results demonstrated that the surface area of H9C2 cells was significantly increased (2.67-fold) after Ang II induction, and the cell surface area in the Ang II + CS group was significantly reduced (2.22-fold) compared to the Ang II group, which was even recovered to the basal level in the Ctrl group. Similarly, the qPCR results further confirmed that CS extract could significantly inhibit the mRNA expression levels of cardiac hypertrophy-related genes in H9C2 as compared with the Ang II group, including ANP (1.42-fold), BNP (2.02-fold), and β-MHC (2.16-fold) (Fig. 1B). To explore the potential mechanism by which CS inhibits cardiac hypertrophy, we examined the changes of intracellular calcium with or without Ang II treatment using Fluo4-AM staining. It is noting that after the Ang II treatment, the intracellular calcium was significantly increased, while this increase was reversed after CS treatment (Fig. 1C). Thus, our result indicated that CS could inhibit Ang II-induced cardiomyocyte hypertrophy in vitro, partially through regulating intracellular calcium.
Fig. 1.
Silicate ions inhibit hypertrophy of cardiomyocytes (H9C2) induced by Ang II in vitro. A Representative and quantification results of immunofluorescence stained with cTnI. n = 4. Scale bar: 20 μm. B qRT-PCR examined the mRNA expression levels of hypertrophy-related genes including atriopeptin natriuretic peptide (ANP), brain natriuretic peptide (BNP), and β-myosin heavy chain (β-MHC) in cardiomyocytes (H9C2). n = 3. C Representative image of intracellular calcium and their quantitative analysis of mean density in cultured H9C2 treated with Ctrl, Ang II or Ang II + CS. n = 4. *p < 0.05, **p < 0.01 or ***p < 0.001
Silicate ions alleviate Ang II-Induced cardiac dysfunction in vivo
Since the Ang II-induced cardiac hypertrophy model was accompanied by cardiac dysfunction, the effect of silicate ions on Ang II-induced cardiac dysfunction was investigated. Silicate ions solution was intravenously injected in mice once 48 h, 7 times over 14 days after Ang II infusion (Fig. 2A). The echocardiographic evaluation showed that Ang II induction for 28 days led to a significant increase in cardiac functional parameters including the left ventricular ejection fraction (LVEF) and the fractional shortening (LVFS) compared with the control group, in which the value of LVEF increased from 60.78 ± 1.66% to 76.29 ± 4.93%, and the value of LVFS increased from 31.93 ± 1.16% to 47.09 ± 5.86% (Fig. 2B). While the treatment of silicate ions significantly decreased the value of LVEF and LVFS (LVEF: 63.32 ± 3.52%, and LVFS: 34.20 ± 2.63%), as compared to Ang II + Saline group. In addition, the systolic left ventricle posterior wall (LVPW; s) was significantly increased compared with the control group (from 1.04 mm to 1.60 mm) (Table 2), which was greatly attenuated after silicate ions treatment (1.17 mm). Furthermore, the heart rate and blood pressure (BP) including the systolic BP, diastolic BP, and mean BP of mice after 7, 14, and 28 days were shown in Table 3, Ang II provoked a remarkable increase in BP but not in heart rate, there was no significant differences in those parameters between Ang II + Saline and Ang II + CS groups, indicating that silicate ions did not affect the BP and heart rate. These results suggested that silicate ions could significantly alleviate Ang II-induced cardiac dysfunction.
Fig. 2.
Silicate ions alleviate cardiac dysfunction of mice induced by Ang II. A Schematic diagram of the experimental procedure. Briefly, mice were perfused with a sustained-release pump containing Ang II for 28 days, and silicate ion-containing saline or saline control were intravenously injected in mice 7 times every other day after Ang II or PBS infusion. B Representative ultrasound images of cardiac function in the M-model in different groups. Quantified C LVEF and D LVFS according to the ultrasound analysis. n = 8 or 9 or 10. *p 0.05
Table 2.
Effect of silicate ions on the cardiac function in Ang II induced hypertrophic mice
| Category | Saline | Ang II | Ang II + CS |
|---|---|---|---|
| LVAW; s (mm) | 1.21 ± 0.007 | 1.60 ± 0.15 | 1.53 ± 0.12 |
| LVAW; d (mm) | 0.80 ± 0.08 | 1.09 ± 0.12 | 1.00 ± 0.09 |
| LVPW; s (mm) | 1.04 ± 0.06 | 1.60 ± 0.13** | 1.17 ± 0.06# |
| LVPW; d (mm) | 0.73 ± 0.03 | 1.00 ± 0.09* | 0.92 ± 0.05 |
| Diameter; s (mm) | 2.75 ± 0.11 | 2.25 ± 0.26 | 2.61 ± 0.19 |
| Diameter; d (mm) | 4.03 ± 0.06 | 3.60 ± 0.23 | 3.83 ± 0.17 |
| Volume; s (μL) | 22.85 ± 2.80 | 13.71 ± 3.83 | 21.61 ± 4.03 |
| Volume; d (μL) | 57.64 ± 5.96 | 48.44 ± 6.37 | 54.93 ± 6.29 |
| Stroke Volume (μL) | 42.93 ± 2.07 | 37.15 ± 3.67 | 38.76 ± 2.25 |
| LV Mass (mg) | 110.66 ± 6.25 | 151.07 ± 14.03* | 147.14 ± 9.87 |
| LV Mass Cor (mg) | 88.53 ± 5.00 | 120.85 ± 11.23* | 117.71 ± 7.90 |
| Cardiac Output (mL/min) | 17.89 ± 1.16 | 15.50 ± 1.67 | 16.34 ± 0.82 |
LVAW; s, the systolic left ventricular anterior wall; LVAW; d, the diastolic left ventricular anterior wall; LVPW; s, the systolic left ventricle posterior wall; LVPW; d, the diastolic left ventricular posterior wall; Diameter; s, left ventricular systolic diameter; Diameter; d, left ventricular diastolic diameter; Volume; s, left ventricular volume contraction; Volume; d, left ventricular volume diastolic; LV Mass, left ventricular mass; LV Mass Cor, left ventricular mass correction. n = 5 ~ 10. *p < 0.05 or **p < 0.05 vs. Saline, #p < 0.05 vs. Ang II + Saline
Table 3.
Effect of silicate ions on the blood pressure in Ang II induced hypertrophic mice
| Category | Saline | Ang II | Ang II + CS |
|---|---|---|---|
| Mean BP (mmHg) | 86.82 ± 2.14 | 115.83 ± 2.96 | 108.33 ± 6.07 |
| SBP (mmHg) | 117.06 ± 2.63 | 159.30 ± 5.75*** | 141.90 ± 7.20 |
| DBP (mmHg) | 71.53 ± 2.67 | 94.04 ± 2.58** | 91.47 ± 5.66 |
| HR (bpm) | 653.25 ± 26.96 | 638.00 ± 18.38 | 607.10 ± 23.38 |
Mean BP, Mean blood pressure; SBP, systolic blood pressure; DBP, diastolic blood pressure; HR, heart rate. n = 8 or 9. **p < 0.01 or ***p < 0.001 vs. Saline
Silicate ions inhibit Ang II-induced cardiac hypertrophy in vivo
To further explore the effect of silicate ions on Ang II-induced cardiac hypertrophy in mice, hearts and body weight of mice in each group were weighed. The results showed that the ratio of the heart weight/body weight increased significantly in Ang II + Saline group (0.71%) as compared with the control group (0.43%), while the treatment of CS extract significantly reduced to 0.60% (Fig. 3A), indicating the therapeutic effect of silicate ions on cardiac hypertrophy. This conclusion was further confirmed by the HE staining (Fig. 3B) and WGA fluorescent staining (Fig. 3C), as the surface area of cardiomyocyte in the Ang II + CS group was significantly lower than that in Ang II + Saline group. In addition, the input of Ang II significantly upregulated the mRNA expression levels of hypertrophy-related genes, including ANP, BNP, and β-MHC in cardiac tissue, which were reduced after silicate ions treatment (Fig. 3D). The above results testified that the silicate ions may mitigate Ang II-induced cardiac hypertrophy in mice.
Fig. 3.
Silicate ions alleviate cardiomyocyte hypertrophy in mouse heart tissue induced by Ang II. A Representative images of the mouse heart and a statistical graph of the ratio of heart weight to body weight (HW/BW) in different groups. n = 5 or 6. Scale bar: 2 mm. B Representative images of HE staining of heart tissue. Scale bar: 500 μm. C Representative images of WGA staining of heart tissue and statistical graph of the cardiomyocytes cross-sectional area in mouse hearts. n = 5 or 6. scale bar: 20 μm. D Statistical plots of mRNA expression levels of ANP, BNP, and β-MHC in cardiac tissues by qRT-PCR. n = 3. *p < 0.05, **p < 0.01 or ***p < 0.001
Silicate ions promote capillary formation in Ang II-induced hypertrophic heart
Some studies have proved that cardiac hypertrophy can lead to an augmented demand for blood supply, which can’t be compensated by the original number of capillaries in hearts, resulting in capillary rarefaction [1]. Therefore, we further explore the effect of silicate ions on capillary formation in the hypertrophic heart. The CD31 immunofluorescence staining results showed that the number of capillaries in the heart tissue induced by Ang II in mice was significantly lower than that in the control group, which was significantly increased under the treatment of silicate ions, as compared to the Ang II group (Fig. 4A). Correspondingly, the mRNA expression levels of angiogenesis-related genes, including VEGF, bFGF, and KDR in Ang II group were significantly lower than that of the control group, and silicate ions treatment could significantly improve the expression of those angiogenesis-related genes, in which VEGF, bFGF, and KDR promoted for 1.37-fold, 2.21-fold and 1.55-fold, respectively (Fig. 4B). The above studies showed that silicate ions could promote capillary formation in Ang II-induced hypertrophic heart (Fig. 5).
Fig. 5.
The schematic diagram of the silicate ions for the treatment of Ang II-induced cardiac hypotrophy. Silicate ions can alleviate cardiac hypertrophy through the reduction of cardiac hypertrophic-related genes, including ANP, BNP, and β-MHC, as well as increase the capillary density by activation angiogenic-related genes including VEGF, bFGF, and KDR, thereby reverse the cardiac functions of LVEF and LVFS in Ang II-induced mice. Figures was created with BioRender.com
Fig. 4.
Silicate ions promote capillary formation in mouse hypertrophic heart induced by Ang II. A Representative CD31 (red) immunofluorescence staining and quantified capillary numbers in cardiac tissue. n = 3. Scale bar: 20 μm. B the mRNA expression levels of the angiogenesis-related genes including bFGF, KDR, and VEGF. n = 3. *p < 0.05, **p < 0.01 or ***p < 0.001
Discussions
Cardiac hypertrophy can be clarified into physiological and pathological, and both are involved with the enlargement of individual cardiomyocytes [1, 21]. Pathological cardiac hypertrophy is characterized by ventricular shrinkage, increased wall thickness, and diastolic dysfunction, which is commonly seen in patients with chronic hypertension, valvular disease, myocardial infarction, and metabolic syndrome [3]. Such pathological changes in the heart are usually triggered by neuroendocrine hormones such as Ang II, which is the main inducement of cardiac hypertrophy and is a commonly used targeting molecule [22].
Previous studies showed that mice treated with Ang II for four weeks had an elevation of LVEF and LVFS [19, 23]. While, on the contrary, some studies showed that the values of LVEF and LVFE were reduced after Ang II induction, which may be determined by different doses, or different induction time of Ang II. In the present study, we found that the value of LVEF and LVFS increased significantly after 4 weeks of Ang II (1000 ng/kg/min) induction, which indicates a compensatory stage. Moreover, both the value of LVEF and LVFS were recovered to normal level after the treatment of silicate ions. One possible reason to explain such beneficial effects is due to the direct inhibition effects of silicate ions on cardiomyocyte hypertrophy, which produced a significantly reduced hypertrophic response to Ang II stimulation, as confirmed by in vitro studies.
More interestingly, unlike conventional strategies to reduce cardiac afterload with ACE inhibitors or ARBs for the treatment of cardiac hypertrophy [24], we found that silicate ions could directly inhibit Ang II-induced cardiac hypertrophy in a manner independent of blood pressure regulation (Table 3), which may be due to its limited vasodilation effect on the aorta. This finding is consistent with the results of a previous study, which showed that the same dose of silicate ions did not affect arterial blood pressure [25].
In addition, we also demonstrated that CS extract could stimulate angiogenesis in Ang II-induced hypertrophic heart and ameliorate the insufficiency of blood supply caused by cardiac hypertrophy, which may attribute to the excellent performance for the promotion of cardiac functions. The powerful pro-angiogenic ability of CS was fully proved by abundant studies [26–29]. Also, the pro-angiogenic ability of CS made an important contribution to the treatment of Ang II-induced pathological myocardial hypertrophy since the balance between the size of cardiomyocyte and angiogenesis is important to reverse cardiac hypertrophy [30, 31].
The underlying mechanism of CS on inhibiting cardiomyocyte hypertrophy may be ascribed to regulative ability of CS on intracellular calcium signaling. As one of the most basic signals of cardiac hypertrophy, calcium signal disturbance in cardiomyocytes was long-term accompanied in pathological progress [32]. Specifically, intracellular calcium increase happened in Ang II induced cardiac hypertrophy as Ang II has been reported to induce the inward flow of calcium ions, which was also observed in our study (Fig. 1C). However, CS treatment significantly reduced the overexpression of calcium signal in cardiomyocytes to a basal level. Thus, it is reasonable to presume that CS could inhibit cardiac hypertrophy by regulating and maintaining intracellular calcium homeostasis of cardiomyocytes. Such speculation has also been proved in our previous study to a certain degree as silicate bioceramic containing materials were found to enhance the calcium signaling traverse between myocardial cells [15].
The contents and concentration of CS extract that we used in this study are known and have been fully detected in our previous study [13, 25, 33]. In detail, the concentration of CS and calcium in 1/16 CS (extracted with DMEM) were 6.66 ± 0.05 μg/mL and 56.19 ± 0.46 μg/mL, respectively. The concentration of silicate ion in CS (extracted with saline) were 77.6 ± 0.16 μg/mL. In addition to the components inherent in the extract medium, pure silicate ceramic powder does not introduce trace elements other than silicate and calcium, as shown in our previous study [13]. In addition, the bio-safety of CS with this injection method have been fully proved to be no toxic in our previous study, since the concentration of silicate ions had returned to pre-injection levels at day 28 in major organs [13, 25].
In conclusion, this study showed that silicate bioceramics extract could ameliorate Ang II-induced cardiomyocyte hypertrophy both in vitro and in vivo. Considering its inherent advantages such as good stability, bioactivity and biocompatibility, it is likely to be practiced as a new therapeutic therapy for the treatment of chronic cardiac remodeling diseases such as cardiac hypertrophy.
Supplementary Information
Below is the link to the electronic supplementary material.
Acknowledgements
This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16010203), the Chengdu Municipal Technological Innovation R & D Project (2021-YF05-01871-SN), the project of Chengdu Municipal Health Commission (2021059), the National Natural Science Foundation of China (82100427), the seed grants from the Wenzhou Institute, University of Chinese Academy of Sciences (WIUCASQD2020013, WIUCASQD2021030). We thank Scientific Research Center of Wenzhou Medical University for consultation and instrument availability that supported this work.
Declarations
Conflict of interest
The authors have no financial conflicts of interest.
Ethical statement
The animal experiment protocol of this study was approved by the Animal Research and Ethics Committee of Wenzhou Institute of University of Chinese Academy of Sciences (Approval Issue No. WIUCAS22021301).
Footnotes
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Contributor Information
Zhihong Dong, Email: zhdong@cdu.edu.cn.
Yumei Que, Email: yumeique@hotmail.com.
References
- 1.Shimizu I, Minamino T. Physiological and pathological cardiac hypertrophy. J Mol Cell Cardiol. 2016;97:245–62. [DOI] [PubMed]
- 2.Huang J, Wang L, Shen Y, Zhang S, Zhou Y, Du J, Liang D, Shi D, Ma H, Li L, Zhang Q, Chen YH. CDC-like kinase 4 deficiency contributes to pathological cardiac hypertrophy by modulating NEXN phosphorylation. Nat Commun. 2022;1:4433. doi: 10.1038/s41467-022-31996-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Nakamura M, Sadoshima J. Mechanisms of physiological and pathological cardiac hypertrophy. Nat Rev Cardiol. 2018;7:387–407. doi: 10.1038/s41569-018-0007-y. [DOI] [PubMed] [Google Scholar]
- 4.Hinkel R, Batkai S, Bahr A, Bozoglu T, Straub S, Borchert T, Viereck J, Howe A, Hornaschewitz N, Oberberger L, Jurisch V, Kozlik-Feldmann R, Freudenthal F, Ziegler T, Weber C, Sperandio M, Engelhardt S, Laugwitz KL, Moretti A, Klymiuk N, Thum T, Kupatt C. AntimiR-132 attenuates myocardial hypertrophy in an animal model of percutaneous aortic constriction. J Am Coll Cardiol. 2021;23:2923–2935. doi: 10.1016/j.jacc.2021.04.028. [DOI] [PubMed] [Google Scholar]
- 5.Ding J, Chen YX, Chen Y, Mou Y, Sun XT, Dai DP, Zhao CZ, Yang J, Hu SJ, Guo X. Overexpression of FNTB and the activation of Ras induce hypertrophy and promote apoptosis and autophagic cell death in cardiomyocytes. J Cell Mol Med. 2020;16:8998–9011. doi: 10.1111/jcmm.15533. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Shirakabe A, Zhai P, Ikeda Y, Saito T, Maejima Y, Hsu CP, Nomura M, Egashira K, Levine B, Sadoshima J. Drp1-dependent mitochondrial autophagy plays a protective role against pressure overload-induced mitochondrial dysfunction and heart failure. Circulation. 2016;13:1249–1263. doi: 10.1161/CIRCULATIONAHA.115.020502. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Groenewegen A, Rutten FH, Mosterd A, Hoes AW. Epidemiology of heart failure. Eur J Heart Fail. 2020;8:1342–1356. doi: 10.1002/ejhf.1858. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Marfella R, D'Onofrio N, Mansueto G, Grimaldi V, Trotta MC, Sardu C, Sasso FC, Scisciola L, Amarelli C, Esposito S, D'Amico M, Golino P, De Feo M, Signoriello G, Paolisso P, Gallinoro E, Vanderheyden M, Maiello C, Balestrieri ML, Barbato E, Napoli C, Paolisso G. Glycated ACE2 reduces anti-remodeling effects of renin-angiotensin system inhibition in human diabetic hearts. Cardiovasc Diabetol. 2022;1:146. doi: 10.1186/s12933-022-01573-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Zhang S, Wang Y, Yu M, Shang Y, Chang Y, Zhao H, Kang Y, Zhao L, Xu L, Zhao X, Difrancesco D, Baruscotti M, Wang Y. Discovery of herbacetin as a novel sgk1 inhibitor to alleviate myocardial hypertrophy. Adv Sci (Weinh) 2022;2:e2101485. doi: 10.1002/advs.202101485. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Bisping E, Wakula P, Poteser M, Heinzel FR. Targeting cardiac hypertrophy: toward a causal heart failure therapy. J Cardiovasc Pharmacol. 2014;4:293–305. doi: 10.1097/FJC.0000000000000126. [DOI] [PubMed] [Google Scholar]
- 11.Dawood AE, Parashos P, Wong RHK, Reynolds EC, Manton DJ. Calcium silicate-based cements: composition, properties, and clinical applications. J Investig Clin Dent. 2017. 10.1111/jicd.12195 [DOI] [PubMed]
- 12.Prati C, Gandolfi MG. Calcium silicate bioactive cements: Biological perspectives and clinical applications. Dent Mater. 2015;4:351–370. doi: 10.1016/j.dental.2015.01.004. [DOI] [PubMed] [Google Scholar]
- 13.Yi M, Li H, Wang X, Yan J, Gao L, He Y, Zhong X, Cai Y, Feng W, Wen Z, Wu C, Ou C, Chang J, Chen M. Ion therapy: a novel strategy for acute myocardial infarction. Adv Sci. 2019;1:1801260. doi: 10.1002/advs.201801260. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Gao L, Yi M, Xing M, Li H, Zhou Y, Xu Q, Zhang Z, Wen Z, Chang J. In situ activated mesenchymal stem cells (MSCs) by bioactive hydrogels for myocardial infarction treatment. J Mater Chem B. 2020;34:7713–7722. doi: 10.1039/D0TB01320J. [DOI] [PubMed] [Google Scholar]
- 15.Wang X, Wang L, Wu Q, Bao F, Yang H, Qiu X, Chang J. Chitosan/calcium silicate cardiac patch stimulates cardiomyocyte activity and myocardial performance after infarction by synergistic effect of bioactive ions and aligned nanostructure. ACS Appl Mater Interfaces. 2019;1:1449–1468. doi: 10.1021/acsami.8b17754. [DOI] [PubMed] [Google Scholar]
- 16.Kolwicz SC, Jr, Purohit S, Tian R. Cardiac metabolism and its interactions with contraction, growth, and survival of cardiomyocytes. Circ Res. 2013;5:603–616. doi: 10.1161/CIRCRESAHA.113.302095. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Wilkins BJ, Molkentin JD. Calcium-calcineurin signaling in the regulation of cardiac hypertrophy. Biochem Biophys Res Commun. 2004;4:1178–1191. doi: 10.1016/j.bbrc.2004.07.121. [DOI] [PubMed] [Google Scholar]
- 18.Patten IS, Rana S, Shahul S, Rowe GC, Jang C, Liu L, Hacker MR, Rhee JS, Mitchell J, Mahmood F, Hess P, Farrell C, Koulisis N, Khankin EV, Burke SD, Tudorache I, Bauersachs J, del Monte F, Hilfiker-Kleiner D, Karumanchi SA, Arany Z. Cardiac angiogenic imbalance leads to peripartum cardiomyopathy. Nature. 2012;7398:333–338. doi: 10.1038/nature11040. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Luo YX, Tang X, An XZ, Xie XM, Chen XF, Zhao X, Hao DL, Chen HZ, Liu DP. SIRT4 accelerates Ang II-induced pathological cardiac hypertrophy by inhibiting manganese superoxide dismutase activity. Eur Heart J. 2017;18:1389–1398. doi: 10.1093/eurheartj/ehw138. [DOI] [PubMed] [Google Scholar]
- 20.Zhou WW, Dai C, Liu WZ, Zhang C, Zhang Y, Yang GS, et al. Gentianella acuta improves TAC-induced cardiac remodelling by regulating the Notch and PI3K/Akt/FOXO1/3 pathways. Biomed Pharmacother. 2022;154:113564. [DOI] [PubMed]
- 21.Gibb AA, Hill BG. Metabolic coordination of physiological and pathological cardiac remodeling. Circ Res. 2018;1:107–128. doi: 10.1161/CIRCRESAHA.118.312017. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Shen Y, Wang X, Yuan R, Pan X, Yang X, Cai J, et al. Prostaglandin E1 attenuates AngII-induced cardiac hypertrophy via EP3 receptor activation and Netrin-1upregulation. J Mol Cell Cardiol. 2021;159:91–104. [DOI] [PubMed]
- 23.Frank D, Kuhn C, van Eickels M, Gehring D, Hanselmann C, Lippl S, Will R, Katus HA, Frey N. Calsarcin-1 protects against angiotensin-II induced cardiac hypertrophy. Circulation. 2007;22:2587–2596. doi: 10.1161/CIRCULATIONAHA.107.711317. [DOI] [PubMed] [Google Scholar]
- 24.Kim S, Yoshiyama M, Izumi Y, Kawano H, Kimoto M, Zhan Y. Effects of combination of ACE inhibitor and angiotensin receptor blocker on cardiac remodeling, cardiac function, and survival in rat heart failure. Circulation. 2001;1:148–154. doi: 10.1161/01.CIR.103.1.148. [DOI] [PubMed] [Google Scholar]
- 25.Que Y, Zhang Z, Zhang Y, Li X, Chen L, Chen P, et al. Silicate ions as soluble form of bioactive ceramics alleviate aortic aneurysm and dissection. Bioact Mater. 2022. 10.1016/j.bioactmat.2022.07.005 [DOI] [PMC free article] [PubMed]
- 26.Fan C, Xu Q, Hao R, Wang C, Que Y, Chen Y, et al. Multi-functional wound dressings based on silicate bioactive materials. Biomaterials. 2022. 10.1016/j.bioactmat.2022.07.005 [DOI] [PubMed]
- 27.Yang C, Ma H, Wang Z, Younis MR, Liu C, Wu C, Luo Y, Huang P. 3D printed wesselsite nanosheets functionalized scaffold facilitates NIR-II photothermal therapy and vascularized bone regeneration. Adv Sci (Weinh) 2021;20:e2100894. doi: 10.1002/advs.202100894. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Xu Q, Jiang F, Guo G, Wang E, Younis MR, Zhang Z, et al. Targeted hot ion therapy of infected wound by glycol chitosan and polydopamine grafted Cu-SiO2 nanoparticles. Nano Today. 2021;41:101330.
- 29.Dong C, Yang C, Younis MR, Zhang J, He G, Qiu X, Fu LH, Zhang DY, Wang H, Hong W, Lin J, Wu X, Huang P. Bioactive NIR-II Light-responsive shape memory composite based on cuprorivaite nanosheets for endometrial regeneration. Adv Sci (Weinh) 2022;12:e2102220. doi: 10.1002/advs.202102220. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Oka T, Akazawa H, Naito AT, Komuro I. Angiogenesis and cardiac hypertrophy: maintenance of cardiac function and causative roles in heart failure. Circ Res. 2014;3:565–571. doi: 10.1161/CIRCRESAHA.114.300507. [DOI] [PubMed] [Google Scholar]
- 31.Hamasaki S, Al Suwaidi J, Higano ST, Miyauchi K, Holmes DR, Lerman A. Attenuated coronary flow reserve and vascular remodeling in patients with hypertension and left ventricular hypertrophy. J Am Coll Cardiol. 2000;35:1654–60. [DOI] [PubMed]
- 32.Wang P, Xu S, Xu J, Xin Y, Lu Y, Zhang H, Zhou B, Xu H, Sheu SS, Tian R, Wang W. Elevated MCU expression by CaMKIIdeltaB limits pathological cardiac remodeling. Circulation. 2022;14:1067–1083. doi: 10.1161/CIRCULATIONAHA.121.055841. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Chen T, Zhang Z, Weng D, Lu L, Wang X, Xing M, Qiu H, Zhao M, Shen L, Zhou Y, Chang J, Li HP. Ion therapy of pulmonary fibrosis by inhalation of ionic solution derived from silicate bioceramics. Bioact Mater. 2021;10:3194–3206. doi: 10.1016/j.bioactmat.2021.02.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.