Alleviation of Myocardial Infarction by Hydrogen Sulfide-Releasing Nanoparticles: Mechanisms and Therapeutic Effects

doi:10.21203/rs.3.rs-4987842/v1

Download PDF

Research Article

Alleviation of Myocardial Infarction by Hydrogen Sulfide-Releasing Nanoparticles: Mechanisms and Therapeutic Effects

https://doi.org/10.21203/rs.3.rs-4987842/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Myocardial infarction (MI), a common and severe disease threatening human health worldwide, results from ischemic and hypoxic-induced necrosis of cardiac tissue due to coronary artery obstruction or rupture. Hydrogen sulfide (H₂S) is a gasotransmitter involved in various physiological and pathological processes. Exogenous supplementation of H₂S is significantly beneficial for the treatment of MI. In this study, a novel H₂S donor - zinc sulfide nanoparticles encapsulated in hyaluronic acid (HA@ZnS NPs), has been developed through a biomimetic mineralization process for the treatment of MI. HA@ZnS NPs can stably release H₂S at the site of myocardial ischemic injury due to the acidic microenvironment. Compared to the MI group, the NP-treated group significantly improved cardiac function, including increased left ventricular ejection fraction and fractional shortening, as well as reduced end-systolic volume. Furthermore, the NPs significantly reduced the size of the myocardial infarction area, improved left ventricular remodeling, and exerted therapeutic effects by promoting angiogenesis and reducing apoptosis in cardiac tissue. In conclusion, HA@ZnS NPs demonstrate potential for treating MI through precise control of H₂S release, providing valuable insights into new therapies for MI and laying the groundwork for the clinical application of H₂S-releasing materials in the future.

myocardial infarction

hydrogen sulfide

nanoparticles

myocardial ischemic injury

Myocardial infarction (MI) occurs due to coronary artery obstruction or rupture, leading to ischemia and hypoxia-induced necrosis in myocardial tissues. It is one of the clinical syndromes of coronary atherosclerotic heart disease (coronary heart disease)(1–4), and has become one of the common severe diseases globally threatening human’s health(2, 5). Coronary heart disease is prevalent in 6.3% of the adults in the United States, and 1 in every 7 deaths is primarily due to coronary heart disease. Moreover, MI is prevalent among 7.9 million adults, with a new case occurring approximately every 40 seconds(4, 6). Given that the risk of premature cardiovascular diseases in young people is increasing(7), the prevention and treatment of MI have become in a huge need.

Recently, emerging fields such as tissue engineering and biomaterials have begun to offer potential therapeutic approaches to address the dilemma(7, 8). For instance, integrating biomaterials with clinical treatment methods could slow the progression of ventricular remodeling and rebuild cardiac function post-infarction(8–10). More recently, gasotransmitters have garnered widespread attention as an emerging therapeutic modality because of their excellent therapeutic efficacy and good biocompatibility. The three recognized gasotransmitters are nitric oxide (NO)(11), carbon monoxide (CO)(12), and hydrogen sulfide (H₂S)(13). These gases are implicated in a wide range of physiological and pathological processes, such as cardiovascular diseases, oxidative stress, and inflammation. Therefore, developing targeted delivery systems capable of releasing therapeutic doses of gasotransmitters represents an ideal tool in modern medicine(13, 14). It is proven that exogenous H₂S plays a vital role in the treatment of MI. Studies have shown that animals treated with H₂S exhibit reduced myocardial infarction area, improved left ventricular fractional shortening in the risk zone, and enhanced microvascular reactivity. Moreover, both short-term and long-term administration of H₂S donors can lower blood pressure(15, 16). Besides, the expression levels of inflammatory factors such as IL-6, IL-8, and TNF-α in myocardial tissue decline following H₂S treatment(17). These experimental results collectively suggest that supplementing H₂S is highly meaningful for treating MI. It should be noted that the H₂S dose-response curve is bell-shaped, with its therapeutic effects limited to a narrow range(18). Therefore, precise dose control is a key issue in the development of H₂S-based therapies for MI treatment.

In the past few years, various types of H₂S donors responsive to different stimuli including light, biological thiols, pH changes, enzymatic activity, and others have been developed(19–22). Despite huge advances have been achieved, most suffered from low water solubility, limited means to modulate release kinetics, and no capacity for targeted delivery, all of which may limit H₂S-based treatments due to the reactive nature of this signaling molecule(23).

To address the aforementioned critical issue, we prepared an H₂S donor-zinc sulfide nanoparticles (NPs) from hyaluronic acid by biomineralization. It is assumed that the H₂S could be steadily released from NPs at the lesion site of myocardial ischemic injury, which prevents the injury from further deterioration. The protective effects of NPs on myocardial ischemic injury were carefully examined on rats (Fig. 1A). And the underlying mechanisms were deciphered both in vitro and in vivo.

2.1. Materials

Sodium hyaluronate (HA, mW ≈ 3 KDa) was purchased from Bloomage Biotech (Beijing, China). Sodium sulfide nonahydrate (Na₂S·9H₂O), zinc acetate (Zn(CH₃COO)₂), 30 wt % hydrogen peroxide solution (H₂O₂), and paraformaldehyde solution (4 wt% PFA) were purchased from Titan Biotechnology (Shanghai, China). HA@ZnS nanoparticles (NPs) were prepared according to the published procedure with a slight modification(21). Ferric chloride hexahydrate (FeCl₃ • 6H₂O) and 4-amino-N, N-dimethylaniline sulfate (DMPD) were purchased from Aladdin Biochem Technology (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM) medium, trypsin, penicillin, and fetal bovine serum (FBS) were purchased from HyClone (Waltham, USA). Dimethyl sulfoxide (DMSO), Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) kit, and 5% normal goat serum were purchased from Thermo Fisher Scientific (Massachusetts, USA), Dulbecco’s phosphate-buffered saline (DPBS) and streptomycin were purchased from Solarbio (Beijing, China). Bicinchoninic acid (BCA) protein assay kit was purchased from Abcam (Cambridge, UK). CCK-8 reagent was purchased from Solarbio (Beijing China). Antibodies: CD86, IL-6, α-smooth muscle actin (α-SMA), Bcl-2, BAX, and Caspase-3 were purchased from Abcam (Cambridge, UK). Secondary antibodies: Alexa Fluor 488 and 594 were purchased from Molecular Probes (California, USA).

2.2. Animals and cells

The procedures of animal experiments in this study were approved by the Institutional Animal Care and Use Committee of the Westchina Hospital, Sichuan University (Chengdu, China) (20230228042). Adult male Sprague-Dawley rats (10 weeks old, 200–250 g) were purchased from the Chengdu Dashuo Biotechnology Co., Ltd and housed in standard cages under standard conditions. All animals were acclimatized for one week before use. Rat cardiomyocytes cells (H9C2) and human umbilical vein endothelial cells (HUVECs) were purchased from American Type Culture Collection (ATCC, Manassas, USA), and they were cultured in DMEM with high glucose supplemented with 10% (v/v) FBS, 100 U/mL of penicillin, and 100 mg/mL of streptomycin at 37°C in a humidified incubator with 95% air and 5% CO₂.

2.3. In vitro cell studies

2.3.1. In vitro fluorescence imaging of H₂S release

H9C2 cells (2 x 10³/ well in 12-well plates) were incubated with 1.5 mL of glucose-free DMEM containing Na2S or HA@ZnS NPs at 37°C. Subsequently, the pH values were adjusted to 5.5. The incubation lasted for 1 h, and the mixture of WSP-5 (50 µM) and CTAB (200 µM) was added after removing the culture medium. The green fluorescence was then observed using the fluorescence microscope.

2.3.2. Cytocompatibility test

To evaluate the cytotoxicity of HA@ZnS NPs, H9C2 cells (8 x 10³/ well in 96-well plates) were incubated with 200 µL of glucose-free DMEM containing different concentrations of HA@ZnS NPs at 37°C for 24 h, then the culture medium was removed and replaced with serum-free DMEM containing 10 wt% of CCK-8 solution. After incubation for 1 h, the absorbance at 450 nm was collected using the Multi-Detection Reader.

2.3.3. ROS assay test

ROS production in H9C2 cells was analyzed using dihydroethidium (DHE). H9C2 cells were incubated with 10 µM of DHE at 37°C for 30 minutes and then washed twice with PBS. DHE staining was then observed using a fluorescence microscope to detect ROS production. Fluorescence was collected at 535 nm excitation and 610 nm emission using a Multi-Detection Reader.

2.3.4. HUVEC tube formation assay

Tube formation assay was performed using tanswell chambers (65 mm PET membrane, 0.4 µm pore size) and 24-well plates. The matrix gel was spread evenly in the 24-well plate and incubated at 37°C for 30 min. After co-culture for 7 days, 1.5x10⁵ HUVECs/well (3 replicates per group) were inoculated into the 24-well plate and cultured using the complete medium. The control group was incubated at 37°C for 12 h, and the NPs group was incubated with 10 µg/mL of HA@ZnS NPs at 37°C for 12 h. The number of lumen formations was counted in the top, bottom, left, right, and center of view, and the average value was taken. The control data were used as one to calculate the relative formation rate of the number of tubules.

2.4. In vivo studies

2.4.1. Establishment of MI model on rats

Adult male Sprague-Dawley rats (SD rats, 10 weeks old, 200–250 g) were used for the studies. Rats were anesthetized with isoflurane, intubated for respiratory support, and maintained under anesthesia using a gas anesthetic machine. The heart was exposed and the left anterior descending coronary artery was ligated to create left ventricular infarction. Then, after ligation for 30 min, NPs or saline (10 injections for 200 µL in total) was injected at the periphery and center of the infarct site, followed by suturing of the surgical wound on the rats' chest. Rats were randomly divided into 5 groups: (1) Sham group; (2) MI group (underwent LAD ligation and saline injection); (3) ZnCl₂ group (underwent LAD ligation and injected with 200 µg/kg of ZnCl₂); (4) Na₂S group (underwent LAD ligation and injected with 200 µg/kg of Na₂S); (5) NPs group (underwent LAD ligation and injected with 200 µg/kg of NPs).

2.4.2. In vivo fluorescence imaging of H2S release

To assess the imaging of NPs in the hearts of MI rats, SD rats were divided into three groups: (1) MI group, untreated MI rats; (2) Sham + NPs group (200 µg/kg), healthy rats injected with NPs; (3) MI + NPs group, MI rats injected with NPs (200 µg/kg). 30 min after NPs administration, rats in Sham + NPs group and MI + NPs group were locally injected with 100 µL of 25 µM WSP-5 (dissolved in DMSO) and 10 µL of 100 µM CTAB (dissolved in ddH₂O) to give the total volume to 200 µL. 30 min after WSP-5 administration, imaging was performed using Spectral Instruments Imaging (Kino, U.S.A) in fluorescence mode (excitation: 500 nm; emission: 525 nm).

To study the relationship between NPs dosage and fluorescence intensity, MI rats received a single local injection of different NPs doses (25, 50, 100, 150, 200 µg/kg) 30 min post-ligation. Control rats were injected with saline. 1 h after injection, rats were euthanized. Hearts were harvested, and ex vivo imaging was conducted using Spectral Instruments Imaging in fluorescence mode.

To study the metabolism and biodistribution of NPS in MI rats, rats received a single local injection of NPS (200 µg/kg) 30 min post-ligation. At each predetermined time point (0.5, 1, 6, 8, 12, and 24 h post-injection), rats were euthanized, and major organs such as the heart, liver, spleen, and kidney were collected for ex vivo imaging.

2.4.3. Echocardiographic assessment of cardiac function

At 7, 14, 21, and 28 days post-surgery, echocardiography was used to evaluate cardiac function in five groups of rats. First, the SD rats were weighed, and chloral hydrate was administered intraperitoneally as an anesthetic at 0.024 g/100 g. Once fully anesthetized and immobilized, the fur at rats' neck and chest was removed using a specialized depilatory cream. The rats were secured on the test table, and ultrasound coupling agent was uniformly applied to the chest for imaging. Echocardiography was conducted using the Vevo 3100 Imaging System (Visual Sonics, Canada). Several parameters including left ventricular ejection fraction (EF), fractional shortening (FS), and end-systolic volume (ESV) were measured and the average values of three separated cardiac cycles were used to represent the cardiac function.

2.4.4. Histology examination

Triphenyltetrazolium chloride (TTC) staining was used to assess infarct size 7 days post-myocardial infarction. The heart was sectioned into four horizontal slices (2 mm thick) and incubated in 1% TTC to differentiate between the infarcted and viable myocardium. The infarct sizes were quantified using Image J.

28 days post-surgery, the SD rats were weighed and anesthetized with an intraperitoneal injection of chloral hydrate at a dosage of 0.024 g/100 g. Once the rats were fully immobilized, their abdomens were incised with a surgical scalpel, and blood was withdrawn from the abdominal aorta using a 10 mL syringe. The excised hearts were thoroughly rinsed with saline and then fixed in a 4% paraformaldehyde solution. The hearts were dehydrated in a graded series of 75% ethanol, embedded in paraffin, and serially sectioned (5 µm thick) from apex to base, perpendicular to the longitudinal axis of the heart, ensuring a complete representative cross-section. Slides were stained with Hematoxylin and Eosin (H&E) and Picrosirius Red, then observed under a light microscope for collagen deposition in the myocardial infarction area and morphological changes in surrounding tissues. With Picrosirius Red staining, collagen appears red and normal myocardial tissue appears yellow. Finally, the slides were quantitatively analyzed to measure the infract size, the left ventricular wall thickness and the left ventricular scar size.

For the immunohistochemical analysis, slides were pretreated with 3% hydrogen peroxide in PBS for 10 min at room temperature to suppress the endogenous peroxidase activity. Subsequently, the slides were incubated in a blocking solution composed of 5% normal goat serum in DPBS for 1 h and then treated with the primary antibody at 4°C. The antibodies employed included CD86, IL-6, α-SMA. The slides were then treated with Alexa Fluor 488 or 594 secondary antibodies. After washing, antifade mounting medium with DAPI (H-1200-10, Vector Labs Inc., Malvern, PA, USA) was applied to the slides. Fluorescent cells were visualized using an Eclipse Ti2 microscope (Nikon, Japan).

For cell apoptosis assays, heart paraffin sections were stained with the TUNEL kit. DAPI was used for nuclear staining. Images were taken by an Olympus Research Slide Scanner VS200.

2.4.5. Western Blot

Protein concentrations were measured with the Bicinchoninic Acid (BCA) Protein Assay Kit. Equal volumes of protein samples were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% non-fat milk for 1 h at room temperature, and then incubated overnight at 4 ^◦C with antibodies specific for the CD86, IL-6, α-SMA, Bcl-2, BAX, and Caspase-3. On the following day, the membranes were washed and incubated with horseradish peroxidase (HRP)-coupled secondary antibodies. The blots were detected by enhanced chemiluminescence and quantified using the Bio-Rad imaging system.

2.4.6. Flow cytological analysis

On one day post-MI, the hearts were perfused with pre-cooled PBS. The injured left ventricular tissues were taken and separated by GentleMACS dissociator. The tissue was digested for 30 min at room temperature at 200 rpm in 5 mL of HBSS buffer containing collagenase II, collagenase IV, and DNase I. The resulting suspension was filtered (70 µm) to produce a single cell suspension. The suspension was centrifuged at 300 ×g for 5 min. Cardiomyocytes were then suspended in DMEM culture medium (with the addition of 10% fetal bovine serum and 1% penicillin-streptomycin) and cultured at 37°C in 5% CO₂ for 2 h. Cells were then washed with PBS and cardiac macrophages were concentrated in adherent cells. Cells were collected and incubated with PerCP-Cy5.5-CD86 and PE-Cy7-CD206 with flow cytometry antibodies for 15 min in the dark at 4°C. After washing with PBS, cardiac macrophage phenotypes were detected by BD FACSCanto II flow cytometry.

2.5. Statistical analysis

Data are presented as mean ± standard deviation (SD) (n ≥ 3). Significance was determined by the student’s t-test or one-way analysis of variance (ANOVA) using GraphPad Prism (version 9). The statistical significance was considered when the P value was less than 0.05.

3.1. Synthesis and characterization of HA@ZnS NPs

HA@ZnS nanoparticles (NPs) were prepared by the one-step biomineralization. In brief, zinc acetate was dropwise added to the premixed solution of HA and Na2S in anaerobic condition. With stirring, insoluble ZnS gradually formed at the aid of HA, affording HA@ZnS NPs. In infarcted cardiac tissue, the expression of CD44 increased. Given that HA can bind specifically to CD44, it is assumed that NPs could accumulate in infarcted cardiac tissues(24, 25). Thus, in this system HA not only acted as the template for ZnS growth and stabilize HA@ZnS nanoparticles in the solution, but also rendered HA@ZnS NPs targeting to CD44 overexpressed tissues. The average size of HA@ZnS NPs measured by dynamic light scattering (DLS) was around 200 nm (Fig. 1B). TEM imaging showed that these NPs were spherical with the diameter comparable to that of DLS (Fig. 1C). Then the H2S release behaviors were investigated in acetate buffer at pH 7.4 and 5.5, respectively. As shown in Fig. 1D, traceable H2S could be detected at pH 7.4, whereas large amount of H2S was liberated in the course of 1 h at acidic pH 5.5, implying that HA@ZnS NPs were pH responsive.

Figure 1. A schematic diagram of HA@ZnS NPs. (A) Synthesis of HA@ZnS NPs and their mechanism of treating myocardial infarction. (B) Representative transmission electron microscope (TEM) image of HA@ZnS NPs. (C) Hydrodynamic size distribution of HA@ZnS NPs in deionized water. (D) H₂S release curves of HA@ZnS NPs in 25 mM acetate buffer (pH 7.4 and 5.5).

3.2. Biocompatibility of HA@ZnS NPs

Nanomaterials intended for biomedical applications require low toxicity or nontoxicity. Cell viability assay (CCK8) was employed to study the toxicity of HA@ZnS NPs against rat cardiomyocytes cells (H9C2) and human umbilical vein endothelial cells (HUVECs). Three concentration gradients of the nanomaterials were selected: 1 µg/mL, 5 µg/mL, and 10 µg/mL. As illustrated in Fig. 2A, no significant cytotoxicity against H9C2 cells or HUVECs was observed within the concentration range of 1–10 µg/mL within 48 hours (p > 0.05). Furthermore, the live/dead staining results on 48 h (Fig. 2B) revealed that most cells (up to 99%) in all groups were stained green and had similar cell morphology, further confirming that HA@ZnS NPs had outstanding cytocompatibility. The migration of endothelial cells plays a crucial role in tissue repair and regeneration(26, 27). Thus, the effects of HA@ZnS NPs on the migration of HUVECs were also explored in the scratch wound model. As revealed in Fig. 2C, cell migration in the NPs group was not significantly affected compared to the control group, demonstrating that HA@ZnS NPs do not impair the migration function of endothelial cells, which is the core of angiogenesis. All results together indicated that the concentrations of HA@ZnS NPs used in subsequent studies were safe for H9C2 cells and HUVECs.

3.3. Fluorescence imaging of H₂S release in vitro and in vivo

We conducted further tests on the fluorescence properties of NPs to explore their potential applications in biomedical imaging. WSP-5 is a fluorescent probe containing active disulfides, specifically used for detecting H₂S in biological samples(28). First, we verified the capacity of NPs and Na₂S to release H₂S intracellularly in H9C2 cells (Fig. 3A). And a single dose of 200 µg/kg HA@ZnS NPs was injected into the hearts of MI rats, followed by the injection of WSP-5 to the same site after 30 min to monitor fluorescence changes (Fig. 3B). It was found that the green fluorescence representing H₂S in the heart gradually intensified and peaked at 1-hour postinjection. Then, the fluorescence slowly diminished in the next 23 h (Fig. 3C-D). Although the intensity become weak at 24-hour postinjection, it was still observable, indicating that NPs could retain at the injection site and ensure steady H₂S release for a relatively long time (Fig. 3C-D). In addition, five different doses of NPs were injecting in MI rats, followed by ex vivo imaging, confirming that the fluorescence intensity detected in the heart was highly correlated with the applied dosage (Fig. 3E-F). Notably, since NPs were locally injected into the myocardium, no fluorescence was observed in other organs after 1 h (Fig. 3G). This not only demonstrated NPs can accumulate in the heart, but also indicated the metabolism and clearance of NPs within the cardiac tissue. Collectively, these results showcased considerable advantages of NPs in in vivo imaging.

3.4. Therapeutic effects of NPs on repairing cardiac function in MI rats

MI refers to the ischemic necrosis of the myocardium, one of severe coronary artery diseases. As the blood flow in the coronary artery is drastically reduced or interrupted, the corresponding myocardium would suffer from severe and persistent acute ischemia, which ultimately leads to the ischemic necrosis of the myocardium(29). When myocardial ischemia occurs, cardiomyocytes undergo necrosis and apoptosis if there is no timely and effective treatment. With the extension of ischemia time, the area of myocardial infarction would continue to expand, ultimately resulting in cardiac insufficiency(29, 30). Therefore, improving cardiac function is one of the important means for the treatment of MI(31). On day 7, 14, 21, and 28 post-NP injection, the cardiac function of the rats was evaluated using echocardiography, focusing on three indices: left ventricular ejection fraction (EF), fractional shortening (FS), and end-systolic volume (ESV). Figure 4A and B displayed the echocardiograms and the corresponding quantitative ultrasonic data for each group of myocardial infarction rats. Both the echocardiograms and statistical data reveal that compared with the sham group, the EF and FS values significantly decreased while the ESV values remarkably increased after myocardial infarction. This is primarily due to the extensive death of myocardial cells post-infarction, which induced abnormal cardiac function, thinning of the ventricular walls, and ventricular dilation. These changes in indices indicated the successful construction of the myocardial infarction model in SD rats. The statistical data further implied that over time the cardiac function of the infarct group exhibited a deteriorating trend, i.e., the values of EF and FS continued to decrease, while those of ESV continued to increase. Due to the absence of therapeutic interventions, the condition of the rats in the MI group progressively worsened, ultimately resulting in heart failure. The indices for both Na₂S group and ZnCl₂ group were almost identical to those of the MI group, i.e., no significant differences were observed. In sharp contrast, the NPs group showed improvements for all indices compared with the MI group, with a surge in EF, suggesting that NPs injection into the infarcted area can ameliorate the function of the damaged myocardium.

3.5. Therapeutic effects of NPs in improving left ventricular remodeling in MI rats

MI in rats was induced by ligation of the left anterior descending coronary artery, leading to gradual thinning of the left ventricular wall as the disease progressed, and ultimately replacement of the necrotic tissue with fibrous tissue(32). We assessed the infract size, left ventricular (LV) wall thickness, and LV scar size in MI rats on day 28 post-infarction using TTC staining, HE staining, and Sirius red staining. Compared with the MI group, NPs treatment significantly reduced the size of the myocardial infarction area (Fig. 4C and D). After NPs treatment, fibrosis in the MI area (red) was remarkably reduced and NPs treatment improved the LV wall thickness and LV scar size (Fig. 4E and F). Notably, rats in the NPs group exhibited the lowest degree of fibrosis and the highest degree of normal myocardial recovery in the infarcted area. In contrast, neither the Na₂S nor ZnCl₂ group showed benefits in reducing infarct size or promoting myocardial recovery. Taken together, these research findings confirm that intramyocardial injection of NPs can effectively lessen the infarct size in myocardial infarction rats and inhibit left ventricular remodeling.

3.6. The mechanism of NPs' therapeutic effects on myocardial ischemic injury

IL-6, an important inflammatory factor, is a small molecular protein secreted by macrophages, and involved in the pathological damage of some autoimmune diseases(33, 34). Following MI, a severe inflammatory response would be induced due to the massive apoptosis of myocardial cells, significantly elevating the expression levels of inflammatory factors. Previous studies have proved that H₂S can effectively suppress the inflammatory response in the myocardial infarction area(35). After treated with H₂S, the expression levels of inflammatory factors such as IL-6, IL-8, and TNF-α are significantly declined in animal models with myocardial infarction(17). The primary source of these inflammatory factors is macrophages activated after injury. CD86 is a specific antigen for macrophages, which could be used as the clue for their presence(36, 37). In this study, we found that the expression of both IL-6 and CD86 cells in the NPs group was much lower than in the MI group (Fig. 5A). This could be attributed to the anti-inflammatory effect of H₂S, effectively suppressing the inflammatory response in the myocardial infarction area.

α-SMA is a characteristic protein of angiogenesis(38). It was observed that the expression of α-SMA was significantly higher in NPs group than in the MI group (Fig. 5A), indicating angiogenesis was enhanced in the damaged cardiac tissue upon the intervention of NPs. This effect was also confirmed in the human umbilical vein endothelial cell tubulation assay, where NPs dramatically increased the number of endothelial cell tubules (Fig. 5B). The anti-inflammatory and angiogenesis-promoting effects of NPs were similarly verified in Western blot, i.e., the expression of the inflammatory indicators IL-6 and CD86 decreased in the NPs group, and the expression of the angiogenesis indicator α-SMA increased in the NPs group.

During MI, the hypoxia of cardiac cells in the ischemic area leads to a reduction of ATP production, consequently causing apoptosis of cardiac cells that highly rely on mitochondrial respiration for ATP generation(39). Compared with MI group, as expected, less TUNEL + apoptotic cells were detected in the group treated by NPs (Fig. 5C), suggesting that NPs can protect ischemic cardiac cells through anti-apoptotic mechanisms.

In addition, previous studies have shown that reactive oxygen species (ROS) play a key role in the progression of atherosclerosis and ischaemic heart disease. And excessive ROS leads to sustained oxidative stress and endothelial dysfunction, which in turn leads to disease progression(40, 41). In the H9C2 hypoxic cell model, we examined the levels of reactive oxygen species using a ROS fluorescence detection probe and showed that NPs were able to significantly weaken ROS levels (Fig. 5D).

The anti-apoptotic effect of NPs was also confirmed in Western blot on rat myocardial tissues (Fig. 6). The Bcl-2 family of proteins are key regulators of apoptosis, including both pro-apoptotic and pro-survival (anti-apoptotic) members, where Bcl-2 exerts an anti-apoptotic effect, while BAX, a member of the Bcl-2 family, plays a pro-apoptotic role(42). In addition, Caspase s are a specific group of enzymes involved in the process of apoptosis, of which the classical Caspase-3 recognizes and disassembles apoptosis-specific sequences and promotes apoptosis(43). Therefore, the expression of Bcl-2, BAX, and Caspase-3 in myocardial tissues of MI rats was detected by Western blot, and it was found that the expression of Bcl-2 was significantly increased in the NPs group, while the expression of BAX and Caspase-3 was significantly decreased, which further validated the anti-apoptotic effect of NPs.

Besides, macrophages in diseased tissues were classified into M1-type (classically activated macrophages) and M2-type (selectively activated macrophages). CD86 is a common marker molecule for M1-type macrophages, which are induced by IFN-γ to produce pro-inflammatory factors. CD206 is a common marker molecule for M2-type macrophages, which are activated by IL-4 to release anti-inflammatory factors(36). By flow cytometric analysis, we examined macrophage polarization in myocardial tissue and found that NPs reduced the proportion of CD86⁺ M1-type macrophages and increased the proportion of CD206⁺ M2-type macrophages (Fig. 7). This further suggests that NPs can improve the function of damaged myocardium through anti-inflammatory effects.

In this study we reported a novel H₂S donor, HA@ZnS NPs, for the treatment of myocardial infarction. Releasing study demonstrated that H₂S release could be triggered by the acidic pH. NPs not only significantly reduced the infarct size but also improved left ventricular remodeling and decreased the levels of inflammatory factors. By promoting angiogenesis in myocardial tissues and reducing cardiomyocyte apoptosis, NPs demonstrated potential in improving cardiac function. These findings provide valuable insights into new therapeutic strategies for myocardial infarction and lay the groundwork for future clinical applications of H₂S-releasing materials.

Ethics approval and consent to participate

The procedures of animal experiments in this study were approved by the Institutional Animal Care and Use Committee of the Westchina Hospital, Sichuan University (Chengdu, China) (20230228042).

Consent or publication

Not applicable.

Availability of data and materials

Not applicable.

Competing interests

The authors declare no competing financial interest.

Funding

This project was supported by National Natural Science Foundation of China (Grant No. 52273090, 22375128, 22105126), the Natural Science Foundation of Sichuan Province (Grant No. 2022NSFSC1395, 2022NSFSC1489), Post-Doctor Research Project, West China Hospital, Sichuan University (Grant No. 20HXBH171, 2021HXBH070), Postdoctoral Science Foundation of China (Grant No. 2021M692282), and the Natural Science Foundation of Shanghai (22ZR1433500).

Authors' contributions

Y.Z.: Methodology; Validation; Visualization; Writing – original draft; Writing – review & editing. X.Z.: Conceptualization; Investigation; Methodology; Software; Validation; Writing – original draft; Writing – review. S.B.: Supervision; Validation; Writing – review. R.L.: Supervision; Validation; Writing – review. Y.G.: Supervision; Validation; Writing – review. J.G.: Funding acquisition; Project administration; Supervision; Validation; Writing – review & editing. Y.W.: Funding acquisition; Project administration; Supervision; Validation; Writing – review & editing.

Acknowledgements

Not applicable.

Authors' information

Y.Z.^a, [email protected]; X.Z.^a, [email protected]; S.B.^b, [email protected]; R.L.^b, [email protected]; Y.G.^c, [email protected]; J.G.^a, [email protected]; Y.W.^c, [email protected]

^a Department of Cardiovascular Surgery, West China Hospital, Sichuan University, Chengdu, 610000, China

^b Department of Burn and Plastic Surgery, West China Hospital, Sichuan University, Chengdu, 610000, China

^cEngineering Research Center of Cell & Therapeutic Antibody, Shanghai Frontiers Science Center of Drug Target Identification and Delivery, National Key Laboratory of Innovative Immunotherapy, School of Pharmaceutical Sciences, Shanghai Jiao Tong University, Shanghai 200240, China

Zheng Z, Tan Y, Li Y, Liu Y, Yi GH, Yu CY, et al. Biotherapeutic-loaded injectable hydrogels as a synergistic strategy to support myocardial repair after myocardial infarction. Journal of Controlled Release. 2021;335:216-36. DOI: 10.1016/j.jconrel.2021.05.023
Reed GW, Rossi JE, Cannon CP. Acute myocardial infarction. Lancet. 2017;389(10065):197-210. DOI: 10.1016/s0140-6736(16)30677-8
Dauerman HL, Ibanez B. The Edge of Time in Acute Myocardial Infarction. Journal of the American College of Cardiology. 2021;77(15):1871-4. DOI: 10.1016/j.jacc.2021.03.003
Lee DJ, Cavasin MA, Rocker AJ, Soranno DE, Meng XZ, Shandas R, et al. An injectable sulfonated reversible thermal gel for therapeutic angiogenesis to protect cardiac function after a myocardial infarction. Journal of Biological Engineering. 2019;13:6. DOI: 10.1186/s13036-019-0142-y
Martin U, Haverich A. Engineering cardiac muscle: new ways to refurbish old hearts? European Journal of Cardio-Thoracic Surgery. 2014;45(2):216-9. DOI: 10.1093/ejcts/ezt490
Mozaffarian D, Benjamin EJ, Go AS, Arnett DK, Blaha MJ, Cushman M, et al. Heart Disease and Stroke Statistics-2016 Update A Report From the American Heart Association. Circulation. 2016;133(4):E38-E360. DOI: 10.1161/cir.0000000000000350
de Ferranti SD, Steinberger J, Ameduri R, Baker A, Gooding H, Kelly AS, et al. Cardiovascular Risk Reduction in High-Risk Pediatric Patients A Scientific Statement From the American Heart Association. Circulation. 2019;139(13):E603-E34. DOI: 10.1161/cir.0000000000000618
Zimmermann WH, Melnychenko I, Wasmeier G, Didié M, Naito H, Nixdorff U, et al. Engineered heart tissue grafts improve systolic and diastolic function in infarcted rat hearts. Nature Medicine. 2006;12(4):452-8. DOI: 10.1038/nm1394
Zhu Y, Matsumura Y, Wagner WR. Ventricular wall biomaterial injection therapy after myocardial infarction: Advances in material design, mechanistic insight and early clinical experiences. Biomaterials. 2017;129:37-53. DOI: 10.1016/j.biomaterials.2017.02.032
Wang XT, Wang LY, Wu Q, Bao F, Yang HT, Qiu XZ, et al. Chitosan/Calcium Silicate Cardiac Patch Stimulates Cardiomyocyte Activity and Myocardial Performance after Infarction by Synergistic Effect of Bioactive Ions and Aligned Nanostructure. Acs Applied Materials & Interfaces. 2019;11(1):1449-68. DOI: 10.1021/acsami.8b17754
Yao XP, Liu Y, Gao J, Yang L, Mao D, Stefanitsch C, et al. Nitric oxide releasing hydrogel enhances the therapeutic efficacy of mesenchymal stem cells for myocardial infarction. Biomaterials. 2015;60:130-40. DOI: 10.1016/j.biomaterials.2015.04.046
Kim I, Han EH, Bang WY, Ryu J, Min JY, Nam HC, et al. Supramolecular Carbon Monoxide-Releasing Peptide Hydrogel Patch. Advanced Functional Materials. 2018;28(47):1803051. DOI: 10.1002/adfm.201803051
Zheng YQ, Yu BC, De La Cruz LK, Choudhury MR, Anifowose A, Wang BH. Toward Hydrogen Sulfide Based Therapeutics: Critical Drug Delivery and Developability Issues. Medicinal Research Reviews. 2018;38(1):57-100. DOI: 10.1002/med.21433
Gemici B, Elsheikh W, Feitosa KB, Costa SKP, Muscara MN, Wallace JL. H2S-releasing drugs: Anti-inflammatory, cytoprotective and chemopreventative potential. Nitric Oxide-Biology and Chemistry. 2015;46:25-31. DOI: 10.1016/j.niox.2014.11.010
Calvert JW, Jha S, Gundewar S, Elrod JW, Ramachandran A, Pattillo CB, et al. Hydrogen Sulfide Mediates Cardioprotection Through Nrf2 Signaling. Circulation Research. 2009;105(4):365-U105. DOI: 10.1161/circresaha.109.199919
Elrod JW, Calvert JW, Morrison J, Doeller JE, Kraus DW, Tao L, et al. Hydrogen sulfide attenuates myocardial ischemia-reperfusion injury by preservation of mitochondrial function. Proceedings of the National Academy of Sciences of the United States of America. 2007;104(39):15560-5. DOI: 10.1073/pnas.0705891104
Sodha NR, Clements RT, Feng J, Liu YH, Bianchi C, Horvath EM, et al. Hydrogen sulfide therapy attenuates the inflammatory response in a porcine model of myocardial ischemia/reperfusion injury. Journal of Thoracic and Cardiovascular Surgery. 2009;138(4):977-84. DOI: 10.1016/j.jtcvs.2008.08.074
Rong F, Wang TJ, Zhou Q, Peng HW, Yang JT, Fan QL, et al. Intelligent polymeric hydrogen sulfide delivery systems for therapeutic applications. Bioactive Materials. 2023;19:198-216. DOI: 10.1016/j.bioactmat.2022.03.043
Powell CR, Dillon KM, Matson JB. A review of hydrogen sulfide (H2S) donors: Chemistry and potential therapeutic applications. Biochemical Pharmacology. 2018;149:110-23. DOI: 10.1016/j.bcp.2017.11.014
Zhu YM, Archer WR, Morales KF, Schulz MD, Wang Y, Matson JB. Enzyme-Triggered Chemodynamic Therapy via a Peptide-H2S Donor Conjugate with Complexed Fe2+. Angewandte Chemie-International Edition. 2023;62(22):e202302303. DOI: 10.1002/anie.202302303
Wang ZX, Ge YX, Liu JQ, Shi PYF, Xue RY, Hao B, et al. Integrating a Biomineralized Nanocluster for H2S-Sensitized ROS Bomb against Breast Cancer. Nano Letters. 2024;24(8):2661-70. DOI: 10.1021/acs.nanolett.4c00347
Ge YX, Rong F, Li W, Wang Y. On-demand therapeutic delivery of hydrogen sulfide aided by biomolecules. Journal of Controlled Release. 2022;352:586-99. DOI: 10.1016/j.jconrel.2022.10.055
Szabo C, Papapetropoulos A. International Union of Basic and Clinical Pharmacology. CII: Pharmacological Modulation of H2S Levels: H2S Donors and H2S Biosynthesis Inhibitors. Pharmacological Reviews. 2017;69(4):497-564. DOI: 10.1124/pr.117.014050
Zhang Q, Chen L, Huang LY, Cheng HX, Wang L, Xu L, et al. CD44 promotes angiogenesis in myocardial infarction through regulating plasma exosome uptake and further enhancing FGFR2 signaling transduction. Molecular Medicine. 2022;28(1):145. DOI: 10.1186/s10020-022-00575-5
Teriete P, Banerji S, Noble M, Blundell CD, Wright AJ, Pickford AR, et al. Structure of the regulatory hyaluronan binding domain in the inflammatory leukocyte homing receptor CD44. Molecular Cell. 2004;13(4):483-96. DOI: 10.1016/s1097-2765(04)00080-2
Hasan A, Khattab A, Islam MA, Abou Hweij K, Zeitouny J, Waters R, et al. Injectable Hydrogels for Cardiac Tissue Repair after Myocardial Infarction. Advanced Science. 2015;2(11):1500122. DOI: 10.1002/advs.201500122
Li RT, Liu K, Huang X, Li D, Ding JX, Liu B, et al. Bioactive Materials Promote Wound Healing through Modulation of Cell Behaviors. Advanced Science. 2022;9(10):e2105152. DOI: 10.1002/advs.202105152
Zhou YZ, Mazur F, Liang K, Chandrawati R. Sensitivity and Selectivity Analysis of Fluorescent Probes for Hydrogen Sulfide Detection. Chemistry-an Asian Journal. 2022;17(5):e202101399. DOI: 10.1002/asia.202101399
Frantz S, Hundertmark MJ, Schulz-Menger J, Bengel FM, Bauersachs J. Left ventricular remodelling post-myocardial infarction: pathophysiology, imaging, and novel therapies. European Heart Journal. 2022;43(27):2549-+. DOI: 10.1093/eurheartj/ehac223
Zhang Q, Wang L, Wang SQ, Cheng HX, Xu L, Pei GQ, et al. Signaling pathways and targeted therapy for myocardial infarction. Signal Transduction and Targeted Therapy. 2022;7(1):78. DOI: 10.1038/s41392-022-00925-z
Shi HT, Huang ZH, Xu TZ, Sun AJ, Ge JB. New diagnostic and therapeutic strategies for myocardial infarction via nanomaterials. Ebiomedicine. 2022;78:103968. DOI: 10.1016/j.ebiom.2022.103968
Hundahl LA, Tfelt-Hansen J, Jespersen T. Rat Models of Ventricular Fibrillation Following Acute Myocardial Infarction. Journal of Cardiovascular Pharmacology and Therapeutics. 2017;22(6):514-28. DOI: 10.1177/1074248417702894
Broch K, Anstensrud AK, Woxholt S, Sharma K, Tollefsen IM, Bendz B, et al. Randomized Trial of Interleukin-6 Receptor Inhibition in Patients With Acute ST-Segment Elevation Myocardial Infarction. Journal of the American College of Cardiology. 2021;77(15):1845-55. DOI: 10.1016/j.jacc.2021.02.049
Sun JY, Du LJ, Shi XR, Zhang YY, Liu Y, Wang YL, et al. An IL-6/STAT3/MR/FGF21 axis mediates heart-liver cross-talk after myocardial infarction. Science Advances. 2023;9(14):eade4110. DOI: 10.1126/sciadv.ade4110
Wu T, Li H, Wu B, Zhang L, Wu SW, Wang JN, et al. Hydrogen Sulfide Reduces Recruitment of CD11b<SUP>+</SUP>Gr-1<SUP>+</SUP> Cells in Mice With Myocardial Infarction. Cell Transplantation. 2017;26(5):753-64. DOI: 10.3727/096368917x695029
Sica A, Mantovani A. Macrophage plasticity and polarization: in vivo veritas. Journal of Clinical Investigation. 2012;122(3):787-95. DOI: 10.1172/jci59643
Ryabov V, Gombozhapova A, Rogovskaya Y, Kzhyshkowska J, Rebenkova M, Karpov R. Cardiac CD68+and stabilin-1+macrophages in wound healing following myocardial infarction: From experiment to clinic. Immunobiology. 2018;223(4-5):413-21. DOI: 10.1016/j.imbio.2017.11.006
Venugopal H, Hanna A, Humeres C, Frangogiannis NG. Properties and Functions of Fibroblasts and Myofibroblasts in Myocardial Infarction. Cells. 2022;11(9):1386. DOI: 10.3390/cells11091386
Wang XW, Guo ZK, Ding ZF, Mehta JL. Inflammation, Autophagy, and Apoptosis After Myocardial Infarction. Journal of the American Heart Association. 2018;7(9):e008024. DOI: 10.1161/jaha.117.008024
Zhang Z, Dalan R, Hu ZY, Wang JW, Chew NW, Poh KK, et al. Reactive Oxygen Species Scavenging Nanomedicine for the Treatment of Ischemic Heart Disease. Advanced Materials. 2022;34(35):e2202169. DOI: 10.1002/adma.202202169
Zhang YX, Murugesan P, Huang K, Cai H. NADPH oxidases and oxidase crosstalk in cardiovascular diseases: novel therapeutic targets. Nature Reviews Cardiology. 2020;17(3):170-94. DOI: 10.1038/s41569-019-0260-8
Singh R, Letai A, Sarosiek K. Regulation of apoptosis in health and disease: the balancing act of BCL-2 family proteins. Nature Reviews Molecular Cell Biology. 2019;20(3):175-93. DOI: 10.1038/s41580-018-0089-8
Riedl SJ, Shi YG. Molecular mechanisms of caspase regulation during apoptosis. Nature Reviews Molecular Cell Biology. 2004;5(11):897-907. DOI: 10.1038/nrm1496

No competing interests reported.

Download PDF

Reviews received at journal
05 Nov, 2024
Reviews received at journal
02 Nov, 2024
Reviewers agreed at journal
01 Nov, 2024
Reviewers agreed at journal
29 Oct, 2024
Reviewers agreed at journal
29 Oct, 2024
Reviewers agreed at journal
02 Oct, 2024
Reviewers invited by journal
02 Oct, 2024
Editor assigned by journal
04 Sep, 2024
Submission checks completed at journal
04 Sep, 2024
First submitted to journal
27 Aug, 2024

You are reading this latest preprint version

Alleviation of Myocardial Infarction by Hydrogen Sulfide-Releasing Nanoparticles: Mechanisms and Therapeutic Effects

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials and methods

2.1. Materials

2.2. Animals and cells

2.3. In vitro cell studies

2.3.1. In vitro fluorescence imaging of H₂S release

2.3.2. Cytocompatibility test

2.3.3. ROS assay test

2.3.4. HUVEC tube formation assay

2.4. In vivo studies

2.4.1. Establishment of MI model on rats

2.4.2. In vivo fluorescence imaging of H2S release

2.4.3. Echocardiographic assessment of cardiac function

2.4.4. Histology examination

2.4.5. Western Blot

2.4.6. Flow cytological analysis

2.5. Statistical analysis

3. Results and discussion

3.1. Synthesis and characterization of HA@ZnS NPs

3.2. Biocompatibility of HA@ZnS NPs

3.3. Fluorescence imaging of H₂S release in vitro and in vivo

3.4. Therapeutic effects of NPs on repairing cardiac function in MI rats

3.5. Therapeutic effects of NPs in improving left ventricular remodeling in MI rats

3.6. The mechanism of NPs' therapeutic effects on myocardial ischemic injury

4. Conclusion

Declarations

References

Additional Declarations

Status:

Version 1

Alleviation of Myocardial Infarction by Hydrogen Sulfide-Releasing Nanoparticles: Mechanisms and Therapeutic Effects

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials and methods

2.1. Materials

2.2. Animals and cells

2.3. In vitro cell studies

2.3.1. In vitro fluorescence imaging of H2S release

2.3.2. Cytocompatibility test

2.3.3. ROS assay test

2.3.4. HUVEC tube formation assay

2.4. In vivo studies

2.4.1. Establishment of MI model on rats

2.4.2. In vivo fluorescence imaging of H2S release

2.4.3. Echocardiographic assessment of cardiac function

2.4.4. Histology examination

2.4.5. Western Blot

2.4.6. Flow cytological analysis

2.5. Statistical analysis

3. Results and discussion

3.1. Synthesis and characterization of HA@ZnS NPs

3.2. Biocompatibility of HA@ZnS NPs

3.3. Fluorescence imaging of H2S release in vitro and in vivo

3.4. Therapeutic effects of NPs on repairing cardiac function in MI rats

3.5. Therapeutic effects of NPs in improving left ventricular remodeling in MI rats

3.6. The mechanism of NPs' therapeutic effects on myocardial ischemic injury

4. Conclusion

Declarations

References

Additional Declarations

Status:

Version 1

2.3.1. In vitro fluorescence imaging of H₂S release

3.3. Fluorescence imaging of H₂S release in vitro and in vivo