The New Nano-Resuscitation Solution (TPP-MR) Attenuated Myocardial Injury in Hemorrhagic Shock Rats by Inhibiting Ferroptosis

Introduction

Hemorrhagic shock, caused by acute blood loss, was characterized by a decrease in actual circulating blood volume, insufficient tissue perfusion, metabolic disorder, and organ dysfunction.¹ According to the World Health Organization, approximately 1.9 million people worldwide died from hemorrhagic shock in 2016 alone.² In the United States, hemorrhagic shock was one of the leading causes of death among people under 46 years old.³ The heart was the organ most commonly implicated in hemorrhagic shock, and once cardiac dysfunction happened, the prognosis further deteriorated.^4,5 Previous studies reported that the mechanisms of myocardial injury in hemorrhagic shock were mainly related to inflammation, autonomic dysfunction, and apoptosis.^6,7 However, the therapeutic efficacy targeted to these mechanisms was not ideal.

Fluid resuscitation was a key emergency treatment for hemorrhagic shock as it could quickly replenish the actual blood volume and improve systemic perfusion.⁸ Lactate Ringer’s solution (LR) was commonly used in fluid resuscitation. However, extensive use of LR might further increase the high lactate levels caused by tissue hypoxia. With acetate as the source of bicarbonate, Acetated Ringer’s solution (AR) could avoid the accumulation of lactic acid. Compared with acetate, malic acid had a longer half-life and thus could continuously produce HCO3⁻ to maintain the acid-base balance. In addition, as a key intermediate metabolite of the tricarboxylic acid cycle and a major source of mitochondrial ATP. Malic acid has been reported to alleviate myocardial ischemia-reperfusion injury, enhance myocardial contractility, reduce the expression of inflammatory factors, and inhibit apoptosis.^9,10 Nevertheless, rapid infusion of a large amount of fluid often increased the burden on the heart and led to abnormal coagulation, cell edema, and mitochondrial dysfunction, thus limiting the effectiveness of resuscitation.¹¹ Developing better resuscitation solution was a key strategy to improve hemorrhagic shock. TPP@PAMAM-MR(TPP-MR) was a novel type of nanocrystal resuscitation fluid, exhibiting a stronger targeted capability towards myocardial mitochondria. TPP-MR was synthesized by replacing the L-malic acid in MR with TPP@PAMAM@MA which was a polymer formed by wrapping L-malic acid with a triphenylphosphine material modified with polyamidoamine (PAMAM).¹² Our previous study indicated that TPP-MR significantly protected against myocardial injury in sepsis.¹³ However, it remained unclear whether TPP-MR offered protection against myocardial injury in hemorrhagic shock.

Ferroptosis was first coined by Scott J. Dixon et al¹⁴ in 2012. As a programmed iron-dependent cell death form, ferroptosis was found to participate in various pathological and physiological conditions, including septic shock, ischemia-reperfusion injury, and targeted therapy of malignant tumors.¹⁵ Caused by the redox imbalance, ferroptosis was usually accompanied by the accumulated reactive oxygen species (ROS), polyunsaturated fatty acid chains in phospholipids, and iron, resulting in the damage of the integrity of the phospholipid bilayer and the rupture of cell membrane and organelle membrane, ultimately leading to oxidative cell death. Previous studies showed a close relationship between ferroptosis and myocardial dysfunction. Animal experiments conducted by Han She et al¹⁶ found that sepsis led to mitochondrial-related ferroptosis and myocardial injury, which could be alleviated through inhibition of voltage-dependent anion channel 2 (VDAC2) malonylation by a mitochondria targeting nano material. Zeng et al¹⁷ proved that inhibiting ferroptosis by resveratrol through the Sirt1/Nrf2 pathway could alleviate myocardial dysfunction induced by sepsis in rats. However, whether the myocardial dysfunction in hemorrhagic shock rats was related to ferroptosis was unclear.

Therefore, we conducted animal and cell experiments to verify our hypothesis that TPP-MR might have the protective effects on myocardial function in hemorrhagic shock, which was related to ferroptosis.

Materials and Methods

Ethical Approval of the Study Protocol

The study protocol was approved by the Laboratory Animal Welfare and Ethics Committee of Army Medical University (Approval No. AMUWEC20224867). The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (8th edition, 2011, National Institutes of Health, Bethesda, Md).

Reagents

Lactate Ringer’s solution was purchased from Sichuan Kelun Pharmaceutical (Sichuan, China). Malate Ringer’s solution was purchased from B. Braun (Melsungen AG, Germany). Sodium chloride, potassium chloride, calcium chloride, magnesium chloride and sodium acetate were purchased from Macklin Biochemical Co Ltd (Shanghai, China). ATP detection kit was purchased from Cell Biolabs, Inc (San Diego, USA). OCR (mitochondrial respiration detection) kit was purchased from Sigma (St. Louis, Missouri, USA). GPX4 (ab125066) and ACSL4 (ab205199) were purchased from Abcam (Cambridge, MA, USA). β-actin (Cat. BM0627) was purchased from Boster (China). COX2 (12,282) was purchased from Cell Signaling Technology (Danvers, Massachusetts, USA). MitoTracker Deep Red (M22426) and Mito-SOX Red (M36008) were purchased from Invitrogen (Carlsbad, CA, USA). MitoBright-Deep Red (MT12) and Mito-FerroGreen (M489) were purchased from Dojindo (Kyushu, Japan). Lipid Peroxidation kit(C10445) was purchased from Thermo Scientific (Waltham, MA, America). JC-1 (C2003S), MDA (S0131M), and Glutathione detection kit (S0053) were purchased from Beyotime Biotechnology (Shanghai, China). DAPI (Cat. ab188804) was purchased from Abcam company (USA). Ferrostatin-1 (Cat. #SML0583) was purchased from Sigma-Aldrich (St. Louis, MO). Triphenyl phosphine (TPP)(T84409) and polyamidoamine(PAMAM) (412,449) were purchased from Sigma (St. Louis, Missouri, USA).

Synthesis of TPP@PAMAM@MA

Triphenylphosphine (TPP, 10 mmol) and 6- bromohexanoic acid (10.5 mmol) were dissolved in anhydrous acetonitrile and refluxed for 16 hours under the protection of nitrogen. After the reaction, the product was recrystallized to obtain TPP-COOH. TPP-COOH (2 mmol), N, N’- dicyclohexylcarbodiimide (2.4 mmol), and N-hydroxysuccinimide (NHS, 2.4 mmol) were mixed in 10 mL of anhydrous DMSO and reacted at room temperature for 12 hours. Then PEG(1 mmol) was added and the reaction continued at room temperature for another 12 hours. After that, the reaction solution was transferred to a dialysis bag (molecular weight cut-off 1000), dialyzed with DMSO for 24 hours and deionized water for 48 hours successively. The dialysate was freeze-dried to obtain an intermediate product (TPP-PEG). Subsequently, TPP-PEG(1 mmol), N, N’- dicyclohexylcarbodiimide (1.2 mmol) and N- hydroxysuccinimide (NHS, 1.2 mmol) were mixed in 5 mL of anhydrous DMSO and reacted at room temperature for 24 hours. Polyamide amine (PAMAM, 3mmol) was added and the reaction continued for 24 hours. The reaction solution was transferred to a dialysis bag (molecular weight cut-off 2000), dialyzed with DMSO for 24 hours and deionized water for 48 hours. Freeze-drying the dialysate to obtain an intermediate product (TPP-PEG-PAMAM). Finally, an equal proportion of malic acid and TPP-PEG-PAMAM solution was gently mixed by vortex for 30 seconds, and left it at room temperature for 30 minutes to form TPP-PEG-PAMAM malic acid polymer (TPP@PAMAM@MA). TPP@PAMAM@MA was dissolved in DMSO and characterized by 1HNMR, particle size distribution, zeta potential and electron microscope.

Organ Toxicity and Cytotoxicity Testing of TPP@PAMAM@MA

TPP@PAMAM@MA was dissolved in sterile water to prepare a 10 mg/mL solution. The solution was injected intravenously into rats at a dose of 30 mg/kg. After 24 hours, the tissues of rat heart, liver and kidney were collected for H&E staining. About 5000 cardiomyocytes (H9C2) were cultured in 96-well plates and treated with 1ug/mL solution at 37°C for 15 minutes. Then cell viability was measured by cell counter.

Preparation of TPP-MR

TPP@PAMAM@MA was synthesized by nano-material synthesis technology. MR was prepared in the following proportions: sodium chloride 6.799 g, potassium chloride 0.2984 g, calcium chloride 0.3675 g, magnesium chloride 0.2033 g, sodium acetate 3.266 g, L-malic acid 0.671 g and sodium hydroxide 0.2 g, and the volume was adjusted to 1000 mL with water for injection. In TPP-MR, L-malic acid in MR was replaced by TPP@PAMAM@MA to form a new resuscitation fluid. The drug load and encapsulation rate of TPP-MR was24.27% and 80.09%, respectively.¹⁸ Based on the drug load and peak load rate, TPP-MR was prepared using an equivalent mass of L-malic acid and TPP@PAMAM@MA.

Metabolism Analysis of TPP-MR in vivo

Thirty SD rats were randomly divided into groups for ten time points, each group consisting of 3 rats. TPP-MR was administered intravenously at a dose of 35mL/kg. Blood was collected from the abdominal main vein at 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, 36 hours, 48 hours, and 72 hours post-administration, and the rats were euthanized immediately after dissection. The 200uL of plasma was mixed with 400uL of methanol, vortexed for 1 minute, centrifuged at 12000r/min for 10 minutes at 4°C, and the supernatant was collected. The supernatant was then freeze-dried, redissolved in 100uL of methanol, centrifuged at 12,000 r/min for 10 minutes, and 10uL of the ultimate supernatant was used for metabolism analysis by the LC-MS/MS system.

Hemorrhagic Shock Model and Groups

Adult SD rats (weight 200–220 g, provided by the experimental animal center of Daping Hospital of Army Medical University) were anesthetized by intraperitoneal injection of sodium pentobarbital (45 mg/kg). SD rats fasted for 8 hours before the operation but drank water freely. Each animal’s right femoral artery and femoral vein were isolated and cannulated with polyethylene tubing (0.9×0.5 mm) for bleeding, monitoring the mean arterial pressure (MAP) and infusion. Blood was withdrawn through the suitable femoral artery catheter to maintain the MAP at 40 mmHg to induce hemorrhagic shock.¹⁹

A total of 198 SD rats were randomly divided into four groups. The sham group was given an incision without bleeding, and the shock group only received the hemorrhagic shock modeling. At 4 hours after HS surgery, the Fer-1 group was infused with Lactated Ringer’s solution (LR, 35mL/kg) and was injected with ferrostatin-1 (Fer-1, ferrostatin-1 0.8mg/kg) in the tail vein at the same time.²⁰ For the animals in the TPP-MR group, LR was replaced by TPP-MR (35mL/kg) at a constant rate of 2.5mL/h. After the infusion, the blood vessels were ligated, and the incision was sutured. Then, the mean survival time and the survival rate of rats within 24 hours were recorded, and the following experiments were carried out.

Cell Culture and Processing

The H9C2 cardiomyocytes were obtained from the American Type Culture Collection (ATCC, Manassas, VA, United States) and cultured in DMEM supplemented with 10% FBS in a humidified, 5% CO₂/ 95% air atmosphere at 37°C. The cells in this state were the normal group. The cells were treated with hypoxia to simulate the state of hemorrhagic shock in animals for mechanism research. H9C2 cells were seeded in a culture dish for 12 hours and then were placed in a sealed tank at 37°C for hypoxia treatment. The cells underwent hypoxia process involved first introducing anoxic gas (95% N2 and 5% CO2) for 15 minutes, then closing the vent valve for 10 minutes, and reopening the vent valve to introduce anoxic gas again. This hypoxia process was repeated four times until the oxygen concentration in the sealed tank fell below 0.2%. The tank was then sealed for 4 hours. Hypoxic cells were randomly divided into 3 groups, including the hypoxia group which only received hypoxia treatment, Fer-1 group and TPP-MR group which received Fer-1 (5uM)²¹+1% volume of LR (1% of the volume of the culture medium in the culture dish) or only 1% volume of TPP-MR solution (1% of the volume of the culture medium in the culture dish) during the tank sealed for 4 h, respectively.

Cardiac Output Measurement and Oxygen Supply and Consumption Testing

In each group, rats underwent catheterization of the jugular vein, with the catheter opening positioned at the right heart atrium. A thermodilution catheter was inserted into the carotid artery up to the proximal end of the aorta, with the depth adjusted until the catheter showed noticeable pulsation with the heartbeat and connected to a cardiac output measuring device. A temperature probe was placed in 0°C ice-salt water until the temperature stabilized. Then, 0.3 mL of ice-salt water was rapidly injected through the venous catheter, and the cardiac output (CO) and heart rate (HR) of each group of rats were measured using a Cardiomax III (USA). The cardiac index [CI = CO ÷ S, body surface area: S = K × W ^(2/3) (cm²), where K = 9.1; W: body weight (g)] and stroke index (SI = CI ÷ HR) were calculated using these measurements. After that, arterial and venous blood samples were collected from the femoral artery and vein for blood gas analysis: oxygen tension (pO2), hemoglobin (Hb), arterial oxygen saturation (SaO₂), and venous oxygen saturation (SvO₂). Tissue oxygen delivery (DO₂ = CI × 13.4 × Hb × SaO₂) and consumption [VO₂ = CI × 13.4 × Hb × (SaO₂ - SvO₂)] were calculated.

Echocardiographic Assessment in Rats

Echocardiographic assessment was conducted using a Vevo2100 small animal echocardiography system and a corresponding MS-400 high-frequency probe. Images of the left ventricular short-axis view were captured in rats under 0.4% pentobarbital sodium intraperitoneal anesthesia. Left ventricular posterior wall thickness in diastole (LVPWTD), left ventricular posterior wall thickness in systole (LVPWTs), left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), left ventricular mass, left ventricular ejection fraction (LVEF), and left ventricular short-axis fractional shortening (FS) were calculated.

Mitochondrial Respiration Measurement

In an XF24 cell culture plate, excluding background correction wells, each well was inoculated with 100 µL of cardiomyocyte suspension at a density of 10K cells/100µL/well. The cells were allowed to adhere for 1 hour before adding 150 µL of high-glucose culture medium to each well. After various treatments, the cell culture plate was incubated for 12 hours in a 37°C CO₂ incubator. Meanwhile, 1 mL XF Calibrant was added to each well of a hydration plate. The entire probe plate assembly was hydrated overnight in a 37°C non-CO₂ incubator. On the day of the experiment, each well of the cardiomyocyte culture plate was washed twice with assay medium, ensuring 500 mL of assay medium in each well, and then incubated in a 37°C non-CO₂ incubator for 60 minutes before testing with the XF Cell Mito Stress Test Kit.

Mitochondrial Membrane Potential Measurement

Cardiomyocytes were seeded onto confocal culture dishes and treated according to group protocols once cell coverage reached about 70%. Added ultrapure water to 50ul of 200x JC-1 to reach a total volume of 8 mL, mixed well, and then added 2 mL of staining buffer (5x) to mix to prepare the JC-1 staining working solution. Each culture dish was incubated with 0.5 mL of JC-1 working solution in a 37°C incubator for 20 minutes, then washed twice with JC-1 staining buffer (1x) and finally replaced with cell culture medium before observing JC-1 expression under a laser confocal microscope. Normal cells should show red fluorescence due to intact mitochondrial membrane potential, while cells with lost membrane potential should exhibit green fluorescence.

Mitochondrial Reactive Oxygen Species Detection

Cardiomyocytes reached to 70% confluency in confocal culture dishes were treated with ROS detection working solution at a final concentration of 10 umol/L and incubated in a 37°C incubator for 30 minutes. The culture dishes were gently shaken every 5 minutes to ensure full contact between the probe and cells. After incubation, cells were washed three times with serum-free culture medium, replaced with pre-warmed fresh cell culture medium at 37°C, and observed under a laser confocal microscope.

Confocal Microscopy Observation of Mitochondrial Morphology

Cardiomyocytes were seeded in confocal culture dishes and treated according to group protocols for 4 hours. Then cells were washed with 37°C PBS and incubated with pre-prepared Mitro Tracker live cell mitochondrial dye (1:10,000) at 37°C for 30 minutes before washing three times with 37°C PBS. Finally, 1 mL of 37°C serum-free culture medium was added to the culture dish, and mitochondrial morphology in cardiomyocytes was observed using a confocal microscope.

Cardiomyocyte Ferrous Ion Detection

Cardiomyocytes were seeded in culture dishes and treated according to group protocols for 30 minutes in a CO₂ incubator before adding 5 µmol/L Mito-FerroGreen and 200 nmol/L MitoBright Deep Red mitochondrial staining probe. Then 100 µmol/L ammonium iron (II) sulfate was added to the cell culture dishes, thoroughly mixed. After 1 hour, the cardiomyocyte ferrous was observed by fluorescence.

Lipid Peroxidation Detection in Cardiomyocytes

Cardiomyocytes were seeded in culture dishes and treated according to group protocols before adding Image iT^® lipid peroxidation reagent to achieve a final concentration of 10µM. The cells were then incubated at 37°C for 30 minutes, washed three times with PBS, and the culture medium was replaced with 1 mL of serum-free one pre-warmed at 37°C. Finally, lipid peroxidation was observed using a confocal microscope.

Statistical Analysis

Statistical analysis was performed using SPSS 20.0 (SPSS Inc., Chicago, IL, USA). Data were presented as mean ± standard deviation. Independent sample t-tests were used to analyze differences between two groups. One-way analysis of variance (ANOVA) and post-hoc tests (S-N-K/LSD) were used for multiple group comparisons. P-values less than 0.05 were considered statistically significant.

Results

Characterization and Toxicity of TPP@PAMAM@MA

TPP@PAMAM@MA was verified by mass spectrometry and NMR. Hydrogen spectrum measurement showed malic acid-CH2-1H NMR characteristics at δ2.01 and δ2.24. PAMAM-COOR 1H NMR characteristics were at δ2.01–2.58. TTP-COOH 1H NMR characteristics were at 7.32–7.58 (Ar-H) (Figure 1A). PEG 1H NMR characteristics were at δ2.14–2.21 (-NHCH2CH2-) (Figure 1B). Electron microscope photos showed that TPP@PAMAM@MA was spherical with regular particles (Figure 1C). The synthesized TPP@PAMAM@MA had a potential of (−5.7±0.6) mV, with an average particle size of (122.3±0.8) nm (Figure 1D and E). The -CH2- peak appeared at 2888.74 cm⁻¹, characteristic of methylene. Additionally, the key active carboxyl group peak of TPP@PAMAM@MA appeared at 1115.02 cm⁻¹. The results indicated that the characteristic functional groups of TPP@PAMAM@MA had characteristic absorption peaks (Figure 1F). HE staining results indicated that TPP@PAMAM@MA did not disperse in the liver, kidney, and heart after administration, suggesting the safety of TPP@PAMAM@MA for in vivo use, with pathological scoring conducted as per the mentioned method²²(Figure 1G and Supplementary Figure 1). In vitro experiments were also conducted to assess the toxicity of TPP@PAMAM@MA to cardiomyocytes. CCK-8 analysis showed that TPP@PAMAM@MA did not affect the survival rate of cardioH9C2 cells (Figure 1H). TPP@PAMAM@MA was formulated into a clear and transparent TPP-MR with MR’s component ratio, meeting the pH and osmotic pressure requirements for infusion (Figure 1I). LC-MS/MS system analysis showed that the peak concentration (Cmax), the elimination phase half-life (HL_Lambda_z), and the apparent distribution volume (Vz_F_obs) of TPP-MR was 472.1±16.2ng/mL, 10.1±1.1h, and 6722.5±1482.1mL/kg, respectively. (Figure 1J).

Figure 1 Characterization of TPP@PAMAM@MA. (A) TPP@PAMAM@MA mass spectrometry. (B) TPP@PAMAM@MA NMR determination. (C) The size of TPP@PAMAM@MA was observed under the electron microscope (bar, 1um). (D) Potential of TPP@PAMAM@MA. (E) Size determination of TPP@PAMAM@MA particle. (F) Infrared spectroscopy of TPP-MR. (G) HE stains of heart, liver, and kidney after TPP@PAMAM@MA treatment in rats(n=6/group). (H) Effect of TPP@PAMAM@MA on the cell viability of H9C2 cells (3 independent experiments). (I and J) Preparation and pharmacokinetics of TPP-MR.

TPP-MR Improved Cardiac Function and Survival Time in Rats with Hemorrhagic Shock

Cardiac function was evaluated using echocardiography, showing a significant decrease in LVEF in rats with hemorrhagic shock. After TPP-MR infusion, LVEF significantly improved, with an increase rate of 27.2% compared to the shock group (Figure 2A and B). Additionally, hemorrhagic shock caused significant reductions in cardiac output (CO), cardiac index (CI), and stroke index (SI), which were improved by TPP-MR infusion with increase rates of 44.5%,52.0%, and 26.1%, respectively (Figure 2C–E). MAP dropped from around 114 mmHg in sham rats to 45 mmHg in rats with hemorrhagic shock, which was increased by 89.8% after TPP-MR infusion (Figure 2F). The HE staining of heart pathology slice showed that in sham myocardial tissue, myocardial fibers were arranged orderly and interstitial tissue was sparse. In shock group, myocardial cells exhibited obvious swelling, with vacuole formation, disordered myocardial fiber arrangement, and expanded interstitial space. In the TPP-MR group, interstitial edema of myocardial cells was significantly reduced, interstitial space narrowed, and disordered arrangement of myocardial fibers notably restored, with pathological scoring conducted as per previous studies (Figure 2G and H). Compared to the shock group, cardiac troponin T (cTnT) and lactate dehydrogenase (LDH) in the TPP-MR group significantly decreased, indicating significant improvement in myocardial injury (Figure 2I and J). Oxygen supply and consumption significantly decreased in rats with hemorrhagic shock, which was significantly increased after TPP-MR infusion (Figure 2K). No rats in the shock group survived beyond 12 hours, while survival time in the TPP-MR group was significantly extended to 18.6±5.4 hours, with a 24-hour survival rate of 31.3% (Figure 2L). These results indicated that TPP-MR significantly improved cardiac function in rats with hemorrhagic shock by reducing myocardial injury, increasing cardiac output, improving perfusion, and thereby extending survival time.

Figure 2 Effects of TPP-MR on cardiac function in hemorrhagic shock rats. Cardiac function including (A) echocardiograms (n=6/group), (B) LVEF (n=6/group), (C) cardiac output (n=8/group), (D) cardiac index(n=8/group) and (E) stroke index (n=8/group) were measured 2 hours after resuscitation treatment. (F) Mean arterial pressure measurement of the hemorrhagic shock rats (n=8/group). (G and H) HE stains of cardiac pathological sections and heart injury scores (bar, 20um, n=8/group). The expressions of (I) cardiac troponin T and (J) lactate dehydrogenase in venous blood (n=8/group). (K) Detection of oxygen supply and consumption of the hemorrhagic shock rats (n=8/group). (L) Survival rate and survival time were observed in different groups (n=16/group). aP<0.05 compared with the sham group, bP<0.05 compared with the shock group.

TPP-MR Improved Mitochondrial Function in Cardiomyocytes of Rats with Hemorrhagic Shock

To explore whether TPP-MR improved mitochondrial function of cardiomyocytes after hemorrhagic shock, we used hypoxia-treated cardiomyocytes and incubated them with TPP-MR to simulate the rat shock model. Mitochondrial ATP production and Oxygen Consumption Rate (OCR) were observed by using Agilent hippocampus XF technology. Results showed that TPP-MR treatment improved the reduction of ATP production and OCR of cardiomyocytes caused by hypoxia (Figure 3A–C). Confocal microscopy was used to observe mitochondrial morphology changes. Mitochondria in normal cardiomyocytes were distributed as continuous long columns. After hypoxia treatment, most mitochondria in cardiomyocytes undergo morphological changes, presenting as dots or spheres, with a significant increase in the number of mitochondria. TPP-MR incubation restored mitochondrial morphology and significantly reduced the number of mitochondria (Figure 3D). Further observation of mitochondrial membrane potential and ROS levels in each group showed that after hypoxia treatment, mitochondrial membrane potential in cardiomyocytes significantly decreased and ROS fluorescence intensity significantly increased. TPP-MR was able to counteract the reduction in mitochondrial membrane potential and the increase in ROS fluorescence intensity induced by hypoxia, indicating that TPP-MR infusion could alleviate the damage to mitochondrial function in cardiomyocytes induced by hypoxia (Figure 3E–H).

Figure 3 Mitochondrial function of cardiomyocytes hemorrhagic shock. (A) Changes in the ATP level (3 independent experiments). (B and C) Mitochondrial maximum respiratory rate assay in H9C2 (3 independent experiments). (D) Representative images of mitochondrial morphology of H9C2 cells (bar, 20um, 3 independent experiments). (E) The mitochondrial membrane potential of H9C2 was observed by confocal microscopy (bar, 20um, 3 independent experiments). (F) Red/green fluorescent (Δψm) quantitative analysis in H9C2. (G) Representative confocal images of MITO-SOX (bar, 10um, 3 independent experiments). (H) ROS fluorescence quantitative analysis in H9C2. aP<0.05 compared with the normal group, bP<0.05 compared with the hypoxia group.

Hemorrhagic Shock-Induced Myocardial Injury is Associated with Ferroptosis

Reduced glutathione (GSH) and oxidized glutathione (GSSG) played important roles in ferroptosis, while GSH, under the action of GPX4, reduced cell polyunsaturated fatty acid phospholipid hydroperoxides (PUFA-PL-OOHs) to non-lethal PUFA phospholipid alcohols (PUFA-PL-OHs), thereby countering cell ferroptosis. To explore whether myocardial injury in rats with hemorrhagic shock was related to ferroptosis, GSH and GSSG levels in myocardial tissues were measured. Results showed that compared to sham rats, GSH levels decreased, GSSG levels increased, and the GSH/GSSG ratio decreased in the shock group. In contrast, the Fer-1 group showed significantly higher GSH levels, reduced GSSG levels, and an increased GSH/GSSG ratio compared to the shock group (Figure 4A–C). Additionally, lipid peroxidation product MDA level in myocardial tissues significantly increased in the shock group compared to the sham group, which was markedly reduced in the Fer-1 group (Figure 4D). At the cellular level, post-hypoxia Fe²⁺ and lipid peroxidation levels significantly increased compared to the normal group, while Fer-1 notably ameliorated these trends (Figure 4E and F). Western blot results showed that hypoxia significantly increased the expression of ferroptosis-related proteins ACSL4 and COX2, but decreased the GPX4 expression in cardiomyocytes. Compared to the hypoxia group, the Fer-1 group showed a significant decrease in ACSL4 and COX2 expression by 21.3% and 20.0%, respectively, and an increase in GPX4 expression by 15.2% (Figure 4G–J).

Figure 4 Hemorrhagic shock and ferroptosis. (A–D) Effects of Hemorrhagic shock on the level of GSH, GSSG, GSH/GSSG and MDA (n=6/group). (E) Representative images of H9C2 cells after treatment with MitoBright-Deep Red and Mito-FerroGreen (bar, 10um, 3 independent experiments). (F) Representative images of H9C2 cells treated with LPO (bars, 10um, 3 independent experiments). (G–J)The effect of TPP-MR on the expression of ACSL4, COX2 and GPX4 proteins (3 independent experiments). aP<0.05 compared with the sham or normal group, bP<0.05 compared with the shock or hypoxia group.

Echocardiographic assessment of cardiac function revealed a significant reduction in LVEF in rats with hemorrhagic shock, which was noticeably improved after using the ferroptosis inhibitor Fer-1 (Figure 5A and B). Additionally, hemorrhagic shock caused significant reductions in CO, CI, and SI, which were all significantly improved in the Fer-1 group (Figure 5C–E). MAP also recovered noticeably in hemorrhagic shock rats after using Fer-1 (Figure 5F). HE staining of heart pathology slice showed that interstitial edema of myocardial cells was significantly reduced and the disordered myocardial fiber was restored in the Fer-1 group, with pathological scoring ameliorated (Figure 5G and H). Oxygen supply and consumption were significantly reduced in hemorrhagic shock rats, which were improved after Fer-1 use (Figure 5I). Besides, the ATP production in the Fer-1 group also significantly increased compared to that in the hypoxia group (Figure 5J). At the cellular level, mitochondrial ATP production and OCR were also measured. Results showed that OCR in cardiomyocytes was significantly reduced under hypoxic conditions but noticeably improved after adding the ferroptosis inhibitor Fer-1 (Figure 5K). No rats in the shock group survived beyond 12 hours. In the Fer-1 group, survival time was extended to 10.3±7.7 hours, with a 24-hour survival rate of 4.2% (Figure 5L).

Figure 5 Effects of Fer-1 on myocardial injury in Hemorrhagic shock. Cardiac function including (A) echocardiograms (n=6/group), (B) LVEF (n=6/group), (C) cardiac output (n=8/group), (D)cardiac index(n=8/group) and (E) stroke index (n=8/group) were measured 2 hours after resuscitation treatment. (F) Mean arterial pressure measurement of the hemorrhagic shock rats (n=8/group). (G) HE stains of cardiac tissue and (H) heart injury score (bar, 20um, n=6/group). (I) Detection of oxygen supply and consumption of the hemorrhagic shock rats (n=8/group). (J) Changes in the ATP level (3 independent experiments). (K) Mitochondrial maximum respiratory rate assay in H9C2 (3 independent experiments). (L) The effects of Fer-1 on survival rate and survival time of hemorrhagic shock rats (n=8/group). aP<0.05 compared with the sham or normal group, bP<0.05 compared with the hemorrhagic shock or hypoxia group.

TPP-MR Significantly Inhibits Ferroptosis

Preliminary animal experiments indicated that TPP-MR significantly improved cardiac function in rats with hemorrhagic shock, and myocardial injury in these rats was closely associated with ferroptosis. Western blot results showed that TPP-MR more significantly inhibited ferroptosis compared to the Fer-1 group, further reducing the expression of ferroptosis-related proteins ACSL4 and COX2 by 32.2% and 15.2%, respectively, but slightly increasing the expression of GPX4 by 6.3% in cardiomyocytes after hypoxia stimulation (Figure 6A–D). Similarly, the Fe²⁺ and lipid peroxidation levels further decreased in the TPP-MR group compared to the Fer-1 group (Figure 6E–H). These results suggested that TPP-MR could reduce ROS to inhibit ferroptosis, improve myocardial mitochondrial function, alleviate myocardial injury, enhance cardiac output, improve perfusion, and thereby extend survival time.

Figure 6 Effect of TPP-MR on H9C2 cells ferroptosis. (A–D) The effect of TPP-MR on the expression of ACSL4, COX2 and GPX4 proteins (3 independent experiments). (E and F) Representative images of H9C2 cells after treatment with MitoBright-Deep Red and Mito-FerroGreen(bar, 10um, 3 independent experiments). (G and H) Representative images of H9C2 cells treated with LPO (bars, 10um, 3 independent experiments). aP<0.05 compared with the sham or normal group, bP<0.05 compared with the hypoxia group, cP<0.05 compared with the Fer-1 group.

Discussion

In this study, we synthesized TPP-MR, a new nano-resuscitation solution for hemorrhagic shock. Our findings demonstrated that TPP-MR protected cardiac function in rats with hemorrhagic shock by improving mitochondrial function and inhibiting cellular ferroptosis. The mechanism might involve the delivery of exogenous malic acid to myocardial cells by the nanomaterial targeting myocardial mitochondria present in TPP-MR. Malic acid, through the malate-aspartate shuttle, enhanced intracellular ATP production, improved electron transport chain efficiency, and thus improved mitochondrial function. Additionally, TPP-MR reduced mitochondrial ROS production by upregulating GPX4, converting cell phospholipid hydroperoxides (PLOOH) to non-lethal phospholipid alcohols (PLOH), thereby inhibiting cellular ferroptosis. It also downregulated ACSL4 and COX2, inhibiting the production of PLOOH in cells and thus further preventing ferroptosis (Figure 7).

Figure 7 Mechanism of TPP-MR Inhibiting the Ferroptosis. The protective mechanism of TPP-MR on myocardial injury in hemorrhagic shock rats.

Hemorrhagic shock was a common critical condition in clinical settings and a major cause of early death in trauma cases.² Cardiac dysfunction was one of the most severe complications of hemorrhagic shock and the key factor leading to poor prognosis.²³ Myocardial injury in hemorrhagic shock mainly manifested as reduced myocardial contractility, leading to insufficient systemic blood perfusion and multi-organ dysfunction.²⁴ Energy supply was crucial for myocardial contractility. In healthy adult hearts, 70% of energy came from fatty acid β-oxidation, with only 10–30% from glucose metabolism and the rest from lactate, ketone bodies, and amino acid metabolism.²⁵ Post-hemorrhagic shock, the metabolism of myocardial cell significantly changed, characterized by reduced fatty acid β-oxidation and enhanced glycolysis, leading to a significant reduction in ATP synthesis, and potentially contributing to cardiac dysfunction. Mitochondria, indispensable for regular myocardial cell activity and energy production, undergo various alterations during hemorrhagic shock-induced myocardial injury, including inner membrane damage, permeability changes, membrane potential alterations, and swelling. These changes led to the uncoupling of oxidative phosphorylation, with accompanying calcium ion and cytochrome c efflux, causing a series of mitochondrial dysfunctions, including oxidative phosphorylation damage, decreased kinase activity, and oxidative damage.²⁶ Therefore, improving mitochondrial function was vital in myocardial injury after hemorrhagic shock.

Fluid resuscitation was the first-line treatment for hemorrhagic shock. Wang et al²⁷ found that bicarbonate Ringer’s solution (BRS) maintained typical glycocalyx structure and mitigated inflammation, maintaining mean arterial pressure in rats with hemorrhagic shock. Zhao et al²⁸ discovered that HES (130/0.4) protected against endothelial injury in hemorrhagic shock rats by downregulating heparanase, hyaluronidase, and neuraminidase expression. Malate Ringer’s (L-malate acetic acid-balanced fluid) was a novel resuscitation fluid containing the indispensable intermediate metabolite L-malate (330mg/500mL). L-malate, via the malate-aspartate shuttle, entered the mitochondrial matrix through the oxaloacetate malate carrier (OMC) channel and was converted to NADH under malate dehydrogenase. NADH reduced oxaloacetate to malate, which penetrated the matrix through the OMC on the mitochondrial inner membrane, regenerating oxaloacetate and NADH. NADH entered the electron respiratory chain to produce ATP. At the same time, matrix-generated oxaloacetate converted to aspartate under the action of glutamate oxaloacetate transaminase, which was then transported out of the matrix by the aspartate glutamate carrier (AGC), continuing the shuttle to provide energy and enhance the body’s oxidative phosphorylation and energy metabolism.²⁹ It’s reported that Malate Ringer’s fluid reduced myocardial injury in septic rats by improving mitochondrial function and inhibiting apoptosis, with L-malate playing a crucial role.¹³ Our TPP@PAMAM encapsulating L-malate to form RPP@PAMAM@MA was confirmed in preliminary studies to target myocardial mitochondria, reducing consumption and delivering more L-malate to myocardial mitochondria, providing more raw materials for ATP synthesis.

Ferroptosis was a form of regulated cell death, closely associated with the production of mitochondrial lipid reactive oxygen species (ROS) and the destruction of the antioxidant system. Jang et al³⁰ found that mitochondria were involved in the onset of ferroptosis, since mitochondrial fragmentation and lipid peroxidation occurred shortly after ferroptosis-related stimuli, leading to inhibition of oxidative phosphorylation and electron transport chain (ETC) complexes, and therefore exacerbating RSL3-induced ferroptosis. Ye et al³¹ discovered that inhibiting the ferroptosis SLC7A11/GPX4 pathway contributed to attenuating mitochondrial damage in the myocardium. Im et al³² found that COX-2 functions as a cellular factor which induces superoxide-mediated cell death in primary cortical neurons. Cortical neurons with enhanced COX-2 expression showed superoxide generation, GSH depletion, and lipid peroxidation in response to low doses of Fe²⁺. We also found substantial increases in MDA, GSSG, and Fe²⁺, but decreases in GPX4 and GSH levels in cardiac tissues after hemorrhagic shock, indicating impaired antioxidant capacity, accumulation of lipid peroxides, and comprehensive disruption of redox homeostasis happened in the hemorrhagic shock-induced rat model. TPP-MR directly reduced mitochondrial ROS levels in myocardial cells, enhanced myocardial mitochondrial energy supply, and improved mitochondrial function. Furthermore, after TPP-MR infusion, the antioxidant capacity improved as the expression of GSH and GPX4 increase, but the expression of ACSL4 and COX2 decreased. By this way, TPP-MR infusion increased MAP and cardiac output, improved myocardial tissue perfusion, and significantly improved survival. Therefore, we hypothesized TPP-MR improved cardiac function in rats with hemorrhagic shock by reducing mitochondrial ROS and improving mitochondrial function via inhibiting ferroptosis.

Although this study observed the protective effects of TPP-MR on myocardial injury in hemorrhagic shock, whether it could protect patients with hemorrhagic shock remained to be further studied. What’s more, the impact of extensive infusion of this fluid on the long-term prognosis of hemorrhagic shock also need further confirmation.

Conclusion

TPP-MR alleviated myocardial injury in rats with hemorrhagic shock by improving mitochondrial function and inhibiting ferroptosis.