Angiotensin II Is Involved in MLKL Activation During the Development of Heart Failure Following Myocardial Infarction in Rats

Abstract

Several reports assume that myocardial necroptotic cell death is induced during the development of chronic heart failure. Although it is well accepted that angiotensin II induces apoptotic cell death of cardiac myocytes, the involvement of angiotensin II in the induction of myocardial necroptosis during the development of heart failure is still unknown. Therefore, we examined the role of angiotensin II in myocardial necroptosis using rat failing hearts following myocardial infarction and cultured cardiomyocytes. We found that administration of azilsartan, an angiotensin II AT₁ receptor blocker, or trandolapril, an angiotensin-converting enzyme inhibitor, to rats from the 2nd to the 8th week after myocardial infarction resulted in preservation of cardiac function and attenuation of mixed lineage kinase domain-like (MLKL) activation. Furthermore, the ratio of necroptotic cell death was increased in neonatal rat ventricular cardiomyocytes cultured with conditioned medium from rat cardiac fibroblasts in the presence of angiotensin II. This increase in necroptotic cells was attenuated by pretreatment with azilsartan. Furthermore, activated MLKL was increased in cardiomyocytes cultured in conditioned medium. Pretreatment with azilsartan also prevented the conditioned medium-induced increase in activated MLKL. These results suggest that angiotensin II contributes to the induction of myocardial necroptosis during the development of heart failure.

INTRODUCTION

Increased hemodynamic overload caused by various cardiac diseases, such as hypertension, myocardial infarction (MI), cardiomyopathy, and myocardial valvular disease, results in myocardial remodeling, which is a histological change characterized by hypertrophy of cardiac myocytes and fibrosis of the cardiac interstitium. Cardiac remodeling is a compensatory mechanism contributing to the preservation of cardiac function against the increased hemodynamic load for the heart. On the other hand, prolonged pressure overload for the heart fails the compensatory mechanism, leading to chronic heart failure. However, the mechanism underlying the failure of this adaptive mechanism during the development of heart failure is still unknown.

The cause of cardiac pump dysfunction is postulated to involve not only contractile dysfunction of cardiomyocytes but also a decrease in the number of cardiomyocytes. Cardiomyocyte depletion is caused by cell death in the myocardium. Apoptosis is defined as a type of programmed cell death with characteristic intracellular changes in morphological and biochemical parameters. Several studies have suggested that cell death due to apoptosis is the major cause of the decrease in myocardial cells during the development of chronic heart failure.^1,2) Recently, a programmed cell death pathway called “necroptosis,” in which a specific intracellular signaling pathway induces necrosis-like cell death, was proposed.^3–6) Necroptosis is induced during impaired apoptosis by ligand-dependent stimulation of cell surface death receptors such as Fas, tumor necrosis factor (TNF) receptor 1, interferon (IFN) receptors, and Toll-like receptors.⁷⁾ More recently, necroptosis has, however, been shown to be activated in inflammatory diseases, neurodegeneration, and cancer, and has been seen under pathological conditions such as ischemic brain injury, myocardial infarction, and chemotherapy-induced cell death.⁸⁾ Furthermore, since the involvement of necroptosis in the development of chronic heart failure has been assumed, it is attracting attention as a new therapeutic target in chronic heart failure.^9–12) In the necroptosis signaling pathway, death receptors trigger the activation of receptor interacting protein kinase 1 (RIP1). Then, RIP1 binds to RIP3, which shares a common motif with RIP1, and these two proteins are interphosphorylated.^13,14) Phosphorylated RIP3 phosphorylates and activates mixed lineage kinase domain-like (MLKL), an executioner of necroptosis. Activated MLKL translocates to the plasma membrane and penetrates the plasma membrane to induce necroptosis.^15,16)

Angiotensin II (Ang II) is one of the major neurohumoral factors involved in the development of heart failure.^17–19) Ang II augments both cardiac preload and afterload by increasing circulating blood volume and elevating vasoconstriction. Furthermore, Ang II directly induces cardiac myocyte hypertrophy.^20,21) Ang II also induces myocardial fibrosis by activating cardiac fibroblasts.²²⁾ Therefore, Ang II AT₁ receptor blockers and angiotensin-converting enzyme inhibitors are widely used for the treatment of chronic heart failure.²³⁾ Furthermore, Ang II promotes the release of proinflammatory cytokines from cardiac fibroblasts.^24,25) Cytokines released from cardiac fibroblasts include TNF-α and IFN-β, which are involved in the induction of necroptosis.^26,27) Therefore, Ang II may be involved in cardiomyocyte necroptosis via the release of humoral factors from cardiac fibroblasts. A recent study reported that Ang II-induced renal tubular cell necroptosis occurs in a mouse with renal failure.²⁸⁾ However, the involvement of Ang II in myocardial necroptosis remains unclear. In this study, we evaluated the role of Ang II in the activation of MLKL during the development of heart failure using rats with MI and cultured cells.

MATERIALS AND METHODS

Animals

We used 96 male Wistar rats (10 weeks old, SLC, Hamamatsu, Japan) as an animal model of heart failure following myocardial infarction. In addition, 70 neonatal Wistar rats (2 d old, SLC) were used as a source of primary cultures of cardiomyocytes and cardiac fibroblasts. These animals were kept at a constant temperature and humidity (23 ± 1 °C, 55 ± 5%) with a light/dark cycle (7:00–19:00 light period/19:00–7:00 dark period). Food and water were available ad libitum according to the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 85-23, revised 2011). This study was approved by the Committee of Animal Use and Welfare of Tokyo University of Pharmacy and Life Sciences (Approval Numbers: P22-01 and P23-03).

Operation and Drug Treatment

The method of coronary artery ligation (CAL) in rats was described previously.^11,29–33) Isoflurane (induction: 5%, maintenance: 3%; Pfizer, New York, NY, U.S.A.) was used as an anesthetic to eliminate pain in the experimental animals, and the surgical field was disinfected with povidone-iodine and the instruments with chlorhexidine gluconate to prevent infection.

Forty-eight Sham and 48 CAL rats each were randomly divided into three groups of 16 rats each and orally gavaged with azilsartan (Azi, Ang II AT₁ receptor blocker, 0.3 mg/kg/d), trandolapril (Tra, angiotensin-converting enzyme inhibitor, 3 mg/kg/d), or vehicle every day from the 2nd to the 8th week after the CAL and Sham operation as described previously.^30,34,35)

Echocardiogram

Echocardiographic parameters of CAL and Sham rats were measured at 8 weeks postoperatively. To subject the animals to echocardiography, rats were immobilized with 30 mg/kg intraperitoneally (i.p.) injected sodium pentobarbital according to a previously described method.^11,31–33)

Cardiac Fibroblasts Culture and Preparation of Conditioned Medium

The culture procedure for cardiac fibroblasts prepared from rat heart was described previously.^29,36,37) The third passages of cardiac fibroblasts were used as cardiac fibroblasts in the experiments. Two days after the last passages, the cells were replaced with Dulbecco’s modified Eagle’s medium (DMEM) containing 1% (v/v) fetal bovine serum (FBS) (98% (v/v) DMEM (Sigma-Aldrich, St. Louis, MO, U.S.A.), 1% (v/v) FBS (Biowest, Nuaillé, France), and 1% (v/v) penicillin–streptomycin (Thermo Fisher Scientific Inc., Waltham, MA, U.S.A.) supplemented with Azi (1 µmol/L) or solvent (phosphate-buffered saline (PBS)). Ang II (1 µmol/L, Peptide Institute, Inc., Osaka, Japan) or solvent (PBS) was treated 1 h later, similar to the method used in our previous study.³⁷⁾ The culture supernatant of cardiac fibroblasts was collected 48 h after AngII treatment and centrifuged (10000 × g, 30 min, 4 °C), and the supernatant was separated and used as conditioned medium. (Fig. 1) To examine effects of blocking antibodies for TNF-α and IFN-β on NRVM necroptosis, anti-TNF-α (rat TNF-α antibody, R&D Systems, Minneapolis, MN, U.S.A., AF-510-NA) and anti-IFN-β (Bioss Antibodies Inc., Woburn, MA, U.S.A., Bs-0787) were added into the conditioned medium at 2.5 µg/mL, respectively.

Fig. 1. Experimental Protocol for Preparing Cardiac Fibroblast Conditioned Medium

Neonatal Rat Ventricular Cardiomyocytes Culture and Necroptosis Induction

The culture procedure for ventricular cardiomyocytes prepared from neonatal rats (NRVMs) was described previously.^29,36) Induction of necroptotic cell death was performed as previously reported.^10,11) NRVMs were cultured in DMEM containing 1% (v/v) FBS supplemented with 20 µmol/L pancaspase inhibitor Z-Val-Ala-Asp(Ome)-CH₂F (Z-VAD, Peptide Institute, Inc.) for 1 h, followed by incubation in DMEM containing 1% (v/v) FBS supplemented with 20 µmol/L Z-VAD and 20 ng/mL TNF-α (PeproTech, Rocky Hill, NJ, U.S.A.) or cardiac fibroblast conditioned medium supplemented in the presence of 20 µmol/L Z-VAD.

Cell Death Assay

Cell death assay was performed as described above using an Apoptotic, Necrotic, and Healthy Cells Quantification Kit (Biotium, Inc., Fremont, CA, U.S.A.).^10,11) The cells were stained 24 h after the addition of Ang II, TNF-α, or cardiac fibroblast conditioned medium. Then, stained cells were observed with a microscope digital camera (DP80, Olympus, Tokyo, Japan).

Western Blotting and Detection of Proteins

Samples for Western blotting were prepared using the method described previously.^36,37) Viable left ventricular tissues isolated at 8 weeks after surgery were used. For in vitro experiments, NRVMs were harvested 8 h after the addition of Ang II, TNF-α, or cardiac fibroblast-conditioned medium. Cardiac fibroblasts were harvested 48 h after the addition of Ang II to the culture medium.

Western blot analysis was performed according to the method described previously.^10,11) In the present study, ECL™ Prime (Cytiva, Tokyo, Japan) or ECL Select™ (Cytiva) was used for chemiluminescent substrates. Blotting images were acquired using a Fusion SOLO 6S EDGE (Vilber Lourmat, Marne-la-Vallée, France). Each protein was labeled with anti-phospho-MLKL (Ser358, Affinity Biosciences, Cincinnati, OH, U.S.A., AF7420, 1 : 1000), anti-MLKL (Affinity Biosciences, DF7412, 1 : 1000), anti-TNF-α (Genetex, Irvine, CA, U.S.A., GTX110520, 1 : 500), anti-IFN-β (Bioss Antibodies Inc., Bs-0787, 1 : 500), or anti-actin (Sigma-Aldrich, A4700, 1 : 3000) antibodies for detection.

Immunostaining

The method of immunostaining of the cardiac tissue section was previously described.³⁶⁾ anti-phospho-MLKL (Ser358, Affinity Biosciences, AF7420, 1 : 50) antibody was used as the primary antibody. Cy3-conjugated anti-rabbit immunoglobulin G (IgG) (Vector Laboratories, Newark, CA, U.S.A.) was used as a secondary antibody. Cell membranes and nuclei were stained with fluorescein isothiocyanate (FITC)-conjugated wheat germ agglutinin (FITC-WGA, Vector Laboratories) and 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich), respectively. To prevent autofluorescence, sections were treated with the TrueVIEW^R autofluorescence quenching kit (Vector Laboratories).

Statistics

The results are presented as the mean ± standard error of the mean (S.E.M.). Statistical significance was assessed by ANOVA followed by Tukey’s multiple comparison test using JMP Pro 17 software (JMP Statistical Discovery LLC., Cary, NC, U.S.A.). p < 0.05 was determined to be significant.

RESULTS

Effects of Tra and Azi on Cardiac Parameters in CAL Rats

Figure 2 shows the tissue and echocardiographic parameters of CAL and Sham rats treated with and without Tra or Azi. The vehicle-treated CAL rats weighed approximately 90% of the Sham rats (Fig. 2A). The heart weight/body weight ratio (HW/BW) of vehicle-treated CAL rats was increased to approximately 140% that of Sham rats (Fig. 2B). On the other hand, the increase was attenuated by the administration of Tra or Azi (Fig. 2B). The infarct size in all CAL groups was similar (Fig. 2C). Echocardiographic parameters were measured under similar heart rate conditions (Fig. 2D). The left ventricular internal dimension at end diastole (LVIDd, Fig. 2E) and the left ventricular internal dimension at end systole (LVIDs, Fig. 2F) of the vehicle-treated CAL rats were approximately 10.08 and 8.90 mm, respectively. These values for the Tra-treated CAL rats were approximately 9.50 and 7.97 mm, and those for the Azi-treated CAL rats were approximately 9.03 and 7.52 mm. Left ventricular fractional shortening (Fig. 2G), a measure of cardiac contractility calculated from LVIDd and LVIDs, was approximately 11.8, 17.0 and 17.5% in vehicle, Tra-, and Azi-treated CAL rats, respectively. E/A ratio, a measure of cardiac diastolic function, (Fig. 2H) of the vehicle-, Tra-, and Azi-treated CAL rats were approximately 2.21, 1.83, and 1.80, respectively. The cardiac output (CO) index values (Fig. 2I) of the vehicle-, Tra-, and Azi-treated CAL rats were approximately 290, 405, and 396 µL/g/min, respectively. These results show that administration of Tra or Azi to CAL animals attenuates the development of heart failure in the animals with myocardial infarction used in this study.

Fig. 2. Tissue and Echocardiographic Parameters of CAL and Sham Rats Treated with and without Tra or Azi

(A) Body weight (BW), (B) heart weight/body weight ratio (HW/BW), (C) infarct size, (D) heart rate, (E) left ventricular internal dimension at end diastole (LVIDd), (F) left ventricular internal dimension at end systole (LVIDs), (G) left ventricular fractional shortening, (H) E/A ratio, (I) cardiac output (CO) index of the CAL (closed column) and Sham rats (open column) at the 8th week (8w) after the operation. Each value represents the mean ± S.E.M. of 8 animals (* p < 0.05).

Effects of Tra and Azi on p-MLKL and MKLK in CAL Rats

Figure 3 shows the changes in p-MLKL and MLKL in CAL and Sham rats with and without Tra or Azi. The p-MLKL contents (Figs. 3A, B) of the vehicle-, Tra-, and Azi-treated CAL rats were approximately 175, 115, and 105%, respectively, of that for the vehicle-treated Sham rats. The MLKL contents (Figs. 3A, C) of the vehicle-, Tra-, and Azi-treated CAL rats were approximately 155, 105, and 105%, respectively, of that for the vehicle-treated Sham rats. Figure 3D shows immunofluorescence staining for p-MLKL in myocardial tissues. Myocardial tissues from vehicle-treated CAL rats showed cardiomyocytes stained with red fluorescence indicative of p-MLKL. Furthermore, high-intensity red fluorescence was observed near the cell membrane (green) and around the nucleus (blue), as indicated by the arrowheads. These results suggest that necroptosis induced in failing hearts after myocardial infarction is alleviated by inhibition of Ang II.

Fig. 3. Viable Left Ventricular p-MLKL and MLKL Contents of the Tra- or Azi-Treated CAL (Closed Column) and Sham (Open Column) Rats

(A) Representative Western immunoblot images. (B, C) Semiquantitative values of left ventricular p-MLKL (B) and MLKL (C) contents. (D) Immunohistochemical images of p-MLKL (red), FITC-WGA (green), and nuclei (blue) in left ventricles. P-MLKL-positive spots are indicated by arrow heads. Each value represents the mean ± S.E.M. of 8 animals (* p < 0.05).

Effects of Ang II on Necroptotic Cell Death in NRVMs

In Fig. 4, NRVMs were used to examine whether Ang II directly induces necroptosis in cardiomyocytes. An exposure of NRVMs to Ang II did not induce the rate of necrosis-like cell death in NRVMs (Figs. 4A, B). Similarly, Ang II did not increase the levels of MLKL and its phosphorylated form in NRVMs (Figs. 4C–E). Pretreatment of the cells with Azi also had no effects on the rate of necroptotic cell death. These results suggest that Ang II does not directly induce necroptosis in cardiomyocytes.

Fig. 4. Effects of Ang II (1 µmol/L) on Necroptosis in NRVMs

(A) Representative images of ethidium homodimer III (EthD-III)-labeled nuclei (red, necrotic cell death) and DAPI-labeled total nuclei (blue) of NRVMs. (B) Values of EthD-III-labeled necroptotic cells among the total cells. (C) Representative Western immunoblot images. (D, E) Semiquantitative values of p-MLKL (D) and MLKL (E) in NRVMs. Each value represents the mean ± S.E.M. of 4 different experiments (* p < 0.05).

Effects of Ang II-Stimulated Cardiac Fibroblast-Conditioned Medium on Necroptotic Cell Death in NRVMs

Figure 5 shows the indirect effects of Ang II on cardiomyocyte necroptosis via cardiac fibroblasts. Figures 5A and B show the effects of conditioned medium prepared after culture of cardiac fibroblasts in the presence of Ang II on changes in the ratio of ethidium homodimer-III (EthD-III)-positive ratio in NRVMs. In NRVMs cultured in DMEM containing 1% (v/v) FBS (medium 1), the ratio of EthD-III-positive NRVMs in the presence of Z-VAD and TNF-α, which are known to induce necroptosis, was approximately 50% compared to approximately 1% in the control group. The EthD-III-positive staining ratio in NRVMs cultured in conditioned medium after culturing with cardiac fibroblasts (medium 2) was approximately 1%, similar to that in the control group, and pretreated with Z-VAD was approximately 3%. In contrast, the EthD-III-positive staining ratio was increased to approximately 20% in NRVMs that were cultured with conditioned medium prepared from cardiac fibroblasts in the presence of AngII (medium 3), and this increase in the stained cells was reversed to approximately 6% by pretreatment with Azi (medium 4). Figures 5C–E show the effects of conditioned medium prepared after culture of cardiac fibroblasts in the presence of Ang II on changes in p-MLKL and MKLK contents in NRVMs. In the presence of both Z-VAD and TNF-α, the levels of p-MLKL (Figs. 5C, D) and MLKL (Figs. 5C, E) were increased in NRVMs cultured in DMEM containing 1% (v/v) FBS (medium 1). These contents in NRVMs were also increased by conditioned medium prepared from cardiac fibroblasts in the presence of Ang II (medium 3) in the presence of Z-VAD. The conditioned medium prepared from Azi-pretreated cardiac fibroblasts (medium 4) attenuated the conditioned medium-induced increase in these two protein contents in NRVMs. These results suggest that Ang II induces the release of some humoral factors from cardiac fibroblasts, leading to an induction of cardiomyocyte necroptosis.

Fig. 5. Effects of Conditioned Medium Prepared from Cardiac Fibroblasts in the Presence of Ang II on Necroptosis in NRVMs

(A) Representative images of ethidium homodimer III (EthD-III)-labeled nuclei (red, necrotic cell death) and DAPI-labeled total nuclei (blue) of NRVMs. (B) Values of EthD-III-labeled necroptotic cells among the total cells. (C) Representative Western immunoblot images. (D, E) Semiquantitative values of p-MLKL (D) and MLKL (E) in NRVMs. Each value represents the mean ± S.E.M. of 4 different experiments (* p < 0.05).

TNF-α Released from Cardiac Fibroblasts is Involved in Necroptosis of NRVMs

Figure 6 shows effects of NRVMs on necroptosis by humoral factors released from cardiac fibroblasts by Ang II. Effects of Ang II on TNF-α and IFN-β levels in cardiac fibroblasts are shown in Figs. 6A–C. In the presence of Ang II, the levels of TNF-α (Figs. 6A, B) and IFN-β (Figs. 6A, C) were increased in cardiac fibroblasts cultured in DMEM containing 1% (v/v) FBS. In contrast, these increases in cytokines were attenuated by pretreatment with Azi. Neutralization of conditioned medium with anti-TNF-α antibody prevented an increase in the number of necrotic cell death (Figs. 6D, E) and a rise in p-MLKL and MLKL levels in NRVMs (Figs. 6F–H). On the other hand, neutralization with anti-IFN-β antibody did not significantly attenuate cell death. These results suggest that the induction of cardiomyocyte necroptosis by Ang II is an indirect effect via the release of TNF-α from cardiac fibroblasts.

Fig. 6. Effect on Necroptosis of NRVMs by Humoral Factors Released from Cardiac Fibroblasts

(A) Representative Western immunoblot images of TNF-α and IFN-β. (B, C) Semiquantitative values of TNF-α (B) and IFN-β (C) in cardiac fibroblasts. (D) Representative images of ethidium homodimer III (EthD-III)-labeled nuclei (red, necrotic cell death) and DAPI-labeled total nuclei (blue) of NRVMs. (E) Values of EthD-III-labeled necroptotic cells among the total cells. (F) Representative Western immunoblot images of p-MLKL and MLKL. (G, H) Semiquantitative values of p-MLKL (G) and MLKL (H) in NRVMs. Each value represents the mean ± S.E.M. of 4 different experiments (* p < 0.05).

DISCUSSION

In this study, since inhibition of the renin-angiotensin system induces cardiac rupture in infarcted animals in the early phase post-MI, we started drug administration at the second postoperative week. Cardiac functional and histological parameters at 2 weeks after myocardial infarction have already been shown in our previous reports.^11,38) In the vehicle-treated rats, a marked decrease in cardiac pump function occurred at 8 weeks after MI. In contrast, treatment of animals with Tra or Azi prevented cardiac dysfunction following MI and preserved cardiac function similar to the level at the onset of the drug treatment. In the vehicle-treated group, an increase in the content of MLKL protein, an executioner of necrotic cell death, and a high phosphorylation level of MLKL were simultaneously observed. Since MLKL is activated by its phosphorylation, phosphorylated MLKL forms pores in the plasma and mitochondrial membranes and then induces necrosis-like cell death. In our previous study, we found that the necroptotic signaling pathway is activated in the failing heart with MI in rats¹¹⁾ and with pressure overload in mice.¹⁰⁾ These findings suggest that necroptotic cell death is induced in various types of failing hearts. In this study, myocardial p-MLKL was observed by immunostaining (Fig. 3D). In myocardial tissue from failing hearts in rats, cardiomyocytes showing strong red fluorescence staining of p-MLKL were observed, and within these cells, high intensity red fluorescence was observed near the cell membrane and around the nucleus.

In a previous study²⁸⁾ using mouse kidneys, administration of Ang II with an osmotic pump was shown to increase renal MLKL expression and induce necroptotic cell death. In this study, we examined the effects of renin-angiotensin system inhibitors, which are widely used in therapy for heart failure, on the activation of MLKL. The results showed that Tra or Azi reduced the rise in p-MLKL levels at the 8th week after MI (Fig. 3). These results suggest that an increase in necroptotic cells in myocardial tissues during the development of heart failure following MI was reversed. Furthermore, we found that the increase in MLKL and p-MLKL levels were reversed in the Tra and Azi groups. Therefore, our findings suggest that Ang II, at least in part, contributes to increased MLKL and p-MLKL levels following myocardial infarction.

To examine whether Ang II directly induces necroptosis in cardiomyocytes, NRVMs were cultured in the presence of Ang II (Fig. 4). However, Ang II did not induce necroptosis in NRVMs. Studies^39,40) on the expression pattern of Ang II receptors in cardiomyocytes and cardiac fibroblasts isolated from neonatal rat hearts showed that the Ang II AT₁ receptor expression level in cardiomyocytes is approximately 20% of that in cardiac fibroblasts. Therefore, it has been suggested that the pathophysiological effects of Ang II in myocardial tissue are primarily exerted via the stimulation of cardiac fibroblasts. Therefore, to examine the effects of Ang II on cardiomyocyte necroptosis, NRVMs were cultured in conditioned medium prepared from cardiac fibroblasts in the presence and absence of Ang II. We found that a marked increase in the ratio of EthD-III-positive NRVMs was induced in conditioned medium from cardiac fibroblasts exposed to Ang II (Fig. 4). The conditioned medium-induced increase in EthD-III-stained cells was attenuated by Azi pretreatment (Fig. 4). Therefore, our findings suggest that Ang II is involved in the genesis and/or development of necroptosis in NRVMs via Ang II AT₁ receptor stimulation of cardiac fibroblasts. In support of these results, p-MLKL was increased in NRVMs exposed to conditioned medium prepared from cardiac fibroblasts in the presence of Ang II, and pretreatment with Azi prevented the conditioned medium-induced increase in p-MLKL (Figs. 5A, B).

The results from in vitro experiments suggest that Ang II may induce the release of several humoral factors from cardiac fibroblasts, which contribute to the genesis and/or development of necroptosis in cardiomyocytes. The levels of TNF-α and IFN-β (Figs. 6A–C), which are known to be involved in necroptosis,^41–43) were increased in cardiac fibroblasts exposed to Ang II. Therefore, these cytokines may have induced cardiomyocyte necroptosis under our experimental conditions. Therefore, we examined whether treatment with anti-TNF-α or anti-IFN-β antibodies would inhibit necroptosis of NRVMs in the conditioned medium (Figs. 6D–H). The results showed that anti-TNF-α antibody inhibited necroptosis of NRVMs in the conditioned medium. In patients with heart failure, TNF-α levels are well known to be increased in both of the blood and myocardial tissue.^44,45) On the other hand, clinical trials investigating the effect of TNF-α inhibitors on heart failure have failed to show their efficacy.^46,47) Therefore, it is suggested that inhibition of TNF-α is part of the effect of renin-angiotensin inhibitors on heart failure in this study. Alternatively, the combination of TNF-α inhibitors with existing heart failure medications, including renin-angiotensin inhibitors, may exert a more beneficial effect.

The in vivo experiments in this study could not clearly demonstrate whether Tra or Azi inhibited cardiac necrosis-like cell death via its effect on cardiac fibroblasts. The renin-angiotensin system exerts various pathophysiological effects during the development of heart failure, including increases in cardiac hypertrophy, cardiac fibrosis, fluid retention, and production of inflammatory cytokines. Therefore, the therapeutic effects of renin-angiotensin system inhibition in heart failure may be diverse. The diverse effects of renin angiotensin system inhibitors may result in an attenuation of signal pathways in necroptosis. Therefore, effects of renin-angiotensin system inhibitors on myocardial necroptosis in failing hearts require further investigation.

Several investigators have been reported that necroptosis may involve in myocardial cell death during the acute phase after myocardial infarction.^48,49) Simultaneously, the blood concentration of Ang II is also increased during the acute phase after myocardial infarction.⁵⁰⁾ However, since no studies have yet clarified the relationship between changes in Ang II and necroptotic cell death, the contribution of necroptosis to compensatory mechanisms for cardiac dysfunction in the acute phase after myocardial infarction is still unknown. Pathophysiological roles of necroptosis in the development of contractile dysfunction after myocardial infarction is further needed to be elucidated.

CONCLUSION

In this study, we investigated the involvement of Ang II in necroptosis during the development of chronic heart failure. The results from this study suggest that Ang II enhances necroptosis by increasing MLKL expression and phosphorylation in the myocardium after MI. Furthermore, we showed an indirect mechanism mediated by cardiac fibroblasts in this study. Our findings suggest a new pathophysiological role of Ang II during the development of heart failure.

Funding

This work was supported by JSPS KAKENHI Grant number: JP22K15297 (Tetsuro Marunouchi, the first author).

Conflict of Interest

The authors declare no conflict of interest.

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