Wild type C57BL/6 mice (male, 8 weeks old) were provided by the Experimental Animal Center of Nantong University (Nantong, China). RIPK3^–/– mice were donated by the Institute of Molecular Medicine, Peking University (Beijing, China). The animals were housed in the Experimental Animal Center of Nantong University at 20°C with a 12-h light/dark cycle, and the mice were fed a standard laboratory diet and tap water. All animal-related procedures complied with the recommendations of the “Guidelines for the Care and Use of Laboratory Animals” (approval number: NTU-20161225) issued by the National Institutes of Health and the Animal Care and Use Steering Committee of Nantong University.

Mice were randomly divided into WT mice group (WT), WT mice receiving TAC (26) treatment group (WT + TAC), RIPK3^–/– mice group (RIPK3^–/–), and RIPK3^–/– mice receiving TAC treatment group (RIPK3^–/– + TAC). After 1 week of adaptive feeding, the mice in the model group underwent TAC after overnight food deprivation. Briefly, the mice were intraperitoneally injected with chloral hydrate and depilated after anesthesia. First, the neck skin was cut along the midline, followed by intubation. Next, the sternum was cut to expose the thymus, the thymus was separated, and then the aortic arch was identified and narrowed with a 28-g needle. The mice in the control group received sham surgery. All groups of mice were given continuous feeding for 6 weeks.

To evaluate the role of RIPK3 in HF, male C57BL/6 mice (8 weeks old) were transfected with Adeno-Associated Virus (AAV)-vector and AAV-RIPK3 shRNAs through the tail vein. The injection dose was 1 × 10^11vg per mouse once, and then TAC operation was performed to establish a mouse model of HF. The operation method was the same as those mentioned above. In addition, all groups of mice were given continuous feeding for 6 weeks.

At 6 weeks after the operation, the mice were anesthetized with 1.5% isoflurane, the left chest hair was removed, and the heart geometry was measured at a probe frequency of 30 MHz from the parasternal long-axis view using a small animal color ultrasonic diagnostic device. The cardiac morphology and function were examined by two-dimensional guided M-mode echocardiography (visual sonic VEVO 2100, Toronto, ON, Canada). The thickness of the interventricular septum (IVS) and left ventricle posterior wall (LVPW) was determined. Subsequently, the ejection fraction (EF) and LV fractional shortening (FS) were calculated based on the average of 10 cardiac cycles. All indexes were measured three times in a row.

The left ventricles were fixed overnight in 4% paraformaldehyde, embedded in paraffin, and then cut into 5-μm sections. The sections were subjected to Hematoxylin and Eosin (H&E) staining and dehydrated with ethanol. Subsequently, the sections were observed and photographed under an optical microscope.

The left ventricles were fixed overnight in 4% paraformaldehyde, embedded in paraffin, and then cut into 5-μm sections. After staining with TdT-Mediated dUTP Nick-End Labeling (TUNEL) (Beyotime, Shanghai, China) at 37°C for 60 min, the sections were washed three times with PBS, then observed, and photographed under an optical microscope. The quantification was performed using ImageJ software.

The left ventricles were fixed overnight in 4% paraformaldehyde, embedded in paraffin, and then cut into 5-μm sections. After staining with Weigert’s iron hematoxylin staining solution and then drip staining with Sirius scarlet staining solution, the sections were conventionally dehydrated to transparent, sealed with neutral gum, and photographed under a microscope.

The left ventricles were fixed overnight in 4% paraformaldehyde, embedded in paraffin, and then cut into 5-μm sections. The paraffin-embedded sections were deparaffinized in water, chromated with potassium dichromate overnight, stained with iron hematoxylin, ponceau acid fuchsin, phosphomolybdic acid, and aniline blue sequentially, then dehydrated, mounted, and examined under a microscope.

The left ventricles were fixed overnight in 4% paraformaldehyde, embedded in paraffin, and then cut into 5-μm sections. Next, the paraffin-embedded sections were deparaffinized in water. After antigen retrieval, the WGA working solution was added dropwise, followed by incubation for 30 min. Subsequently, the sections were stained for nuclear mounting and examined under a microscope.

Before sacrifice, whole blood was collected from the orbital vein of each mouse and centrifuged at 3,000 g for 20 min. Serum was kept at −80°C for other analyses. Corresponding commercial kits were used to detect the lactate dehydrogenase (LDH) activity and the contents of creatine kinase (CK), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in mouse serum.

Superoxide production in myocardial tissue was detected using dihydroethidium (DHE) staining under a fluorescence microscope. Briefly, myocardial tissue sections (5 μm) were prepared and then incubated (30 min, 37°C) in Krebs-HEPES buffer (mM components: NaCl 99, KCl 4.7, MgSO₄ 1.2, KH2 PO₄ 1.0, CaCl₂ 1.9, NaHCO₃ 25, glucose 11.1, Na HEPES 20; pH 7.4) containing 2 μM DHE in a dark room. The slides were examined with a Nikon TE2000 inverted microscope (Nikon, Tokyo, Japan) at excitation and emission wavelengths of 480 and 610 nm, respectively.

The thiobarbituric acid method (Beyotime) was used to detect the level of malondialdehyde (MDA) in the myocardium. The total antioxidant capacity (T-AOC) of the myocardium was measured by the T-AOC detection kit using the plasma iron reduction capacity method (Beyotime). The activities of total superoxide dismutase (SOD) in the myocardium were determined using the WST-1 (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-)-2 Htetrazole) method (Beyotime).

The fresh myocardia were cut into three pieces (1 mm) and fixed with 4% glutaraldehyde and 1% osmium acid. The samples were dehydrated with acetone, embedded in Epon812, stained with toluidine blue, cut into 70-nm sections, and stained with uranyl acetate and lead citrate. A transmission electron microscope (JEM-1230) was used to examine the ultrastructure of myocardial tissue. A visible image consisting of 15 randomly selected areas per slice was taken to determine the structure and number of mitochondria. The volume of mitochondria was calculated, and the number of mitochondria was counted.

Total RNA was extracted from myocardial tissue using Trizol reagent (Takara, Kyoto, Japan), and then the purified RNA was reversely transcribed into cDNA using PrimeScript™ RT Master Mix Kit (Takara). SYBR Green Fast qPCR mix (Takara) and ABI 7500 real-time PCR system (ABI, Carlsbad, CA, United States) were used to perform Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). 18S was used as a housekeeping gene. Each experiment was performed in triplicate. The relative expressions of target genes were calculated using the 2^–ΔΔCt method.

The proteins extracted from the myocardial tissue were subjected to SDS-PAGE and transferred onto the PVDF membranes (Millipore, Billerica, MA, United States). After blocking with TBST buffer (Tris-HCl 10 mmol⋅L-1, NaCl 120 mmol⋅L-1, Tween-20 0.1%; pH 7.4) containing 5% skimmed milk (v/v) at room temperature for 2 h, the membranes were incubated with primary antibodies at 4°C overnight, including anti-ANP (1:1,000, Abcam, Cambridge, United Kingdom), anti-BNP (1:1,000, Abcam, Cambridge, United Kingdom), anti-ox-CaMKII (1: 1,000, Millipore, Kenilworth, NJ, United States), anti-CaMKII (1: 1,000, Abcam, Cambridge, United Kingdom), anti-caspase 3, anti-cleaved caspase 3, anti-MLKL, anti-p-MLKL, and anti-RIPK1 (1: 1,000, Cell Signaling Technology, Danvers, MA, United States); anti-p-CaMKII (1: 1,000, Thermo Fisher Scientific, Rockford, IL, United States), anti-GAPDH (1: 5,000, Sigma-Aldrich, St. Louis, MO, United States), and anti-β-tubulin. Subsequently, the blots were incubated with horseradish peroxidase (HRP-)-conjugated secondary antibody at room temperature for 1.5 h. The immunoreactive bands were visualized by enhanced chemiluminescence (ECL) (Thermo Fisher Scientific, Rockford, IL, United States). GAPDH or β-tubulin was used as the loading control.

All data were expressed as mean ± standard error (SEM). Comparisons between two groups were conducted by the unpaired Student’s t-test. One-way analysis of variance analysis (ANOVA) and Student-Newman-Keuls (SNK) test statistics were used for comparisons among multiple groups. P < 0.05 was considered statistically significant.

Echocardiography showed that compared with the control group, HF mice had diastolic and systolic dysfunctions, the levels of EF, FS, and E/A were decreased (Figures 1A–C), and the expressions of hypertrophy and HF indexes ANP and BNP were significantly increased (Figures 1D,E), indicating that an HF model was successfully established in this study. CaMKIIδ alternative splicing disorder easily promotes cardiomyocyte dysfunction and ultimately leads to heart disease (27). Since there is no specific antibody for CaMKIIδ splice variants, qRT-PCR was used to detect the expressions of CaMKIIδ A, CaMKIIδ B, and CaMKIIδ C at the mRNA level. In HF mice, the expressions of CaMKIIδ A and CaMKIIδ B were significantly decreased, while the expression of CaMKIIδ C was significantly increased (Figure 1H), indicating CaMKIIδ alternative splicing disorder in HF mice. In addition, the oxidation and phosphorylation of CaMKII were also increased in the HF group (Figures 1F,G). Several studies have shown that RIPK3 is involved in necroptosis. Our study confirmed that RIPK3 was significantly increased in the heart of HF mice (Figure 2A). Meanwhile, the expression of RIPK1 downstream of RIPK3 and the phosphorylation of MLKL were also up-regulated in the myocardium of HF mice (Figures 2B,C). Necroptosis is a particular form of cell death. TUNEL staining and the expression of cleaved-caspase 3 (Figures 2D–F) showed significantly more apoptotic cardiomyocytes in HF mice compared with the control group. All these data showed that cardiac dysfunction, CaMKII δ activity, alternative splicing disorder, and apoptosis were enhanced in HF mice.

We used RIPK3^–/– mice to verify the role of RIPK3 in the development of HF. Echocardiography showed that depletion of RIPK3 significantly increased EF and FS in HF mice compared with the control group (Figures 3A–C), indicating that systolic and diastolic functions were improved. By detecting the expressions of ANP and BNP at the protein level, we found that depletion of RIPK3 had no significant effect on hypertrophy and expressions of HF genes in HF mice (Figures 3D,E). The cross-sectional area of mouse cardiomyocytes was determined by WGA staining, indicating that cardiomyocytes of WT and RIPK3^–/– mice were significantly enlarged after TAC operation, and depletion of RIPK3 could significantly reduce the size of cardiomyocytes (Figures 4A,B). H&E staining showed that depletion of RIPK3 could reduce the distortion and disorder of cardiomyocytes in HF mice (Figures 4C,D). To evaluate the degree of myocardial fibrosis in mice, we applied Sirius red staining (Figures 5A,B) and Masson staining (Figures 5C,D). The results showed that depletion of RIPK3 significantly improved the degree of collagen deposition and myocardial fibrosis. After TAC operation, the serum LDH and CK levels of RIPK3^–/– mice were significantly lower compared with WT mice (Figures 6D,E). Depletion of RIPK3 improved the degree of myocardial injury in HF mice. Moreover, we found that the inflammatory level was significantly decreased in the myocardial tissue in the RIPK3^–/– + TAC group (Figures 6F,G), indicating that the myocardial injury was improved to a certain extent. These data suggested that although depletion of RIPK3 could not significantly inhibit the occurrence and development of HF, it could significantly ameliorate the central dysfunction, myocardial injury, myocardial fibrosis, and inflammatory response in HF.

Our results showed that depletion of RIPK3 could reduce the oxidation and phosphorylation of CaMKII in the myocardium of HF mice (Figures 6A,B). The expression of CaMKIIδ was detected by qRT-PCR. We found that depletion of RIPK3 improved CaMKIIδ alternative splicing disorder to some extent (Figure 6C), which alleviated cardiomyocyte disorder. In addition to RIPK3, studies have shown that RIPK1 is also involved in necroptosis, and the phosphorylation of MLKL is an essential effector molecule of necroptosis (28). In the present study, the expression of RIPK3 was significantly increased in the myocardium of HF mice (Figure 7A), while the RIPK1 expression and MLKL phosphorylation were also increased. However, the RIPK1 expression and MLKL phosphorylation were reduced in RIPK3^–/– HF mice (Figures 7B,C). TUNEL staining and the expression of cleaved caspase 3 (Figures 7D–F) showed that apoptosis was significantly improved in RIPK3^–/– HF mice. In addition, oxidative stress plays an essential role in the occurrence and development of heart disease. Reactive oxygen species (ROS) accumulation is the main factor in myocardial necrosis, which may reduce the survival of myocardial cells and aggravate myocardial damage. We selected MDA, T-AOC, and T-SOD kits to evaluate the AOC of mouse myocardial tissue, and the results showed that the myocardial tissue’s ability to scavenge oxygen free radicals was enhanced in RIPK3^–/– mice, and the accumulation of ROS was decreased (Figures 7G–I). We used DHE staining to evaluate the level of ROS in tissues. We found that at 6 weeks after the TAC surgery, the red fluorescence intensity of myocardial tissue in WT mice was increased, while the red fluorescence intensity of myocardial tissue in RIPK3^–/– mice was weaker compared with WT mice, indicating that depletion of RIPK3 reduced the accumulation of ROS in myocardial tissue (Figures 8A,B). The ultrastructure of myocardial mitochondria in the left ventricle of mice with myocardial hypertrophy was observed by a transmission electron microscope. However, in contrast to WT and RIPK3^–/– mice without TAC, mitochondria in the RIPK3^–/– + TAC group were relatively intact and began to exhibit cristae fragmentation, which was much improved compared with the WT + TAC group (Figures 8C,D).

To further evaluate the relative role of RIPK3 in myocardial hypertrophy, we injected recombinant AAV-carrying RIPK3 shRNA directly into the tail vein of mice to interfere with the expression of RIPK3. TAC operation was performed on the pretreated mice, and the control group received sham surgery. Echocardiography showed no significant changes in IVS and LVPW in HF mice after interfering with the expression of RIPK3, while the EF and FS values of this group were increased significantly (Figures 9A–C), suggesting that the systolic cardiac function was improved. The expressions of hypertrophy and HF-related genes (ANP and BNP) in TAC-treated mice were increased significantly (Figures 9D,E). In addition, the cross-sectional area of cardiomyocytes in the model group was increased significantly, while the model group interfered with AAV-RIPK3 shRNA was improved significantly (Figures 10A,B). Meanwhile, H&E staining showed that after interfering with AAV-RIPK3 shRNA, the distortion and disorder of cardiomyocytes were reversed (Figures 10C,D). Sirius red staining (Figures 11A,B) and Masson staining (Figures 11C,D) showed that after interfering with AAV-RIPK3 shRNA, the deposition of central muscle collagen in HF mice was reduced, and the situation of myocardial fibrosis was improved. The levels of CK, LDH, IL-6, and TNF-α in mice were detected by corresponding kits (Figures 12D–G), and we found that myocardial tissue injury and inflammatory response were also reversed, indicating that interference with AAV-RIPK3 shRNA could indeed reduce myocardial injury in HF mice.

After injection of the recombinant AAV through the caudal vein, the oxidation and phosphorylation levels of its downstream necroptosis-related effector CaMKII were inhibited (Figures 12A,B). In addition, the alternative splicing disorder of CaMKIIδ was corrected (Figure 12C). Moreover, due to interference with the expression of RIPK3, the up-regulation of RIPK3 in HF mice was inhibited (Figure 13A). In HF mice, after injection of AAV, the expression of RIPK1, a downstream related protein of RIPK3, and the phosphorylation of MLKL were inhibited (Figures 13B,C). TUNEL staining and the expression of cleaved caspase 3 showed improved apoptosis (Figures 13D–F). In addition, the ability of myocardial tissue to scavenge oxygen free radicals was enhanced, and ROS accumulation was reduced (Figures 13G–I). Besides, DHE staining (Figures 14A,B) and transmission electron microscopy (Figures 14C,D) showed that after RIPK3 interference, the oxidative stress level and mitochondrial ultrastructural abnormalities in HF mice were improved.