Gasdermin D Mediates Endoplasmic Reticulum Stress by FAM134B to Regulate Cardiomyocyte Autophagy and Apoptosis in Doxorubicin-induced Cardiotoxicity

doi:10.21203/rs.3.rs-1749975/v1

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Gasdermin D Mediates Endoplasmic Reticulum Stress by FAM134B to Regulate Cardiomyocyte Autophagy and Apoptosis in Doxorubicin-induced Cardiotoxicity

https://doi.org/10.21203/rs.3.rs-1749975/v1

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published 26 Oct, 2022

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Cardiomyocyte pyroptosis and apoptosis play a vital role in the pathophysiology of several cardiovascular diseases. Our recent study revealed that Gasdermin D (GSDMD) can promote myocardial I/R injury via caspase-11/GSDMD pathway. Furthermore, we also found that GSDMD deletion attenuated myocardial I/R and MI injury by reducing cardiomyocyte apoptosis and pyroptosis. However, how GSDMD mediates cardiomyocyte apoptosis and protects myocardial function remains unclear. We here evaluated that doxorubicin (DOX) treatment resulted in significantly increased apoptosis instead of pyroptosis in cardiomyocytes. Interestingly, GSDMD overexpression promoted cardiomyocyte apoptosis, and GSDMD knockdown attenuated cardiomyocyte apoptosis. Notably, GSDMD overexpression exacerbated DOX-induced cardiotoxicity (DIC) injury, impaired cardiac function in vitro and in vivo, and enhanced DOX-induced cardiomyocyte autophagy. Mechanistically, GSDMD regulated the activity of FAM134B, an endoplasmic reticulum autophagy receptor, by pore formation on the endoplasmic reticulum membrane via its N-terminus, thus activating endoplasmic reticulum stress. In turn, FAM134B interacted with autophagic protein LC3, thus inducing cardiac autophagy, promoting cardiomyocyte apoptosis and aggravating DIC. These results suggest that GSDMD promotes autophagy and induces cardiomyocyte apoptosis by modulating the reaction of FAM134B and LC3, thereby promoting DIC injury. Targeted regulation of GSDMD may be a new target for the prevention and treatment of DIC.

Gasdermin D

Apoptosis

Autophagy

Doxorubicin

FAM134B

Pyroptosis is a novel and cryptic form of programmed cell death, identified as cell swelling until membrane rupture, leading to the release of cell contents, and triggering a pronounced inflammatory response [1, 2]. Gasdermin D (GSDMD) is a protein closely related to pyroptosis [1–4]. According to some recent studies, GSDMD-mediated pyroptosis inhibits apoptosis [5–7], while other studies suggest that GSDMD-mediated pyroptosis is irrelevant to apoptosis [8, 9]. Hence, the manner of cell death mediated by GSDMD remains controversial.

Pyroptosis and apoptosis both occur in myocardial ischemia reperfusion (I/R) and myocardial infarction (MI), and GSDMD plays an important role in I/R, MI and atherosclerosis [10–12]. Specifically, GSDMD inhibition reduces cardiomyocyte pyroptosis in I/R and MI, as well as apoptosis. We have previously demonstrated that cardiomyocyte pyroptosis is regulated by caspase-11/GSDMD pathway under oxidative stress conditions [12]. However, whether GSDMD is involved in cardiomyocyte apoptosis remains unclear, as also its role in Doxorubicin (DOX)-induced cardiotoxicity (DIC).

Doxorubicin, the first anthracycline antitumor drug, is used extensively in a broad range of malignancies in children and adults [13]. Despite its wide clinical application, DOX treatment is largely restricted by its accumulative cardiotoxicity. According to a published retrospective study, the incidence of cardiomyopathy and the subsequent heart failure is high when the cumulative DOX dose used exceeds 550 mg per square meter of body-surface area [14]. However, no definitive evidences for the specific underlying mechanism between DOX and cardiomyocyte pyroptosis were revealed.

According to several studies, DOX affects cardiomyocyte autophagic flux and the autophagosome to autolysosome ratio [15–17]. Autophagy is a conserved process of degradation and recovery of cytoplasmic components. It is regarded as a protective mechanism that maintains cellular homeostasis. However, dysregulation of autophagy may cause cardiac dysfunction, and even lead to irreversible degenerative cardiomyopathy and congestive heart failure [18]. Recent studies indicate that autophagy is initiated at omegasome, a cup structure on the endoplasmic reticulum (ER) [19, 20]. Further, some stimulis that induce ER stress (ERS) also cause autophagy. Degradation of specific ER components, lipid membranes, and large protein aggregates in or at ER occur during ER-phagy [21]. The selective destruction is driven by the recognition of autophagy receptors and their LC3-interactingregion (LIR) domains [22]. ERS triggers non-selective macroautophagy and also directly induces ER-phagy [23]. Hence, there is considerable interest in understanding the role of ER in DIC.

In the present study, we investigated the role of GSDMD in DIC, and the underlying molecular mechanisms. Results show that GSDMD is essential for DIC. Specifically, GSDMD causes ER oxidative stress, activating FAM134B, an ER autophagy receptor, which then interacts directly with LC3, leading to cardiomyocyte autophagy and aggravating DIC. Hence, GSDMD may be a novel target for the prevention and treatment of DIC.

Methods Overview

Molecular signaling events in cardiac tissue and cardiomyocytes were determined by immunoblotting, immunostaining, and RNA sequencing. Cardiac function and autophagy flow were investigated by echocardiography and mRFP-GFP-LC3 probing, respectively. Detailed experimental descriptions are provided in Supplemental Methods.

Data Availability

Upon reasonable request, the corresponding author will provide data to support the results of this study. Please refer to the main resources in Supplemental Material.

Animals

All animal experiments were performed under specific sterile barrier conditions in accordance with institutional guidelines, and the experimental protocols were approved by the Ethics Committee of Animal Experimentation of Fudan University. Detailed descriptions of these experiments are provided in Supplemental Methods.

Statistical Analysis

Statistical analyses were conducted using GraphPad Prism 8.0 software (GraphPad Software, San Diego, CA). The value of P<0.05 was considered to indicate statistical significance. All experiments were performed at least three times. Details of statistical analysis are provided in Supplemental Methods.

GSDMD is Essential for Myocardial I/R Injury and Myocardial Infarction

Cardiac I/R and MI are associated with pyroptosis and apoptosis [10, 12, 24]. We confirmed that in I/R and MI model in mouse, the expression of pyroptosis-related proteins and apoptosis-related proteins was significantly increased, but decreased after GSDMD knockout (GSDMD-KO) (Figure S1A–S1D). Echocardiography verified that GSDMD loss reduced the impairment of cardiac function in I/R or MI mice (Figure S1E–S1H). Importantly, GSDMD deficiency decreased serum lactate dehydrogenase (LDH) release (Figure S1I), oxidative stress [reactive oxygen species (ROS) levels, Figure S1J)], and the number of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive cardiomyocytes (Figure S1K). Taken together, these observations indicated that GSDMD-related pyroptosis and apoptosis play an important role in I/R and MI. Our previous studies have confirmed that GSDMD can induce cardiomyocyte pyroptosis through the Caspase-11/GSDMD pathway. However, how GSDMD mediates cardiomyocyte apoptosis is unknown. Previous studies have shown that DIC model is difficult to induce cardiomyocyte pyroptosis. Therefore, we subsequently investigated whether cardiomyocytes undergo pyroptosis and apoptosis in DIC, and the underlying mechanisms.

DOX Treatment Exacerbates GSDMD mediated Cardiomyocyte Injury

The role of GSDMD mediated-pyroptosis and apoptosis in I/R and MI (Figure S1) indicated that GSDMD participated in the myocardial injury. DIC was also a kind of myocardial injury. To investigate the effect of DOX on cardiomyocytes in vivo, WT mice were injected intraperitoneally with DOX to induce acute cardiotoxicity. Sham mice were injected with normal saline (NS), as a control group. Samples were collected 7 d after DOX treatment. The survival rate, heart weight, and body weight (BW) of DOX-treated mice were lower than those of sham mice (Figure S2A and S2B). Consistently, DOX administration significantly impaired cardiac function (Figure S2C and S2D) and triggered an evident cardiomyocyte contractile dysfunction (Figure S2E and S2F), detected as reduced maximal velocity of shortening/relengthening (+dL/dt), peak shortening (PS), and time to peak shortening (TPS) (all at P<0.001) without affecting time to 90% relengthening (TR90). Moreover, DOX-induced myocardial injury obviously upregulated the expression of cleaved caspase-3 (CC3) and BAX, and decreased the levels of Bcl-2 (Figure S2G and S2H).

We used wheat germ agglutinin (WGA) to visualize cardiomyocyte diameter. Further, since DOX is thought to act as an electron acceptor to generate ROS, we also assessed ROS levels in mouse tissues. WGA staining revealed cardiomyocyte diameter reduction after DOX administration and ROS staining suggested more oxidative stress, as indicated by high fluorescence intensity of dihydroethidium (DHE) in cardiac tissues of the DOX-treated group (Figure S2I). We also used TUNEL assay to analyze left ventricular tissue sections 7 d post DOX administration. Compared with the sham control, the numbers of TUNEL-positive cells were increased in DOX-treated mice (Figure S2J and S2L).

The above results indicated that the DIC model was successfully constructed and DOX administration triggered adverse effects in cardiomyocytes. Finally, we probed GSDMD involvement in DIC. We found that the levels of full-length GSDMD and N-terminus of GSDMD were increased in the DOX-treated group (Figure S2K). Collectively, these observations confirmed that GSDMD plays a role in DIC.

GSDMD Deficiency Alleviates DIC and GSDMD Overexpression Aggravates DIC

To explore the biological function of GSDMD in acute animal models of DIC, we established GSDMD-deficient mice. GSDMD-KO mice and control mice underwent DOX treatment. DOX injection led to a high mortality among WT mice, and GSDMD-KO mice further declined (Fig 1A). Further, the BW reduced lower in DOX-treated KO mice than those in WT mice and DOX-treated KO mice exhibited significantly increased ratios of heart weight, accordingly (Figure S3A and S3B). The DOX-increased serum serum creatine kinase-MB (CK-MB) levels and cardiac troponin T (cTnT) activity were further enhanced in WT mice, compared with the levels in the GSDMD-KO mice (Figure 1B). Further, cardiac function reduction upon DOX injection was significantly improved in GSDMD-KO mice, with an increased ejection fraction (EF) and fractional shortening (FS) in DOX-injected GSDMD-KO mice (Figure 1C, and Figure S3C–S3E). Decreased +dL/dt max and -dL/dt min represent systolic dysfunction and diastolic dysfunction, respectively [25], and we observed that DOX exacerbated cardiac function in WT mice but the effect was not pronounced in KO mice (Figure S3G and S3H). Other contractile indexes displayed a similar tendency (Figure S3F and S3H). Consistently, WGA staining revealed that cardiomyocyte diameter decreased upon DOX treatment, with GSDMD deficiency suppressing the DIC shrinkage (Figure 1C, Figure S3I). Interestingly, LDH levels in WT and KO groups were not significantly different (Figure 1F), indicating low or no pyroptosis in DIC combining with the inapparent protein levels of IL-18 and IL-1β (Figure 1E, Figure S4I). Collectively, these observations demonstrate that GSDMD downregulation mitigates cell death after DOX administration.

Next, we infected adult mice with an adenovirus-associated serotype 9 GSDMD-overexpression construct (AAV9-GSDMD-OE) or an adenovirus-associated normal control construct (AAV-NC) to further verify the role of GSDMD in DOX cardiotoxicity. DOX treatment resulted in increased mortality, cardiac injury, as determined by serum biomarker analysis, and cardiac dysfunction of AAV9-GSDMD-OE mice compared with those in WT mice (Figure 1H–1J, Figure S3P, and Figure S4A). Furthermore, ROS levels in DOX-treated GSDMD-overexpressing mice were enhanced compared with those in the control (Figure 1J, Figure S4E). Compared with the control, the number of TUNEL-positive cardiomyocytes in GSDMD-KO mice treated with DOX was reduced (Figure 1D, Figure S3M), however, this effect was reversed by AAV9-GSDMD-OE infection (Figure 1K, Figure S4H). Further, the levels of apoptosis-related proteins BAX and CC3 were significantly increased in DOX treatment groups, and GSDMD loss attenuated this effect (Figure 1E, Figure S3K and S3L). The decrease in protein levels caused by DOX in GSDMD-KO mice was reversed by GSDMD overexpression (Figure 1L, Figure S4F and S4G). Importantly, cardiomyocyte analysis indicated that the occurrence of cell death was reduced after GSDMD deficiency but significantly increased upon GSDMD overexpression (Figure S6E). In summary, these observations indicated GSDMD involvement in DOX-related cardiomyopathy.

Interestingly, the expression of N-GSDMD was significantly increased after DOX treatment, but the level of cardiomyocyte pyroptosis was not changed. GSDMD deficiency alleviates cardiomyocyte apoptosis whereas GSDMD overexpression aggravates cardiomyocyte apoptosis. Taken together, these results demonstrate that GSDMD-mediated cardiomyocyte apoptosis plays an important role in DIC.

Cardiomyocyte-specific GSDMD Deficiency Alleviates Acute and Chronic DIC

Figure 1 suggested that GSDMD played a vital role in regulating the DOX-associated myocardial injury. Numerous lines of evidence indicate that GSDMD induces cell death [1, 3]. However, whether the same is true in the cardiac context remains unknown. Accordingly, to investigate this, we established mice with cardiomyocyte-specific GSDMD KO (GSDMD-CKO). GSDMD-CKO totally abolished the expression of GSDMD protein (Figure 2M). Cardiac GSDMD deficiency decreased the impairment of DOX-induced myocardial injury. After DOX intervention, compared with the control group, the survival rate of GSDMD-CKO mice was significantly prolonged, BW and heart weight loss were decreased, and the levels of myocardial injury markers CK-MB and cTnT were remarkably decreased (Figure 2A–2C, and Figure 2E). The damage of cardiac function in DOX-treated GSDMD-CKO mice was reduced, with a greater increase in ejection fraction, fractional shortening, and contractile indexes than those seen in control mice (Figure 2D, Figure 2F–2K, Figure S5A). In addition, following DOX treatment, cardiomyocytes in GSDMD-CKO mice were bigger than those in GSDMD^(flox/flox) mice (Figure 2L). ROS staining based on DHE fluorescence revealed that ROS levels in DOX-treated GSDMD-CKO mice were lower than those in GSDMD^(flox/flox) littermates (Figure 2L). Cardiomyocyte apoptosis after DOX administration in GSDMD-CKO mice was reduced compared to that in GSDMD^(flox/flox) mice, as indicated by decreased CC3 and BAX protein levels (Figure 2M and 2O) and the number of TUNEL-positive cells (Figure 2N and 2P).

We next repeated the above experiments in a 6-week model of chronic DOX cardiotoxicity. Consistently with the observations in the acute model, DOX-treated GSDMD-CKO mice had an improved survival rate, and with higher heart weight and BW at the end of the observation period than those in control mice (Figure S5B–S5D). Echocardiography at weeks 2 and 4 did not reveal any significant differences between DOX-treated GSDMD^(flox/flox) and GSDMD-CKO mice, however, cardiac function notably improved by week 6 in CKO mice compared with GSDMD^(flox/flox) DOX-treated controls (Figure S5E and S5F). Western blotting revealed a remarkable reduction of the levels of apoptosis-related proteins in CKO heart after DOX treatment (Figure S5G and S5H). Collectively, these observations indicated that GSDMD KO in the heart is protective against DIC in both, acute and chronic models of DOX cardiomyopathy.

GSDMD Deficiency Causes Apoptosis in Cardiomyocytes and Exacerbates DIC

We next repeated the experiments investigating DIC in vitro, using cultured adult cardiomyocytes. Cardiomyocytes were treated with 1 mM DOX and analyzed over time. Western blotting revealed that GSDMD protein levels, either full-length or its N-terminus, peaked 6 h after DOX treatment and began to decline 12 h after the treatment (Figure 3A). The same changes were observed for apoptosis-related proteins (Figure 3B). We therefore selected 12 h as the time point for the ensuing detailed analysis. Similar effects of GSDMD deletion on DOX-treated damage of cardiomyocytes were accordantly observed as in vivo. GSDMD KO in cardiomyocytes reduced DOX-induced increase of GSDMD N-terminus, CC3, and BAX protein levels (Figure 3C and 3D), and the number of dead cardiomyocytes, as assessed by propidium iodide staining (Figure 3E and 3F). All these effects were reversed by adenovirus-mediated GSDMD overexpression (Figure 3G–3J). Collectively, these observations confirmed that GSDMD causes apoptosis in cardiomyocytes, thus exacerbating DIC.

GSDMD Promotes DOX-induced Myocardial Autophagy

The contribution of autophagy to DOX-induced cardiac injury is well-documented [15]. To evaluate the effect of DIC on autophagy in GSDMD inhibition hearts, we first performed time-point analysis of hearts 1, 3, 5, and 7 d after DOX injection. Western blotting revealed the same trends in the levels of GSDMD and microtubule-associated protein-1 light chain 3-II (LC3-II), a marker of autophagy. Specifically, protein levels began to increase 1 d after DOX intervention, peaked after 3 d, and began to decline after 5 d (Figure 4A and 4B). We then determined the expression of several autophagy-associated genes, including ATGs, Beclin-1, LC3B, and Sqstm 1/p62 genes. The expression of most genes showed a similar trend to that shown on the protein level (Figure 4C), reflecting the response to DOX. Therefore, we selected day 3 as the time of cardiac intervention in mice for subsequent exploration of autophagy.

We quantified the levels of LC3-II protein in mouse heart. While the levels were increased by DOX administration, the increase was reduced in the GSDMD-CKO background, and partially rescued by AAV9-GSDMD-OE infection (Figure 4D and 4E). LC3-II level increase could be associated with the initiation of autophagy or prevention of LC3-II degradation. To distinguish between these two possibilities, we used bafilomycin A1 (BAFA1), a lysosomal inhibitor of late-stage autophagy that blocks the degradation of LC3-II [26]. We observed that LC3-II levels were obviously increased in DOX-treated heart and further increased upon BAFA1 treatment (Figure 4F). However, the increase was much lower in DOX-treated GSDMD-CKO heart than that in the GSDMD^(flox/flox) DOX-treated heart (Figure 4F). Further, the reduced LC3-II levels in GSDMD-deficient heart were rescued by GSDMD overexpression (Figure 4G). These observations suggest that LC3-II accumulation in GSDMD-overexpressing heart and DOX-treated heart is caused by its blocked degradation.

We next used transmission electron microscopy (TEM) to investigate the autophagosomes and autolysosomes in the heart of DOX-treated mice. GSDMD-CKO mice were relatively resistant to DOX injection, with a reduced number of autophagosomes and autolysosomes (Figure 4H). However, a marked increase of autophagosomes and autolysosomes was observed in AAV9-GSDMD-OE DOX-treated mice, compared with control mice (Figure 4I). This indicated that GSDMD promotes DOX-induced myocardial autophagy.

DOX Activates Cardiomyocyte Autophagic Flux and GSDMD Aggravates Myocardial Autophagy Levels in Adult Cardiomyocytes

In vivo studies verified the relationship between GSDMD and autophagy. We next explored whether this effect reproduced in cultured cardiomyocytes. Consistent with the cell experiments described in Figure 3A, the levels of LC3-II protein in DOX-treated cardiomyocytes first increased and then fell, reaching a peak 6–12 h after DOX intervention (Figure 5A and 5B). We therefore chose 12 h as the time point for cardiomyocyte autophagy studies. DOX markedly induced the accumulation of autophagy-associated proteins in the WT group, but the levels were nore reduced in cardiomyocytes isolated from adult GSDMD-KO mice (Figure 5C and 5D). By contrast, LC3-II accumulated in GSDMD-overexpressing cardiomyocytes (Figure 5C and 5E). We concluded that DOX promotes autophagy induction, contributing to a high level of LC3-II expression, with GSDMD enhancing this effect.

To identify the specific stages of autophagic flux induced by DOX, we transfected cardiomyocytes with a tandem double-labeled probe, monomeric red fluorescent protein–green fluorescent protein–LC3 (mRFP-GFP-LC3). The cells were then incubated with DOX for 12 h. Since GFP protein is sensitive to low pH, only red fluorescence is detected upon GFP fluorescence after autophagosome and lysosome fusion, as mRFP fluorescence is stable [26]. This allows the assessment of autophagic flux, distinguishing autophagosomes (yellow) from autolysosomes (red). Accordingly, red puncta are more numerous than yellow puncta upon autophagy induction, with the opposite upon autophagy inhibition.

We observed a remarkable increase in the number of autolysosomes and autophagosomes in DOX-treated WT cardiomyocytes, with the former more significant than the latter (Figure 5F). GSDMD deficiency reduced the autolysosome to autophagosome ratio compared with that in the DOX-treated WT cardiomyocyte group (Figure 5F and 5H), whereas GSDMD overexpression had the opposite effect (Figure 5G and 5H). This suggested that downregulation of GSDMD reduces autophagy. These observations, together with immunoblot analysis (Figure 5A–5E), indicated that DOX treatment promotes autophagy, and that GSDMD overexpression enhances this effect.

To verify the above conclusions, we used BAFA1 to prevent the fusion of autophagosomes with lysosomes. The enhanced expression of LC3-II protein upon DOX treatment further increased in the presence of BAFA1 (Figure 5I–5K). By contrast, as anticipated, LC3-II levels were decreased in GSDMD-KO cardiomyocytes (Figure 5I and 5K), and were significantly increased in GSDMD-overexpressing cells (Figure 5J and 5K). We then repeated the autophagic flux analysis using the mRFP-GFP-LC3 reporter system introduced by AAV infection. BAFA1 drastically reduced the number of autolysosomes in WT, GSDMD-KO-CM, and GSDMD-OE-CM groups compared with the controls, with and without DOX treatment, but increased the number of autophagosomes (Figure 5L–5N). In other words, the autolysosome to autophagosome ratio significantly decreased upon BAFA1 treatment, suggesting that the autophagic flux was inhibited, especially in GSDMD-KO cells. Together with western blot data (Figure 5I–5K), these observations confirmed that BAFA1 induces the aggregation of LC3-II by inhibiting LC3-II degradation at the late stage of autophagy, and that downregulation of GSDMD expression weakens this effect.

GSDMD Deletion Ameliorates DIC by Inhibiting Autophagy

The role of autophagy reduction by GSDMD inhibition was further investigated by using autophagy inhibitor 3-methyladenine (3-MA) and autophagy agonist rapamycin (Rapa) in vivo. 3-MA inhibits the formation of autophagosomes and Rapa is an inhibitor of mechanistic target of rapamycin (mTOR) [27]. As anticipated, intraperitoneal injection of 3-MA and Rapa decreased BW in DOX-treated groups, but the weight loss was more pronounced in Rapa-treated groups and declined to a lesser extent in DOX-treated CKO mice than in other mice, while the heart weight to tibia length (HW/TL) ratio showed the opposite trend (Figure S7A). Cardiac function improved after 3-MA treatment, with an increased ejection fraction and fractional shortening, while Rapa treatment exacerbated cardiac function (Figure S7B, S7E, and S7F). Echocardiography readings for the DOX-treated CKO group were significantly better than those for the control group (Figure S7C). Furthermore, 3-MA markedly decreased the LC3-II levels, regardless of DOX treatment (Figure S7D). Meanwhile, Rapa induced accumulation of LC3-II and apoptosis-related proteins, and fully abolished 3-MA–induced decrease of these protein levels upon DOX administration, these protein levels were slightly decreased in CKO mice (Figure S7D, S7G–S7I). Hence, the downregulation of GSDMD levels in the heart during DIC ameliorates the impairment of autophagy.

RNA-sequencing (RNA-seq) Reveals Potential Target Genes of GSDMD

The above experiments suggest that GSDMD deficiency can play a protective role in the myocardium by inhibiting cardiac autophagy. However, how GSDMD mediates cardiac autophagy remains unclear. To further investigate it, we explored the GSDMD-mediated regulatory genes by using RNA-seq. The analysis revealed a significant change of the expression of ER-related genes between DOX-treated GSDMD CKO mice and control mice (Figure 6A and 6B). ERS can lead to the accumulation of unfolded or misfolded proteins in the ER lumen, and induce autophagy in different ways. Western blot analysis indicated that ERS-related proteins were significantly downregulated after GSDMD inhibition (Figure 6C and 6D), further suggesting that GSDMD may induce cardiac autophagy by regulating ERS. Among these ER injury-related genes, FAM134B might be a special one. FAM134B is a common receptor in ER autophagy, and mediates the recognition and removal of ER by autophagosomes [28, 29]. Downregulation of FAM134B leads to ER-phagy, causing ER expansion, ERS, and cell death [28]²⁸. Real-time PCR and western blotting indicated upregulation of ER-regulatory protein Retreg1 (FAM134B) after DOX intervention and its downregulation during GSDMD deficiency (Figure 6C–6E). We observed that ER marker FAM134B co-located with GSDMD, and presented as positive spots upon immunofluorescence staining (Figure 6F). This revealed that GSDMD and FAM134B were expressed in the cardiomyocyte ER, with FAM134B downregulation upon GSDMD deletion (Figure 6F). GSDMD and LC3 (Figure 6G), FAM134B and Bip (Figure 6H), FAM134B and LC3 (Figure 6I) also have co-location in cardiomyocyte. Taken together, these results suggested that GSDMD may cause cardiac autophagy and cardiomyocyte apoptosis by regulating ER-phagy receptor FAM134B.

GSDMD Promotes Autophagy by Promoting ERS to Activate FAM134B, Thus Aggravating Cardiomyocyte Apoptosis

To determine whether GSDMD plays a role in regulating ERS, we used ERS inhibitor 4-phenylbutyric acid (4PBA) to inhibit ERS and observed changes in cardiac autophagy and cardiomyocyte apoptosis . 4PBA inhibited ERS and autophagy in GSDMD^(flox/flox) mice, and neutralized the effects of GSDMD deficiency on ERS and autophagy (Figure 7D and 7E). Further, 4PBA reduced GSDMD^(flox/flox) mouse mortality (Figure 7A), improved cardiac function (Figure 7B and 7C), and decreased cardiomyocyte apoptosis (Figure 7D and 7E). In vitro data were consistent with the in vivo observation that 4PBA reduces ERS and apoptosis (Figure 7F–7H). These findings confirmed that GSDMD loss inhibits cardiac autophagy, reduces myocardial apoptosis, improves cardiac function, and alleviates DIC by mediating ERS in cardiomyocytes.

Furthermore, the expression of FAM134B in GSDMD^(flox/flox) mice was remarkably downregulated after 4PBA administration, further downregulated in the CKO group (Figure 7D), demonstrating that FAM134B is the key downstream target gene of GSDMD regulating ERS. In summary, the presented results indicate that GSDMD-N activates ERS by promoting ER perforation, stimulating FAM134B to fragment ER, thus promoting autophagy, aggravating cardiomyocyte apoptosis, and ultimately aggravating DIC (Figure 7I).

To the best of our knowledge, the present study provides the first direct evidence that GSDMD is upregulated in DIC, exacerbates cardiac injury, and impairs cardiac function by promoting cardiomyocyte apoptosis and autophagy. Here, we found that GSDMD-N is significantly upregulated in DIC. However, we did not observe any differences in the levels of other pyroptosis indicators, which suggests that pyroptosis is rare in DIC. GSDMD-N attaches to the ER and forms pores, resulting in increased ROS production, which activates the expression of FAM134B and induces ERS. Subsequently, FAM134B binds to LC3 to induce autophagy (Figure 7I). These findings thus reveal the previously undiscovered roles of GSDMD and FAM134B during DIC, and provide novel insights into autophagic regulation mechanisms.

DOX exerts pleiotropic effects in cardiotoxicity via multiple mechanisms, including myocardial apoptosis, autophagy, oxidative stress, mitochondria damage, and Ca⁺ disturbance [30, 31]. Apoptosis is characterized by chromatin condensation, DNA fragmentation, cell shrinkage, and formation of apoptotic bodies [32, 33]. We observed a significant elevation of myocardial apoptosis and deterioration of cardiac function in the constructed DIC models, suggesting that DOX causes apoptosis. Pyroptosis is another form of programmed cell death, with GSDMD playing a key role. In pyroptosis, the inflammasome is activated after cell stimulation, following which activated caspase-1 cleaves the N and C domains of GSDMD. The released N-terminal fragment of GSDMD then recognizes and binds to phospholipid molecules of the cell membrane, and forms holes in the membrane, leading to changes in cell osmotic pressure and, ultimately, cell membrane lysis and pyroptosis [1, 3]. We have previously reported that GSDMD inhibition significantly reduces cardiomyocyte pyroptosis and I/R-induced myocardial injury, and that cardiomyocyte pyroptosis is mainly regulated by caspase-11/GSDMD signaling pathway under oxidative stress [12]. The NF-kB/GSDMD axis is also important in bridging the oxidative stress response and cardiomyocyte pyroptosis after MI [34]. Similar, in the current study, we found that pyroptosis plays an important role in I/R and MI. However, the role of GSDMD in DIC remained elusive. We found that DOX treatment leads to an increase in GSDMD levels, suggesting that GSDMD may be involved in the occurrence of tumor heart disease. Accordingly, GSDMD-KO and AAV9-GSDMD-OE mice were obtained to explore the specific underlying mechanism. We observed that GSDMD deletion alleviates DIC, and that the mortality and cardiac injury increase after GSDMD overexpression in mice. At the same time, GSDMD deficiency did not impact IL-18 and IL-1β levels, indicating that pyroptosis might not be the decisive factor in DIC. Although cardiomyocytes account for a large proportion of heart tissue, which is composed of a variety of cells, we could not exclude experimental interference from other cells. Hence, to investigate whether GSDMD plays a role in DIC, we constructed DIC models in GSDMD-CKO mice. In vitro and in vivo analyses showed that cardiomyocyte-specific GSDMD KO exacerbated apoptosis and DIC.

Autophagy is a process whereby the cell degrades damaged organelles and macromolecules using lysosomes, and is regulated by autophagy-related genes [35]. Previous studies have reported on the relationship between DOX and autophagy, but the issue remains controversial. According to a recent study, DOX blocks cardiomyocyte autophagic flux mainly by driving lysosomal acidification and lysosomal function [15]. However, in a study from the Mayo Clinic, early autophagy flux in adult zebrafish heart after DOX intervention is not blocked but upregulated [36]. How autophagy regulates and protects cardiomyocyte function in DIC is still controversial, and the outcomes mainly depend on the DOX dose and observation time. Considering the lack of clarity on the relationship between autophagy and DOX, we here used autophagic regulatory drugs (BAFA1, 3-MA, and Rapa) to clarify the mechanism regulating autophagy in DIC. Accordingly, 3-MA significantly decreased the expression of LC3-II in DOX-treated mice, while BAFA1 and Rapa significantly increased the levels of LC3-II protein, which confirmed that BAFA1 and 3-MA inhibited autophagy by blocking the autophagosome–lysosome fusion and autophagosome formation, respectively. Further, experiments using the mRFP-GFP-LC3 probe revealed an increase of autophagic flux upon DOX treatment. These observations suggest enhanced cardiac autophagic flow and aggravated cardiac autophagy upon DOX intervention. We also found that autophagy is downregulated by GSDMD deficiency but enhanced by GSDMD overexpression, compared with the control group, suggesting that GSDMD deficiency may play a protective role in the myocardium by blocking myocardial autophagy.

The ER is an organelle in which proteins fold to function. Different pathological conditions associated with ER dysfunction lead to the accumulation of misfolded proteins and activation of a complex mechanism known as the unfolded protein response (UPR), which is mainly regulated by ERK, IRE1α, and ATF6α [37, 38]. These three proteins bind to the protein GRP78 and ER pressure sensor under normal conditions, thus blocking their stimulation. High expression of GRP78 at baseline is considered to be a marker of cellular ER stress [37-39]. Furthermore, enhanced expression of GRP78 in ischemic cardiomyopathy and heart failure was reported [40-42]. We showed here that the expression of GRP78 was significantly upregulated in DIC and the expression of Hsp90, another ERS marker, was also increased. The expression of both these proteins was decreased after GSDMD deletion, indicating that DOX caused ERS and that GSDMD deletion alleviated this effect.

The N-terminal domain of GSDMD specifically binds to phosphorylphosphatidylinositol or cardiolipin, which are unique to eukaryotic and prokaryotic cell membrane, respectively, and forms pores [2]. Since the ER membrane contains phosphatidylinositol, we speculated that GSDMD-N could bind to ER membrane and perforate it, leading to ER oxidative stress. Indeed, TEM analysis confirmed the presence of multiple pores in the ER membrane after DOX intervention. ERS suppression resulted in decreased cardiomyocyte apoptosis and improved mice survival after GSDMD deletion compared with control mice. Our study demonstrated that GSDMD could mediate ER membrane perforation and activate ERS to promote cardiomyocyte apoptosis, while inhibition of ERS could alleviate cardiomyocyte apoptosis.

Finally, ERS can trigger autophagy. ER-phagy, a selective autophagy degradation of the ER, is a regulatory mechanism maintaining ER homeostasis under stress conditions and can reduce ERS [43]. ER-phagy is regulated by several receptors, namely, reticular phagocytic regulator 1 (RETREG1, also known as FAM134B or JK1), reticulon 3 (RTN3), preprotein translocus factor SEC62 and cell cycle progression 1 (CCPG1), testis-expressed gene 264 (TEX264), and Atlastin GTPase3 (ATL3) [44-46]. FAM134B is the first identified selective ER phagocytic receptor protein anchored at the ER, and is considered to be the most characteristic ER phagocytic receptor in mammalian cells. It mediates the recognition and removal of the ER by autophagosomes [28]. According to previous studies, downregulation of FAM134B eliminates ER-phagy, leading to ER expansion, ERS, and cell death of the human nerve cell [28]. Further, FAM134B has two specific domains: reticular homologous domain (RHD, N-terminal) and LIR motif (C-terminal) [28]. LIR binds to LC3/GABARAP family proteins and accumulates autophagy. In the current study, we showed that ER oxidative stress activates FAM134B, which causes ER fragmentation and its LIR motif binding to LC3 to induce autophagy, while inhibiting FAM134B reduces autophagy, thus reducing DIC. We also showed that ERS is caused by GSDMD forming pores in the ER.

Collectively, in the present study, we revealed a new interaction between GSDMD and FAM134B in DIC. On the one hand, GSDMD-N causes ER oxidative stress by forming pores in the ER membrane. Oligomerization of activated FAM134B causes ER membrane fragmentation, which is subsequently recognized and enveloped by autophagosomes after a direct interaction between FAM134B and LC3, further leading to the cardiomyocyte autophagy. On the other hand, DOX directly causes ERS and induces cardiomyocyte pyroptosis. Together, these data suggest that GSDMD may be a potential biomarker and therapeutic target for DIC, and inhibition of GSDMD provides new insights into the treatment of tumor heart disease.

-dL/dt	Maximal velocity of shortening
+dL/dt	Maximal velocity of relengthening
3-MA	3-Methyladenine
4PBA	4-phenylbutyric acid
AAV9	Adenovirus-associated serotype 9
BAFA1	Bafilomycin A1
BW	Body weight
CC3	Cleaved caspase-3
CK-MB	Creatine kinase-MB
cTnT	Cardiac troponin T
DHE	Dihydroethidium
DIC	Doxorubicin-induced cardiotoxicity
DOX	Doxorubicin
ER	Endoplasmic reticulum
ERS	Endoplasmic reticulum stress
GFP	Green fluorescent protein
GSDMD	Gasdermin D
GSDMD-CKO	Cardiomyocyte-specific GSDMD knockout
GSDMD-KO	GSDMD knockout
HW/TL	Heart weight to tibia length ratio
IL	Interleukin
I/R	Ischemia reperfusion
LDH	Lactate dehydrogenase
LIR	LC3-interacting region
LVEF	Left ventricular ejection fraction
LVFS	Left ventricular fractional shortening
MI	Myocardial infarction
mRFP	Monomeric red fluorescent protein
NS	Normal saline
PS	Peak shortening
Rapa	Rapamycin
RHD	Reticular homologous domain
RNA-seq	RNA sequencing
ROS	Reactive oxygen species
TEM	Transmission electron microscopy
TPS	Time to peak shortening
TR90	Time to 90% relengthening
TUNEL	Terminal deoxynucleotidyl transferase dUTP nick-end labeling
UPR	Unfolded protein response
WGA	Wheat germ agglutinin
WT	Wild-type

Acknowledgments

This work was supported by the National Natural Science Foundation (grant No.

81870268 and 82000333), National Key Research and Development Program of China (grant No.2018YFC0116305) and Shanghai Clinical Research Center for Interventional Medicine (grant No.19MC1910300).

Author Contribution Statement

Y.N.Q. and R.F.G. conceived and designed the study and prepared the manuscript. Y.N.Q, R.F.G. and X.W. completed the studies in vivo. Y.N.Q. and R.F.G. completed the studies in vitro. X.L.S., K.Y., H.R.S. and Y.G. helped the studies in vivo. S.Y.H., Y.W.W. and J.E.Yhelped analyze and collect data. All authors edited and collectively approved the final version of this manuscript. J.B.G., F.Z. and A.J.S provided critical suggestions.

Conflict of Interest Statement

The authors declare that they have no competing interests

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SIMATERIAL.docx
SupplememtaryFigure1.tif
Supplementary Figure 1. GSDMD is essential for myocardial I/R injury and myocardial infarction.A,B Western blotting detection and quantification of GSDMD-FL, GSDMD-N, Cleaved-caspase 3, BAX, IL-18, IL-1β, and β-actin proteins expressions in the heart of control and I/R mice. N=6 per group. C,D Western blotting detection and quantification of GSDMD-FL, GSDMD-N, Cleaved-caspase 3, BAX, IL-18, IL-1β, and β-actin proteins expressions in the heart of control and MI mice. N=6 per group. E-H Representative echocardiography photographs depicting left ventricular function in the different groups of mice and analysis of the effects of GSDMD knockout on parameters of cardiac function in I/R and MI mice. N=6-7 per group. I LDH (lactate dehydrogenase) release in cardiomyocytes in I/R and MI groups. N=7-11 per group. J I/R or MI-induced reactive oxygen species formation (by dihydroethidium [DHE] staining). Scale bar, 200 μm. N=3 per group. K Representative images of TUNEL stainings in I/R and MI hearts. Scale bar, 200 μm. N=3 per group. Data are depicted as the mean ± SEM. Statistical significance was determined by one-way and two-way ANOVA with a post-hoc Holm-Sidak test, ns, not significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
SupplememtaryFigure2.tif
Supplementary Figure 2. DOX treatment exacerbates cardiac injury.A Cumulative survival rates of control and DOX-treated groups. N=10 per group. B Heart weight/tibia length ratios (HW/TL) and body weights (BW) were measured at 7 days after DOX administration. N=7-9 per group. C Plasma levels of CK-MB and cTnT, respectively, measured by Elisa assay. N=4-6 per group. D Representative images of echocardiographs and statistics of heart rate (HR), ejection fraction (EF) and fractional shortening (FS). N=10 per group. E,F Resting cell length, maximal velocity of relengthening (+dL/dt), maximal velocity of shortening (-dL/dt), peak shortening (PS, normalized to resting cell length), Time-to-PS (TPS), Time-to-90% relengthening (TR90). G,H Protein levels of Cleaved-caspase 3, BAX, BCL-2 using western blot assay. N=6 per group. I The cardiac myocyte sizes measured by wheat germ agglutinin (WGA) and epifluorescence microscopy of reactive oxygen species (ROS). Scale bar, 100 μm for WGA and 200 μm for ROS. N=11 for WGA groups and n=12 for ROS groups. J Apoptosis of myocardium co-stained with cTnT determined by TUNEL staining. Scale bar, 100 μm. K Protein levels of GSDMD-FL and GSDMD-N using western blot assay. N=6 per group. L The graph of TUNEL staining. N=11 per group. Data are depicted as the mean ± SEM. Statistical significance was determined by one-way and two-way ANOVA with a post-hoc Holm-Sidak test, ns, not significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
SupplememtaryFigure3.tif
Supplememtary Figure 3. Deficiency or overexpression of GSDMD alleviates or aggrevates DOX-induced cardiotoxic injury.A,B Body weights (BW) and heart weight/tibia length ratios (HW/TL) in WT and GSDMD-KO mice. N=8-10 per group. C,D Statistics of heart rate (HR) and fractional shortening (FS) of WT and GSDMD-KO mice treated with DOX or not. N=7-9 per group. E Representative images of echocardiographs of WT and GSDMD-KO mice. F-H Resting cell length, maximal velocity of relengthening (+dL/dt), maximal velocity of shortening (-dL/dt), peak shortening (PS, normalized to resting cell length), Time-to-PS (TPS), Time-to-90% relengthening (TR90). I Cell sizes measured by WGA staining, compared with WT-control group. N=10 per group. J Quantitative data of epifluorescence microscopy of control and DOX-treated hearts in WT and KO mice for ROS. N=11 per group. K,L Analysis of GSDMD (FL and N teminals), Cleaved-caspase 3, and BAX proteins levels in control and GSDMD-KO hearts. N=6 per group. M Quantification of TUNEL-positive nuclei per total nuclei. N=11 per group. N,O Body weights (BW) and heart weight/tibia length ratios (HW/TL) in AAV9-NC and AAV9-GSDMD-OE mice. N=8-9 mice per group. P Representative images of echocardiographs of mice infected with AAV9-NC or AAV9-GSDMD-OE with or without DOX treatment. Data are depicted as the mean ± SEM. Statistical significance was determined by one-way and two-way ANOVA with a post-hoc Holm-Sidak test, ns, not significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
SupplememtaryFigure4.tif
Supplememtary Figure 4. Deficiency or overexpression of GSDMD alleviates or aggrevates DOX-induced cardiotoxic injury.A Analysis of heart rate and fractional shortening in AAV9-NC and AAV9-GSDMD-OE mice. N=8-10 per group. B,C Resting cell length, maximal velocity of relengthening (+dL/dt), maximal velocity of shortening (-dL/dt), peak shortening (PS, normalized to resting cell length), Time-to-PS (TPS), Time-to-90% relengthening (TR90) of AAV9-NC and AAV9-GSDMD-OE mice. D,E Analysis of cell sizes and reactive oxygen species production levels measured by WGA and dihydroethidium (DHE) staining, respectively. N=10 for WGA groups and n=11 for ROS groups. F,G Analysis of GSDMD (FL and N teminals), Cleaved-caspase 3, and BAX proteins levels in AAV9-NC and AAV9-GSDMD-OE mice. N=6 per group. H Quantification of TUNEL-positive nuclei per total nuclei in AAV9-NC and AAV9-GSDMD-OE mice. N=10 per group. I Analysis of IL-1β and IL-18 proteins levels between WT mice and KO mice. N=6 per group. Data are depicted as the mean ± SEM. Statistical significance was determined by one-way and two-way ANOVA with a post-hoc Holm-Sidak test, ns, not significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
SupplememtaryFigure5.tif
Supplememtary Figure 5. Cardiomyocyte-specific GSDMD deficiency alleviates acute and chronic DIC. A Analysis of heart rate and fractional shortening in GSDMD^(flox/flox) and CKO mice. N=6 per group. B Kaplan-Meier survival analysis showing improved survival in CKO mice compared with GSDMD^(flox/flox) mice both in control and DOX-treated groups after 6 weeks follow-up. N=10-17 per group. C Percentages of body weight change at different time point (2W, 4W, 6W after DOX injfection) in four groups. N=6-10 per group. D Heart weight/tibia length ratios (HW/TL) of GSDMD^(flox/flox) and CKO mice 6 weeks after saline or DOX administration. N=8-10 per group. E,F Representative images of echocardiographs of GSDMD^(flox/flox) and GSDMD-CKO mice in baseline and 2W, 4W, 6W after DOX treatment. Statistics of heart rate, ejection fraction and fractional shortening at different times. N=7-10 per group. G,H Representative western blot data and quantitative analysis of Cleaved-caspase 3 and BAX from vehicle-treated control and DOX-treated hearts in the absence and presence of cardiac GSDMD. N=6 per group. Data are depicted as the mean ± SEM. Statistical significance was determined by one-way and two-way ANOVA with a post-hoc Holm-Sidak test, ns, not significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
SupplememtaryFigure6.tif
Supplememtary Figure 6. GSDMD promotes myocardial autophagy levels and GSDMD deficiency alleviates DIC.A,B Analysis of LC3II protein expression in GSDMD^(flox/flox) and GSDMD-CKO or AAV9-NC and AAV9-GSDMD-OE hearts in acute DOX model. N=6 for GSDMD^(flox/flox) and GSDMD-CKO mice group, n=3 for AAV9-NC and AAV9-GSDMD-OE groups. C,D Statistics of LC3II protein expression in different mice groups treated with DOX and/or BafA1. N=3 per group. E Dynamic changes of cardiomyocytes treated with DOX at different time points (0h, 18h, 21h, 23h, 24h). Data are depicted as the mean ± SEM. Statistical significance was determined by one-way and two-way ANOVA with a post-hoc Holm-Sidak test, ns, not significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
SupplememtaryFigure7.tif
Supplememtary Figure 7. GSDMD aggregates DIC by promoting myocardial autophagy.A Body weight (BW) changes and heart weight/tibia length ratios (HW/TL) in GSDMD^(flox/flox) and CKO mice treated with DOX, 3MA, RAPA or not. N=6 per group. B,C,E,F Representative images of echocardiographs with different treatments (DOX, 3MA, RAPA) in GSDMD^(flox/flox) and GSDMD-CKO mice. Statistics of heart rate, ejection fraction (EF) and fractional shortening (FS) in various groups. N=6 per group. D,G-I Representative western blot and analysis of LC3, Cleaved caspase 3, and BAX protein levels in GSDMD^(flox/flox) and CKO mice treated with DOX, 3MA, RAPA or not. N=3 per group. Data are depicted as the mean ± SEM. Statistical significance was determined by one-way and two-way ANOVA with a post-hoc Holm-Sidak test, ns, not significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
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Gasdermin D Mediates Endoplasmic Reticulum Stress by FAM134B to Regulate Cardiomyocyte Autophagy and Apoptosis in Doxorubicin-induced Cardiotoxicity

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