Resveratrol reduces cardiac NLRP3-inflammasome activation and systemic inflammation to lessen doxorubicin-induced cardiotoxicity in juvenile mice

Abstract

Doxorubicin (DOX) is a very effective anticancer agent that is widely used in pediatric cancer patients. Nevertheless, DOX is known to have cardiotoxic effects that may progress to cardiomyopathy later in life. We have recently shown that cotreatment of resveratrol (RES) with DOX in juvenile mice attenuates late-onset hypertension-induced cardiomyopathy. However, the molecular mechanism responsible for these changes remains unknown. Herein, we show that the cardiac NLRP3 inflammasome plays a crucial role in regulating cardiac injury in a DOX -treated juvenile mouse model and the detrimental effects of hypertension in these mice later in life. We further demonstrate that RES significantly reduces systemic inflammation to contribute to the improvements observed in DOX -induced cardiac injury in young mice and late-onset hypertension-induced cardiomyopathy.

Anthracyclines, such as doxorubicin (DOX), are very effective anticancer agents that are widely used in pediatric cancer patients [1]. Nevertheless, anthracyclines are known to have subclinical cardiotoxic adverse effects that can go undiagnosed but lead to cardiomyopathy later in life [1]. Surprisingly, these subclinical cardiotoxic effects are rarely investigated in preclinical animal models. Therefore, there is an obvious clinical need to understand how the anticancer treatments damage the heart and then devise treatments that may protect the pediatric population from adverse cardiac outcomes that can occur later in life. To this end, we have recently developed a mouse model of anthracycline-induced cardiotoxicity in juvenile mice using clinically relevant doses of DOX [2]. Utilizing this model, we previously demonstrated that DOX treatments in juvenile mice increase the susceptibility of these mice to the damaging effects of angiotensin II (Ang II)–induced hypertension later in life [2]. In addition, we showed that cotreatment of juvenile mice with resveratrol (RES) and DOX attenuates late-onset overt hypertension-induced cardiomyopathy [2]. However, the molecular mechanism responsible for the pathophysiological changes in early and delayed DOX-induced cardiotoxicity remains largely unknown.

Growing evidence suggests a central role of the cardiac nucleotide-binding domain-like receptor protein-3 (NLRP3) inflammasome and systemic inflammation in the development of myocardial damage induced by anthracyclines such as DOX [3–5]. In some instances, reducing activation of the cardiac NLRP3 inflammasome and systemic inflammation has shown promise in the mitigation of DOX-induced cardiac injury [3,6,7]. Based on this, we investigated whether inflammation is also implicated in early and delayed DOX-induced cardiotoxicity in our juvenile mouse model. Since we have previously demonstrated the protective effects of RES in this model [2], we also tested the hypothesis that RES protects against juvenile DOX-induced cardiotoxicity, as well as late-onset overt hypertension-induced cardiomyopathy through the suppression of inflammation.

Materials and methods

Experimental design and treatment protocol

All protocols involving rodents were approved by the University of Alberta Institutional Animal Care and Use Committee and conform to the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (eighth edition; revised 2011). The University of Alberta adheres to the principles for biomedical research involving animals developed by the Council for International Organizations of Medical Sciences and complies with the Canadian Council on Animal Care guidelines.

Male C57Bl/6N mice were purchased from Charles River Laboratories. All mice were housed under standard conditions (25 °C, 12:12-h light/dark cycle) with ad libitum access to food and water. At 4 weeks of age, mice were randomly assigned into groups and were fed an AIN93G regular chow diet (Dyets Inc., Bethlehem, PA, USA) or AIN93G regular chow diet containing 0.4% RES, a dosage that is equivalent to ~ 320 mg resveratrol·kg⁻¹·day⁻¹, as described previously [2,8]. Importantly, we did not observe any undesirable consequences from supplementing the diets with 0.4% RES, and thus, we assume that mice were treated with a relatively equivalent dose of RES. While we did not quantify the actual RES dose with each animal in this study, we have shown previously that the plasma level of RES with this dose was within the therapeutic values from 10 to 20 μM [9]. The dosage of RES was consistent with previous studies [10,11]. After 1 week of RES treatment, mice were intraperitoneally injected with a low dose of DOX (4 mg·kg⁻¹) or saline (Control) once a week for 3 weeks. The mice were fed chow containing 0.4% RES 1 week before, during, and 1 week after the DOX administrations (Fig. 1). Following a 5-week recovery period at the age of 12 weeks, the mice were infused with Ang II (1.4 mg·kg⁻¹·day⁻¹) for 2 weeks via subcutaneously implanted mini-osmotic pumps (ALZET; Durect Corp, Cupertino, CA, USA) to induce hypertension as described previously [2,8]. Some of the same mice from our previous publication [2] were used in the current study, but measurements and analysis of subsequent molecular pathways are unique to the present study.

Fig. 1.

Scheme of study design for investigating the protective effects of RES on DOX-induced cardiotoxicity in juvenile mice and late-onset overt hypertension-induced cardiomyopathy.

Echocardiography

Transthoracic echocardiography was performed using a Vevo 770 high-resolution imaging system equipped with a 30-MHz transducer (Visual Sonics, Toronto, ON, Canada), as described previously [12].

Blood pressure measurement

Blood pressure was measured by noninvasive tail-cuff method using an IITC blood pressure system (IITC Life Science, Woodland Hills, CA, USA) at 1 week after the last DOX administration and 2 weeks after Ang II infusion, as described previously [2]. Briefly, the mice were maintained at 32 °C and subjected to four cycles of blood pressure measurements after a 15-min acclimation, as we have previously described [13]. Blood pressure was analyzed using PowerLab 4/30 Acquisition and LabChart 7 software (ADInstruments, Colorado Springs, CO, USA).

RNA extraction, cDNA synthesis, and quantification of transcript levels by Quantitative Real-time Polymerase Chain Reaction (real- time PCR)

Total RNA was extracted from 20-mg frozen heart tissue using TRIzol reagent (Invitrogen®, Carlsbad, CA, USA) according to the manufacturer’s instructions as described previously [14,15]. First-strand cDNA synthesis was performed using 5X All-In-One RT MasterMix, according to the manufacturer’s instructions (Applied Biological Materials, Richmond, BC, Canada). Quantification of gene expression was performed by real-time PCR LightCycler® 480 white 384-well reaction plates in the LightCycler® 480 System (Roche Life Science, Mississauga, ON, Canada), as described previously [14,15]. Primer sequences of myosin heavy chain6 (Myh6), Myh7, Nlrp3, thioredoxin-interacting protein (Txnip), interleukin-1β (Il-1β), Il-18, tumor necrosis factor-α (Tnf-α), Il-6, galectin-3, F4/80, Cd68, periostin (Postn), collagen 1a1 (Col1a1), Col4a1 and Rpl32 were summarized in Table S1. While primers were synthesized by Integrated DNA Technologies (IDT, Coralville, IA, USA), the mouse Il-1α primer pair was purchased from Sino Biological (Beijing, China; MP200144). The real-time PCR data were analyzed using the relative gene expression (^ΔΔCt) method, as described previously [14,15].

Histology

Paraffin-embedded hearts were sliced into 5-μm thick sections and used for subsequent histological analyses. Hematoxylin and eosin (H&E) and picrosirius red staining of paraffin-embedded heart sections were visualized using a Leica DMLA microscope (Leica Microsystems, Wetzlar, Germany) equipped with a Retiga 1300i FAST 1394 CCD camera (OImaging, Surrey, BC, Canada) as described previously [2]. Representative images from each sample are shown. For wheat-germ-agglutinin (WGA) staining, sections were treated with WGA conjugated with Alexa Fluor 488 (Thermo Fisher Scientific, Mississauga, ON, Canada); then, nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) using ProLong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific). The cross-sectional area of cardiomyocytes was quantified using imagej software (National Institutes of Health, Bethesda, MD, USA) as described previously [16].

Immunohistochemistry

Paraffin-embedded heart specimens were sectioned in 5 μm thickness and used for subsequent histological analyses. Sections were incubated with a primary antibody directed against galectin-3 (1 : 500; Cedarlane, Burlington, ON, Canada) followed by the appropriate secondary antibody conjugated with Alexa Fluor 594 (1 : 750; Thermo Fisher). Nuclei were stained with DAPI using ProLong Gold Antifade Mountant with DAPI (Thermo Fisher). Images randomly selected fields of the heart were taken.

Measurement of systemic cytokines and chemokine multiplex

Blood samples were collected in evacuated blood collection tubes; then, the samples were allowed to clot by leaving them undisturbed at room temperature for 15 min. Subsequently, the serum was separated by centrifugation of blood samples at 2000 g for 10 min at 4 °C. Following centrifugation, the serum was transferred into clean polypropylene tubes and stored at −80 °C. Systemic cytokines and chemokine multiplex were performed by Eve Technologies (https://www.evetechnologies.com) (Calgary, AB, Canada). Serum IL-18 level was determined using the Abcam IL-18 Assay Kit (Abcam, Cambridge, UK) based on the manufacturer’s instructions.

Statistical analysis

Results are shown as means ± SEM. Statistical analysis was carried out using GRAPHPAD PRISM software (version 7.04) (GraphPad Software, Inc., La Jolla, CA). One-way analysis of variance (ANOVA) followed by Tukey–Kramer’s post hoc multiple comparison test was carried out. A probability value obtained < 0.05 is considered significant.

Results

Resveratrol protects against subclinical DOX-induced cardiotoxicity in juvenile mice and lessens late-onset overt hypertension-induced cardiomyopathy

We examined whether RES cotreatment with DOX could protect against the development of molecular signs of cardiac injury in our juvenile mouse model. To do this, mice were fed a regular chow diet or chow containing 0.4% RES 1 week before, during, and 1 week after the DOX administrations. Consistent with our previous finding [2], while DOX treatment prevented compensated cardiac hypertrophy as demonstrated by the significantly reduced heart weight (HW) despite the elevation of blood pressure (Fig. 2A,E), RES co-administration with DOX restored normal blood pressure and HW in our juvenile mouse model (Fig. 2A,E). However, echocardiography and WGA staining demonstrated that neither DOX nor RES affects the left ventricular posterior wall thickness in diastole (LVPWd) (Fig. 2B,C), LV internal dimension in diastole (LVDd) (Fig. 2B,D) or cardiomyocyte size (Fig. 2F,G), suggesting that our protocol of DOX treatment did not display overt changes in cardiac morphology in mice. Since previous studies have shown that the increase in the relative ratio of Myh7 to Myh6 is a molecular sign of subclinical DOX-induced cardiotoxicity [17,18], we examined whether RES prevented the increase in Myh7 to Myh6 ratio induced by juvenile DOX treatment. Of interest, while DOX upregulated the cardiac expression level of Myh7 (Fig. 2I), RES co-administration with DOX completely normalized Myh7 and upregulated Myh6 in our juvenile mouse model suggesting reduced cardiotoxic injury, at least at the molecular level (Fig. 2H–J).

Fig. 2.

RES protects against subclinical DOX-induced cardiotoxicity in juvenile mice and lessens late-onset overt hypertension-induced cardiomyopathy. (A) Systolic blood pressure. (B) Representative echocardiography motion mode. (C) LVPWd tibia length (TL). (D) LV internal dimension in diastole (LVDd) to TL. (E) HW to TL. (F-G) Representative images and quantification of cardiomyocyte cross-sectional area. (H, I, J) Quantification of transcript levels; (H) Myh6, I: Myh7, and (J) Myh7/Myh6 ratios that were normalized to Rpl32 in control and vehicle or RES-treated DOX mice (n = 10). (K) Systolic blood pressure. (L) Representative echocardiography motion mode. (M) LVPWd to TL. (N) LVDd to TL. (O) HW to TL. (P-Q) Representative images and quantification of cardiomyocyte cross-sectional area. (R, S, T) Quantification of transcript levels; (R) Myh6, (S) Myh7. and (T) Myh7/Myh6 ratios that were normalized to Rpl32 in Ang II and vehicle or RES cotreatment with DOX after 2 weeks of continuous Ang II infusion (n = 6–10). Results are shown as means ± SEM. Comparisons between three groups were made by one-way ANOVA with Tukey–Kramer’s post hoc multiple comparison test. +P < 0.05 vs vehicle control group or Ang II group. *P < 0.05 vs DOX vehicle group or DOX-Ang II group.

In a manner similar to what we have shown previously [2], at 2 weeks after Ang II infusion following a 5-week recovery period (Fig. S1), DOX-treated mice exhibited a lack of cardiac hypertrophic compensation with significantly reduced LVPWd and LVDd and decreased HW and cardiomyocyte size despite the enhanced Ang II-induced hypertension in DOX-treated mice (Fig. 2K–Q). Furthermore, DOX-treated mice following Ang II infusion demonstrated an increased in the cardiac expression level of Myh7 suggesting enhanced cardiac injury (Fig. 2S). Importantly, much earlier cotreatment of RES with DOX restored the hypertrophic compensation, downregulated Myh7, and upregulated Myh6 in Ang II-treated mice (Fig. 2R–T). Collectively, these findings indicate that RES demonstrated a protective effect against DOX-induced cardiac injury in young mice and prevented late-onset overt hypertension-induced cardiomyopathy.

Resveratrol protects against subclinical DOX-induced cardiac fibrosis in juvenile mice and late-onset overt hypertension-induced cardiomyopathy

Since cardiac fibrosis is another major factor shown to induce negative cardiac remodeling [19], we also examined whether RES cotreatment with DOX could protect against the development of cardiac fibrosis in our juvenile mouse model. Using picrosirius red staining of histological cardiac sections and real-time PCR, we found that RES significantly reduced DOX-induced perivascular fibrosis in the heart (Fig. 3A,C) as well as cardiac transcript levels of Postn (Fig. 3F), a hallmark of fibrosis. However, there was no significant difference in intestinal fibrosis and transcript levels of Col1a1 and Col4a1 (Fig. 3B,D,E), suggesting that the dose of DOX administered in this study did not induce overt signs of cardiotoxicity. We also examined whether RES co-administration with DOX at an early age could protect the mice from developing cardiac fibrosis later in life after the administration of Ang II. Interestingly, cotreatment of RES with DOX reduced interstitial and perivascular fibrosis (Fig. 3G–I) as well as transcript levels of Col1a1, Col4a1 and Postn (Fig. 3J–L) in the heart of Ang II-treated adult mice. Overall, these findings indicate that RES ameliorated DOX-induced cardiac fibrosis in young mice and in DOX-treated mice following Ang II treatment later in life.

Fig. 3.

RES protects against subclinical DOX-induced cardiac fibrosis in juvenile mice and late-onset overt hypertension-induced cardiomyopathy. (A) Representative images of formalin-fixed interstitial and perivascular heart sections stained with picrosirius red. (B-C) Quantification of interstitial and perivascular percentage of collagen. (D, E, F) Quantification of transcript levels; (D) Collagen 1a1 (Col1a1) and, (E) Col4a1, (F) Periostin (Postn) that were normalized to Rpl32 in control and vehicle or RES -treated DOX mice (n = 10). (G) Representative images of formalin-fixed interstitial and perivascular heart sections stained with picrosirius red. (H-I) Quantification of interstitial and perivascular percentage of collagen. (J, K, L) Quantification of transcript levels; (J) Col1a1 and, (K) Col4a1, (L) Postn that were normalized to Rpl32 in Ang II and vehicle or RES cotreatment with DOX after 2 weeks of continuous Ang II infusion (n = 6–10). Results are shown as means ± SEM. Comparisons between three groups were made by one-way ANOVA with Tukey–Kramer’s post hoc multiple comparison test. +P < 0.05 vs vehicle control group or Ang II group. *P < 0.05 vs DOX vehicle group or DOX-Ang II group.

The protective effect of resveratrol against early DOX-induced cardiotoxicity and late-onset overt hypertension-induced cardiomyopathy is associated with the suppression of the NLRP3 inflammasome

Since the NLRP3 inflammasome is known to play critical roles in DOX-induced cardiotoxicity [3,6,7] and previous studies have shown that RES can suppress NLRP3 inflammasome activation in the heart [20–22], we hypothesized that RES may suppress the NLRP3 inflammasome and protect against DOX-induced cardiac injury in young mice as well as prevent a late-onset overt hypertension-induced cardiomyopathy. To test this, we measured the effect of RES on the cardiac expression and serum levels of NLRP3 inflammasome markers in DOX-induced cardiac injury in young mice and DOX-treated mice following Ang II infusion. Our results show that juvenile mice administered DOX and DOX-treated mice following Ang II infusion demonstrated an upregulation of the cardiac expression level of the Nlrp3 and Il-18 transcripts (Fig. 4A,D,I,L) and the serum levels of IL-1β, IL-1α, and IL-18 proteins (Fig. 4E,F,G,M,N,O), implicating activation of the NLRP3 inflammasome in DOX-induced cardiac injury in young mice and late-onset overt hypertension-induced cardiomyopathy. However, no significant changes were observed in the cardiac levels of the Il-1β and Il-1α transcripts (Fig. 4B,C,J,K), suggesting that DOX is mainly involved in the activation and the release of IL-1α and IL-1β. Of importance, we also showed that RES reduced the increase in the levels of the Nlrp3, Il-18 transcripts in the heart (Fig. 4A,D,I,L) as well as the serum levels of IL-1β, IL-1α, and IL-18 in juvenile DOX mice and in DOX-treated mice following Ang II infusion (Fig. 4E,F,G,M,N,O). Consistent with the previous observation [23], we also showed that the inhibitory effect of RES on the NLRP3 pathway is associated with the suppression of the cellular redox regulator, Txnip (Fig. 4H), mainly in the hearts of young mice. Overall, these findings indicate that the beneficial effect of RES is mediated, at least in part, by preventing the activation of the NLRP3 inflammasome in response to DOX treatment.

Fig. 4.

RES suppresses the NLRP3 inflammasome in early and delayed DOX-induced cardiotoxicity. (A, B, C, D, H) Quantification of transcript levels; (A) Nlrp3, (B) Il-1β, (C) Il-1α, (D) Il-18, (H) Txnip that were normalized to Rpl32. (E, F, G) Quantification of serum level of IL-1. (E) IL-1β, (F) IL-1α, (G) IL-18 in control and vehicle or RES -treated DOX mice (n = 6). (I, J, K, L, P) Quantification of transcript levels; (I) Nlrp3, (J) Il-1β, (K) Il-1α, (L) Il-18, (P) Txnip that were normalized to Rpl32. (M, N, O) Quantification of serum level of IL-1. (M) IL-1β, (N) IL-1α, (O) IL-18 in Ang II, and vehicle or RES cotreatment with DOX after 2 weeks of continuous Ang II infusion (n = 6–10). Results are shown as means ± SEM. Comparisons between three groups were made by one-way ANOVA with Tukey–Kramer’s post hoc multiple comparison test. +P < 0.05 vs vehicle control group or Ang II group. *P < 0.05 vs DOX vehicle group or DOX-Ang II group.

Resveratrol treatment suppresses cardiac and systemic inflammation in early DOX-induced cardiotoxicity and prevents late-onset overt hypertension-induced cardiomyopathy

Given that DOX is known to cause cardiac and systemic inflammation in experimental animal models and patients [24–26], we investigated whether the protective effect of RES in our juvenile mouse model and late-onset overt hypertension-induced cardiomyopathy is also associated with the suppression of cardiac and systemic inflammation. Using H&E staining of cardiac sections, we show that DOX treatment induced infiltration of inflammatory cells into cardiac tissue as well as increased the number vacuolated cardiomyocytes, two markers of inflammatory lesion in the heart (Fig. 5A). However, we also show that RES co-administration with DOX ameliorated inflammatory lesions in the heart in our juvenile mouse model (Fig. 5A). We further confirmed the effect of RES on DOX-induced cardiac inflammation by measuring infiltration of macrophages into cardiac tissue as well as examining the cardiac expression level of macrophage infiltration and cardiac inflammatory markers. Using immunofluorescence and real-time PCR, we found that RES reduced the number of galectin-3-positive cells (Fig. 5B) and significantly downregulated transcript levels of macrophage infiltration markers, galectin-3 and Cd68 (Fig. 5C,E), and the cardiac inflammatory marker, Tnf-α in DOX-treated mice (Fig. 5G).

Fig. 5.

RES treatment suppresses cardiac inflammation in early DOX-induced cardiotoxicity. (A) Representative images of formalin-fixed heart sections stained with H&E. (B) Repres entative images of heart sections with immuno-staining for galectin-3. (C-G) Quantification of transcript levels. (C) Galectin-3, (D) F4/80, (E) Cd68, (F) Il-6, (G) Tnf-α that were normalized to Rpl32 in control and vehicle or RES -treated DOX mice (n = 10). (F) Representative images of formalin-fixed heart sections stained with H&E. (G) Representative images of heart sections with immuno-staining for galectin-3. (H, I, J, K, L) Quantification of transcript levels. (H) Galectin-3, (I) F4/80, (J) Cd68, (K) Il-6, (L) Tnf-α that were normalized to Rpl32 in Ang II and vehicle or RES cotreatment with DOX after 2 weeks of continuous Ang II infusion (n = 6–10). Results are shown as means ± SEM. Comparisons between three groups were made by one-way ANOVA with Tukey–Kramer’s post hoc multiple comparison test. +P < 0.05 vs vehicle control group or Ang II group. *P < 0.05 vs DOX vehicle group or DOX-Ang II group. Yellow big arrow indicates infiltration of inflammatory cells, and white small arrow indicates the vacuolated cardiomyocyte.

Of importance, 2 weeks after Ang II infusion, DOX-treated mice exhibited a substantial increase in the infiltration of inflammatory cells and the number vacuolated cardiomyocytes (Fig. 5F). Moreover, DOX-treated mice following Ang II infusion demonstrated an increased infiltration of macrophages into cardiac tissue as indicated by the upregulation of galectin-3-positive cells (Fig. 5G). Additionally, there was a significant induction of cardiac transcript levels of macrophage infiltration markers, galectin-3, F4/80, and Cd68 (Fig. 5H–J), in addition to the upregulation of cardiac inflammatory markers, Il-6 and Tnf-α (Fig. 5K,L) in these mice. Interestingly, co-administration of RES with DOX in young mice abolished the effect of Ang II on cardiac inflammation later in life (Fig. 5F–L). Overall, these findings suggest that the beneficial cardiac effect of RES in early and delayed DOX-induced cardiotoxicity is associated with reducing macrophage infiltration in the heart and cardiac inflammation.

In addition to cardiac inflammation, measures of systemic cytokines and chemokines revealed that DOX increased the serum levels of TNF-α, IL-5, IL-6, IL-20, CXCL9, and CXCL10 (Fig. 6A–F) and decreased the serum level of an anti-inflammatory cytokine, erythropoietin, in young mice (Fig. 6H). Notably, 2 weeks after Ang II infusion later in life, DOX-treated mice exhibited an upregulation of serum levels of TNF-α, IL-5, IL-20, CXCL9, CXCL10, and CCL22 (Fig. 6I,J,L,M,N,O), and a decrease in the serum level of erythropoietin (Fig. 6P). Together, these findings suggest that early and delayed DOX-induced cardiotoxicity was associated the activation of systemic inflammation. Interestingly, RES suppressed the serum levels of TNF-α, IL-5, IL-6, and CXCL10 (Fig. 6A,B, C,F) in DOX-induced cardiac injury in young mice and abolished the effect of Ang II on serum cytokine and chemokine levels later in life (Fig. 6I,J,L,M,N,O, P). Collectively, these data provide evidence that RES may reduce cardiac injury and late-onset overt hypertension-induced cardiomyopathy through the suppression of cardiac and systemic inflammation (Fig. 7).

Fig. 6.

RES treatment suppresses systemic inflammation in early DOX-induced cardiotoxicity. (A-H) Quantification of circulating cytokines and chemokines in the blood of mice. (A) TNF-α, (B) IL-5, (C) IL-6, (D) IL-20, (E) CXCL9, (F) CXCL10, (G) CCL22, and (H) erythropoietin (Epo) in control and vehicle or RES -treated DOX mice (n = 6). (I-P) Quantification of circulating cytokines and chemokines in the blood of mice. (I) TNF-α, (J) IL-5, (K) IL-6, (L) IL-20, (M) CXCL9, (N) CXCL10, (O) CCL22, and (P) Epo in Ang II with vehicle or RES cotreatment with DOX after 2 weeks of continuous Ang II infusion (n = 6–10). Results are shown as means ± SEM. Comparisons between three groups were made by one-way ANOVA with Tukey–Kramer’s post hoc multiple comparison test. +P < 0.05 vs vehicle control group or Ang II group. *P < 0.05 vs DOX vehicle group or DOX-Ang II group.

Fig. 7.

Schematic of the anti-inflammatory effects of RES and the protection against early and delayed DOX-induced cardiotoxicity.

Discussion

As subclinical cardiotoxic effects of DOX are rarely investigated in preclinical juvenile animal models, we exposed mice to a low dose of DOX at the age that is relevant to the age of the pediatric cancer population. Importantly, while the dose of DOX administered in this study did not result in overt signs of cardiotoxicity, the DOX-induced cardiotoxicity was clearly revealed in response to Ang II-induced hypertension later in life. Thus, we assumed that the DOX-induced subclinical cardiotoxicity remains undetected (akin to clinically silent in humans) at young ages but that changes at the molecular level make the heart more vulnerable to insults occurring later in life. In addition, we demonstrated that cotreatment of juvenile mice with RES and DOX attenuates late-onset overt hypertension-induced cardiomyopathy [2]. However, the molecular mechanism responsible for the pathophysiological changes in early and delayed DOX-induced cardiotoxicity remains largely unknown.

In the present study, we show that the NLRP3 inflammasome is implicated in the pathophysiological changes in early DOX-induced cardiotoxicity and late-onset overt hypertension-induced cardiomyopathy. Our findings support the hypothesis that the secretion of pro-inflammatory cytokines such as IL-1β, IL-1α, and IL-18 as a consequence of the activation of the NLRP3 inflammasome may contribute to the pathogenesis of chemotherapy-induced cardiac injury, and conversely, that inhibition of NLRP3 inflammasome may be an approach to lessening and/or treating chemotherapy-induced cardiotoxicity [3,6,7,27]. In agreement with this hypothesis, we also provide evidence suggesting that the beneficial effects of RES on subclinical DOX-induced cardiotoxicity in juvenile mice and the detrimental effects of Ang II on these mice later in life were associated with reducing the activation of the cardiac NLRP3 inflammasome. This finding is consistent with recent reports showing that RES exhibits anti-inflammatory effects and suppresses the NLRP3 inflammasome in numerous animal models of myocardial injury [20–22]. These findings are also in agreement with our previous work showing that reducing the activation of the NLRP3 inflammasome plays a role in treating cardiac injury [28]. Thus, it is likely that RES may contribute to reducing cardiac injury in our juvenile mouse model and lessening late-onset overt hypertension-induced cardiomyopathy by reducing the NLRP3 inflammasome-mediated signaling pathway.

The present study also sheds light on the contribution of systemic inflammation to both DOX-induced myocardial injury in young mice and the susceptibility of DOX administration to the detrimental effects of hypertension later in life. This finding is congruent with the concept that systemic inflammation plays a vital role in chemotherapy-induced cardiotoxicity [24–26] and the progression of cardiac disease later in life [29]. Given the fact that: (a) the activation of the NLRP3 inflammasome and the resulting secretion of IL-1 in response to cardiac injury causes systemic inflammation [30,31], and (b) DOX failed to induce systemic inflammation in IL-1 receptor-deficient mice [25], we speculate that the activation of systemic inflammation in our juvenile DOX model is attributed to, at least in part, the activation of the NLRP3 inflammasome and IL-1 secretion. Equally important is that RES reduces systemic inflammation in the juvenile DOX-treated mice and abolishes the effect of Ang II on cardiac and serum cytokine and chemokine levels in these mice later in life. However, it is unclear if this effect of RES on systemic inflammation is due to the anti-inflammatory effect of RES [32,33] or results from a direct effect on the NLRP3 inflammasome [20–22]. Nevertheless, our findings demonstrated that the beneficial cardiac effect of RES in early and delayed DOX-induced cardiotoxicity is associated with reducing inflammation.

A limitation of this study is that it is unclear how RES demonstrated a long-lasting protection against overt-onset hypertension-induced cardiomyopathy in adult mice pretreated with DOX at a young age. However, we found that RES treatment of young mice at the time of DOX administration protects against DOX-induced cardiac and systemic inflammation and thus prevented detrimental Ang II-induced cardiac effects later in life. This finding suggests that the anti-inflammatory effect of RES reduces occult DOX-induced cardiotoxicity in young mice and thus allowed the DOX-treated mice to more appropriately respond to adult-onset hypertension. In support of this, RES demonstrated anti-inflammatory effects and confers a long-lasting protection in several different organisms [20–22,32–34]. Collectively, our findings suggest that targeting inflammation may have significant clinical utility in pediatric cancer patients receiving DOX to prevent cardiac complications later in life.

In summary, our results indicate that increased cardiac NLRP3 inflammasome activity and systemic inflammation play a crucial role in DOX-induced cardiotoxicity in juvenile mice and contribute to the detrimental effects of Ang II on these mice later in life. We also show that the beneficial effect of RES is mediated by reducing cardiac NLRP3 inflammasome activation and systemic inflammation (Fig. 5). Given that: (a) there is a lack of novel pharmacological targets that can mitigate the detrimental cardiac effects of DOX without reducing its antitumor activity [35] and (b) the NLRP3 inflammasome and systemic inflammation are implicated in cancer proliferation, metastasis, and chemoresistance of tumor cells to DOX [36,37], strategies that target the NLRP3 inflammasome and systemic inflammation, such as administration of RES, may help mitigate the cardiotoxic effect of DOX while maintaining and/or even enhancing its anticancer activity.

Supplementary Material

Supporting Materials

Fig. S1. DOX treatment lacks overt cardiotoxicity in mice at 5 weeks after the last injection.

Table S1. Primer’s sequence used for qRT- PCR reactions.

Results S1. DOX treatment lacks overt cardiotoxicity in mice at 5 weeks after the last injection.

Abbreviations

Ang II

angiotensin II

Col

collagen

DAPI

4′,6-diamidino-2-phenylindole

DOX

doxorubicin

H&E

hematoxylin and eosin

HW

heart weight

IL

interleukin

LVDd

left ventricular internal dimension in diastole

LVPWD

left ventricular posterior wall thickness in diastole

NLRP3

nucleotide-binding domain-like receptor protein-3

Postn

periostin

Real-time PCR

quantitative real-time polymerase chain reaction

RES

resveratrol

TL

tibia length

Tnf-α

tumor necrosis factor-α

Txnip

thioredoxin-interacting protein

WGA

wheat-germ-agglutinin

Data accessibility

The data that support the findings of this study are available in the figures and the Supporting information of this article.