Combined Therapy of Low-Dose Angiotensin Receptor–Neprilysin Inhibitor and Sodium–Glucose Cotransporter-2 Inhibitor Prevents Doxorubicin-Induced Cardiac Dysfunction in Rodent Model with Minimal Adverse Effects

Although cancer-therapy-related cardiac dysfunction (CTRCD) is a critical issue in clinical practice, there is a glaring lack of evidence regarding cardiotoxicity management. To determine an effective and suitable dosage of treatment using angiotensin receptor–neprilysin inhibitors (ARNI) with sodium–glucose cotransporter 2 inhibitors (SGLT2i), we adopted a clinically relevant rodent model with doxorubicin, which would mimic cardiac dysfunction in CTRCD patients. After the oral administration of drugs (vehicle, SGLT2i, ARNI, Low-ARNI/SGLT2i, ARNI/SGLT2i), several physiologic parameters, including hemodynamic change, cardiac function, and histopathology, were evaluated. Bulk RNA-sequencing was performed to obtain insights into the molecular basis of a mouse heart response to Low-ARNI/SGLT2i treatment. For the first time, we report that the addition of low-dose ARNI with SGLT2i resulted in greater benefits than ARNI, SGLT2i alone or ARNI/SGLT2i combination in survival rate, cardiac function, hemodynamic change, and kidney function against doxorubicin-induced cardiotoxicity through peroxisome proliferator-activated receptor signaling pathway. Low-dose ARNI with SGLT2i combination treatment would be practically beneficial for improving cardiac functions against doxorubicin-induced heart failure with minimal adverse effects. Our findings suggest the Low-ARNI/SGLT2i combination as a feasible novel strategy in managing CTRCD patients.

ARNI, a combined drug of valsartan and sacubitril, is currently used for managing patients with heart failure [8]. However, the up-titration of ARNI is largely limited by symptomatic hypotension and can even prompt discontinuation of the drug [9]. Furthermore, we do not have sufficient evidence regarding the management of low blood pressure in concomitant medical therapy, such as ACE inhibitors, β-adrenoreceptor blockers, mineralocorticoid receptor antagonists, diuretics and SGLT2-inhibitors.
SGLT2i, a class of glucose-lowering agents, beneficially improves outcomes in patients with type II diabetes and a worsening heart failure event [10,11]. Quagliariello et al., reported that SGLT2i improves the myocardial strain by reducing fibrosis and proinflammatory cytokine levels in a rodent model of doxorubicin-induced cardiotoxicity [12]. Consistently, SGLT2i straighten up cardiac function by elevating the levels of ketone bodies in the same experimental model [13].
Although ARNI and SGLT2i have been proven to be the drugs of choice in treating heart failure [6,8], the underlying mechanisms of these two classes of drugs remain largely undefined. Efforts to fully define the molecular mechanism of the cardioprotective effect beyond the mode of action are ongoing [14][15][16]. Thus, investigating the interactions between the two drugs, which are known to have different cardioprotective mechanism, is required to develop a novel therapeutic strategy for CTRCD.
A recent retrospective study also showed that a combination of SGLT2i with ARNI was more effective in improving cardiac function and reducing the risks of hospitalization for heart failure and cardiovascular death in diabetic patients with heart failure with reduced ejection fraction compared with a single regimen of each drug [17]. However, it remains unclear whether the combined treatment with ARNI and SGLT2i exerts a synergistic or addictive effect on cardiac function of CTRCD.
Doxorubicin is frequently used in many types of cancers and is most widely-known for its association with CTRCD [18][19][20][21]. In this study, we adopted a rodent model with doxorubicin injection as a reliable preclinical model for patients with CTRCD to provide an effective and suitable dosage for ARNI with SGLT2i for improvement of cardiac dysfunction. Here, we demonstrated that the low-dose ARNI in addition to SGLT2i markedly improves doxorubicin-induced cardiac dysfunction with minimal adverse effects.

Tail-Cuff Plethysmography
Blood pressure was measured in conscious mice by tail-cuff plethysmography using a two-channel BP-2000 system (Visitech System, Apex, NC, USA). To minimize stress, each mouse was habituated to a blood pressure platform before the experiment began. Each session involved 20-30 measurements; the initial 10 cycles were acclimation cycles that were not included in the analysis.

Tail-Cuff Plethysmography
Blood pressure was measured in conscious mice by tail-cuff plethysmography using a two-channel BP-2000 system (Visitech System, Apex, NC, USA). To minimize stress, each mouse was habituated to a blood pressure platform before the experiment began. Each session involved 20-30 measurements; the initial 10 cycles were acclimation cycles that were not included in the analysis.

Histopathological Analysis
Cross-sections of the mid-myocardium and kidney were fixed with buffer containing 10% formalin for 24 h and embedded in paraffin. Myocardial tissue sections were stained with hematoxylin and eosin (H&E), and kidney sections were stained with periodic acid-Schiff (PAS) reagent. To analyze the glomerular volume, 20-30 randomly selected superficial glomeruli from each kidney were measured, and calculations were performed using the Weibel-Gomez technique [24].

RNA Isolation and Real-Time qPCR Analysis
Total RNA was extracted from heart tissues using TRIzol ® reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), and 2 µg of total RNA was reverse-transcribed onto complementary DNA (cDNA) with Transcriptor First Strand cDNA Synthesis Kit (Roche Life Sciences, Indianapolis, IN, USA). Quantitative real-time polymerase chain reaction (PCR) containing SYBR Green PCR Master Mix (Applied Biosystems, Austin, TX, USA) was performed using CFX Connect Real-Time PCR Detection System (Bio-Rad). PCR primers used in this study are listed in Supplementary Table S1.

RNA-Sequencing and Analysis of Transcriptome Data
Total RNA was extracted from frozen LV tissues using the RNeasy ® Mini Kit (Qiagen, Hilden, Germany). RNA samples (RNA integrity number > 8) were used for library preparation and sequencing. Poly(A)-enriched cDNA libraries were prepared using the Illumina TruSeq RNA Library Preparation kit. Library templates were then sequenced on NovaSeq 6000 sequencing system at a read length of 151 nt paired end, with a depth of approximately 50 million per sample (Illumina, San Diego, CA, USA). Four to five mice per drug-treated condition were used for subsequent data analysis.

Statistical Analysis
Quantitative data were analyzed using GraphPad Prism version 9.0 (San Diego, CA, USA). Between-group comparisons were performed using one-way or two-way analysis of variance followed by Tukey's post hoc test for multiple comparisons based on the design of the experiment. Log-rank test was used to compare survival distributions. In the gene set enrichment analysis (GSEA), adjusted p-values corresponding to each normalized enrichment score (NES) were applied by Benjamini-Hochberg method. Statistical significance was set at p < 0.05.

Combined Treatment with Low-ARNI/SGLT2i Prolongs the Overall Survival of Acute Doxorubicin-Injected Mice
To evaluate the beneficial effect of ARNI plus SGLT2i on chemotherapy-induced cardiomyopathy, we administered experimental drugs in rodent model of acute doxorubicin heart failure ( Figure 1A). In pilot study for determining the appropriate combined dosages, we found that high doses of ARNI (136 mg/kg/day) aggravated the status of mice rather than protecting them. Drug down-titration is common for agents with a narrow therapeutic index to minimize the risk of adverse effects [25]. Based on the toxic side effects of high-doses of ARNI, we decided to add 50% of ARNI (34 mg/kg/day) to our experimental model.
In acute doxorubicin experiment, the drug-treated groups (ARNI, SGLT2i, Low-ARNI/SGLT2i, ARNI/SGLT2i treatment) showed improved survival rates compared with doxorubicin + vehicle group. Kaplan-Meier survival curves showed significant mortality (75%) in doxorubicin + vehicle group at experiment completion compared with that in the control mice. Additionally, the median survival time in doxorubicin + vehicle group was only 10 days ( Figure 1B). In contrast, mortality in doxorubicin + Low-ARNI/SGLT2i group was significantly lower than that in doxorubicin + vehicle group (doxorubicin + Low-ARNI/SGLT2i: 14.2% vs. doxorubicin + vehicle: 75%, p < 0.05). Moreover, the survival rate of other groups, including SGLT2i only, ARNI only, and ARNI/SGLT2i combination, in acute doxorubicin-injected mice increased up to 66.7%, 50%, and 66.7%, respectively, compared with that of doxorubicin + vehicle group. However, Low-ARNI/SGLT2i group showed the highest survival (85.7%) compared with other three groups. Furthermore, an improvement in survival rate accompanied with weight gain in drug-treated groups (Supplementary Figure S1). Treatment with doxorubicin + vehicle resulted in significant body weight loss (−23.8%) during the early course of study. Besides improved survival rate, treatment with Low-ARNI/SGLT2i markedly attenuated body weight loss (−15.9%) in doxorubicin-injected mice. Taken together, these results suggest that combined treatment with Low-ARNI/SGLT2i improves both survival rate and body weight in acute model of doxorubicin-induced heart failure.
Due to the anti-hypertensive effect of ARNI, blood pressure and pulse rate were measured to determine hemodynamic changes after ARNI and SGLT2i treatment in acute heart failure model. At 4 days after bolus injection of doxorubicin, systolic blood pressure of doxorubicin + vehicle group was lower than that of control mice (control: 114.8 ± 5.4 mmHg vs. doxorubicin + vehicle: 98.9 ± 5.1 mmHg, p < 0.05) ( Figure 1D). Unexpectedly, treatment with ARNI showed dramatic dipping in systolic blood pressure to 66.7 ± 6.3 mmHg compared with other groups in acute model of doxorubicin-induced heart failure. Nonetheless, the dipping of blood pressure after combined treatment with SGLT2i, with either Low-ARNI (94.3 ± 9.3 mmHg) or ARNI (80.6 ± 14.5 mmHg), was alleviated in the presence of SGLT2i. Despite the hypotensive effect of ARNI in acute model of doxorubicin heart failure, the addition of ARNI along with SGLT2i significantly improved heart rate compared with that in the doxorubicin + vehicle group (control: 531 ± 37 bpm; doxorubicin + vehicle: 391 ± 44 bpm vs. doxorubicin + ARNI: 476 ± 27 bpm, p < 0.05; doxorubicin + vehicle vs. doxorubicin + Low-ARNI/SGLT2i: 482 ± 46 bpm, p < 0.05; doxorubicin + vehicle vs. doxorubicin + ARNI/SGLT2i: 487 ± 65 bpm, p < 0.05; Figure 1E). These observations indicate that combining of ARNI with SGLT2i help to maintain proper blood pressure and heart rate rather hypotension and bradycardia shown in acute doxorubicin-injected mice.
We further measured the expression of cardiac structural proteins, including β-myosin heavy chain (βMHC) and myosin-binding protein C3 (MyBPC3). As shown in Figure 3G, a decrease in expression of βMHC and MyBPC3 after doxorubicin was recovered by treatment with Low-ARNI/SGLT2i. These data suggest that combined treatment with Low-ARNI/SGLT2i efficiently attenuates the pathological remodeling of cardiac structure in rodent model of doxorubicin-induced heart failure.

Effects of Combined Treatment with Low-ARNI/SGLT2i on Cardiac Energy Metabolism during Doxorubicin-Induced Cardiac Dysfunction
We performed bulk RNA-sequencing to obtain the mechanical insights of heart in response to ARNI and SGLT2i in chronic doxorubicin-injected mice. Principal component analysis of differentially expressed genes (DEGs) showed that the control, doxorubicin + vehicle, doxorubicin + ARNI, doxorubicin + SGLT2i, and doxorubicin + Low-ARNI/SGLT2i samples were clustered closely, indicating their similarity (Supplementary Figure S2A) [26,27]. Similarly, hierarchical clustering analysis also indicated that these samples were well grouped with each other (Supplementary Figure S2B) [28,29]. To understand the implications of global gene expression changes affected by combined treatment with Low-ARNI/SGLT2i, we performed GSEA [30]. GSEA showed that gene set was positively enriched with PPAR signaling pathway (NES 1.75, p.adjust = 0.008) and fatty acid degradation pathway (NES 1.67, p.adjust = 0.037) in doxorubicin + Low-ARNI/SGLT2i group compared with doxorubicin + vehicle group ( Figure 5A). Genes in the k-means cluster 3 in hierarchical clustering, which are strongly upregulated in combined treatment with Low-ARNI/SGLT2i (228 genes), uncovered significant over-representation of DEGs in fatty acid metabolic process. The two dominant pathways were PPAR signaling (Angptl4, Cpt1a, Cpt1b, Hmgcs2, Plin4, Scd4, and Slc27a1) and fatty acid degradation (Aldh9a1, Cpt1a, and Cpt1b) in Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis ( Figure 5B). Based on enrichment analysis, we examined the gene expression patterns of doxorubicin + Low-ARNI/SGLT2i groups according to Kyoto Encyclopedia of Genes and Genomes terms ("PPAR signaling pathway") by comparing the control and doxorubicin + vehicle groups. Heat maps of relative mRNA expression of gene sets in PPAR signaling pathway are shown in Figure 5C. Fatty acid transport-related genes (Slc27a1, Slc27a6, Acsbg1, Acsl3, Acsl4, Fabp4, and Fabp5), fatty acid oxidation-related genes (Cpt1a, Cpt1b, Acaa1a, and Ehhadh), ketogenesis-related genes (Hmgcs2), and gluconeogenesis-related genes (Pck1, Pck2, and Aqp7) were upregulated in the Low-ARNI/SGLT2i group. Furthermore, recovery of dysregulated genes (Rxrb, Acsl5, Dbi, Plin1, Acaa1a, Acox3, Sorbs1, Apoa2, and Slc27a4) after doxorubicin injection was observable in doxorubicin + Low-ARNI/SGLT2i group. Thus, our transcriptome analysis suggests that combined treatment with Low-ARNI/SGLT2i improves metabolic flexibility in doxorubicin-injected heart through PPAR signaling pathway. ARNI/SGLT2i group was significantly different (p < 0.05) ( Figure 5E). Since glucose is derived either exogenously or from glycogen stored intracellularly in heart, we assessed the level of glucose transporter type 4 (GLUT4) and glycogen synthase kinase 3β (GSK3β). GLUT4 expression was increased whereas inactivation of GSK3β through the protein kinase B (PKB/Akt) signaling pathway was up-regulated in the heart tissues from doxorubicin + Low-ARNI/SGLT2i group (Supplementary Figure S3). To further validate the functional relevance of identified genes associated with cardiac metabolism from RNA-sequencing data, we studied energy utilization in doxorubicininduced cardiac injury. Initially, we examined renal glycosuria by measuring urine glucose levels of mice to assess the pharmacologic effects of SGLT2i. As expected, treatment with SGLT2i significantly increased glucose levels in urine ( Figure 5D). Furthermore, to understand the resultant metabolic changes, we measured plasma glucose following drug administration. Treatment with doxorubicin resulted in a relatively low level of plasma glucose compared with the control (control: 244.7 ± 19 mg/dL vs. doxorubicin + vehicle: 190 ± 18.7 mg/dL, p < 0.001). Since SGLT2i is an anti-hyperglycemic agent that acts through glycosuria, it showed the lowest glucose levels among experimental groups. However, glucose level in SGLT2i group was not statistically significant compared with that of doxorubicin + vehicle group (doxorubicin + vehicle: 190 ± 18.7 mg/dL vs. doxorubicin + SGLT2i: 186 ± 20.6 mg/dL, p = 0.9). Interestingly, ARNI-treated group showed relatively higher levels of plasma glucose than doxorubicin + vehicle group (doxorubicin + ARNI: 209 ± 22.5 mg/dL, doxorubicin + Low-ARNI/SGLT2i: 222 ± 11.1 mg/dL, doxorubicin + ARNI/SGLT2i: 218 ± 20.2 mg/dL). However, only Low-ARNI/SGLT2i group was significantly different (p < 0.05) ( Figure 5E). Since glucose is derived either exogenously or from glycogen stored intracellularly in heart, we assessed the level of glucose transporter type 4 (GLUT4) and glycogen synthase kinase 3β (GSK3β). GLUT4 expression was increased whereas inactivation of GSK3β through the protein kinase B (PKB/Akt) signaling pathway was up-regulated in the heart tissues from doxorubicin + Low-ARNI/SGLT2i group (Supplementary Figure S3).
To ascertain whether the cardioprotective effect observed in the Low-ARNI/SGLT2i treatment was related to fatty acid metabolism, we evaluated the expression of proteins involved in fatty acid metabolism, including PPAR alpha (PPARα), fatty acid transporter (CD36), and carnitine palmitoyl transferase (CPT1α), from heart tissues. Immunoblot analysis showed that the expression of PPARα, CD36, and CPT1α was markedly increased in the Low-ARNI/SGLT2i group compared with doxorubicin + vehicle group ( Figure 5G). Furthermore, quantitative RT-PCR revealed that the expression of genes, including Cpt1a, Ehhadh, Acadl, and Hmgcs2, involved in β-oxidation and ketogenesis was significantly upregulated after Low-ARNI/SGLT2i treatment ( Figure 5H). Altogether, combined treatment with Low-ARNI/SGLT2i exerts protective effects on heart by modulating metabolic pathway, including glucose, ketone bodies, and fatty acids, in doxorubicin-induced model of heart failure.

Discussion
To the best of our knowledge, this study is the first observation to report the efficacy and suitable dosage of ARNI combined with SGLT2i in a preclinical model of CTRCD, and to show that low-dose ARNI in addition to SGLT2i exhibits a favorable effect on hemodynamic changes and enhanced myocardial metabolism through PPAR signaling pathway.
In our experiment, along with better survival, Low-ARNI/SGLT2i-treated group exhibited remarkable weight loss after doxorubicin injection (Supplementary Figure S1). Early weight loss in patients with cancer negatively affects clinical prognosis [31]. Thus, we assumed that improved survival by Low-ARNI/SGLT2i may also be related to maintenance of weight and muscle mass. Although there is a limit in terms of inability to measure food and water intake, an increase in the plasma glucose levels of Low-ARNI/SGLT2i group may support this possibility. Since blood glucose levels vary according to dietary intake, Low-ARNI/SGLT2i groups may have taken sufficient nutrition to have increased blood glucose levels more than doxorubicin + vehicle group ( Figure 5E).
In addition, reduced myocardial mass loss, augmented myofibril thickness, and an improvement of cardiac function by Low-ARNI/SGLT2i may be a result of either reduced degradation or enhanced synthesis of heart structural proteins, including βMHC and MyBPC3 ( Figure 3G). In patients with CTRCD, cardiomyocytes are injured by the direct toxicity of chemotherapeutic reagents [32]. To overcome this issue, Low-ARNI/SGLT2i combination may support the possibility of regulating cardiac myocyte protein turnover for cardioprotection [33]. In this regard, it is important to elucidate the underlying molecular mechanisms through in-depth research.
Heart failure shares many risk factors and numerous pathophysiological pathways with acute and chronic kidney disease through hemodynamic, neurohormonal, and cardiovascular disease-associated mechanisms [34]. The most well-established heart-derived hormones are ANP and BNP, which regulate the whole-body balance of water and electrolytes through target tissues [35]. As the kidney is a main target organ for natriuretic peptides, we tested PAS staining and serum NGAL level to assess kidney injury. In our study, the renal protective effect was obvious in combined Low-ARNI/SGLT2i treatment than in single treatment of either ARNI or SGLT2i ( Figure 4E-I). In contrast to Low-ARNI/SGLT2i combination, ARNI/SGLT2i treatment induced marked renal impairment compared with others. Clinical trials such as Dapagliflozin and Prevention of Adverse Outcomes in Chronic Kidney Disease (DAPA-CKD), and Prospective Comparison of ARNI with ARB Global Outcomes in HF with Preserved Ejection Fraction (PARAGON-HF) reported that beneficial outcomes of ARNI and SGLT2i on kidney function. However, adverse event cases of acute kidney injury associated with SGLT2i and even ARNI/SGLT2i combination were also reported [36,37]. Major declines in kidney function under SGLT2i are often accompanied by acute illnesses such as diarrhea and/or sepsis [38]. Inappropriate ARNI doses induce a hemodynamically unstable condition and SGLT2i may lead to acute kidney injury by excessive intravascular volume depletion, due to diuresis and induction of renal medullary hypoxic injury [39]. Hence, we propose that combined administration of these two drugs in suitable doses would preserve organ function during chemotherapy through cardiorenal crosstalk.
Due to the anti-hypertensive effect of ARNI, extreme blood pressure dipping with ARNI was observed in acute doxorubicin-induced model of cardiac dysfunction ( Figure 1D). Although the dosage of ARNI was a human equivalent dose and comparable to that in previous preclinical studies, these results may need to be interpreted under highly virulent conditions induced by a single high-dose bolus injection of doxorubicin. Acute doxorubicin-induced model of heart failure mimics acute decompensated heart failure and similarly shows a vulnerable physiological state; therefore, even the modest amounts of anti-hypertensive agents can cause large drops. In Comparison of Sacubitril-Valsartan versus Enalapril on Effect on NT-proBNP in Patients Stabilized from an Acute Heart Failure Episode (PIONEER-HF) trial, ARNI group showed a higher tendency for symptomatic hypotension than ACE inhibitor group, although the difference was not statistically significant (ARNI: 15% vs. ACEi: 12.7%) [40]. These comparable rates of symptomatic hypotension may have critically affected the non-significant differences in secondary efficiency and safety outcomes in the PIONEER-HF trial. Therefore, ARNI dose modulation should be conducted with extreme caution in intra-hospital drug management of acute decompensated heart failure.
Furthermore, SGLT2i combined with ARNI reduced the risk of hypotension in acute doxorubicin model, implicating a clinical benefit for hemodynamically unstable patients with heart failure. Our result was consistent with the findings of Empagliflozin Outcome Trial in Patients with Chronic Heart Failure with Reduced Ejection Fraction (EMPEROR-Reduced), demonstrating a slight early increase in placebo-corrected systolic blood pressure among patients in the "<110 mmHg systolic blood pressure" group during the early phase of the trial [7]. In addition to the study by Böhm et al. [41], our study reassures the hemodynamic tolerability of SGLT2i, which seems to be a novel therapeutic intervention for managing low blood pressure.
The heart, an organ with a high metabolic rate, utilizes a wide range of substrates, including fatty acids, and glucose. During end-stage heart failure, the efficiency of fatty acid oxidation is significantly reduced [42]. To compensate for and increase the total energy supply, glucose and ketone bodies may be used for cardiac metabolism [43]. Transcriptome analysis employed in this study suggested that potential mechanisms in the Low-ARNI/SGLT2i group involve modulating cardiac metabolism through PPAR signaling pathway, mainly through fatty acid catabolism. PPARs are fatty acid sensors that regulate whole-body energy metabolism [44]. PPARα plays a crucial role in the body's adaptive response to fasting by modulating fatty acid transport, oxidation, and ketogenesis [45]. PPARβ/δ activate both lipid and glucose metabolism, and are associated with body mass index, fasting glucose levels, and insulin resistance [46]. PPAR activation induces the expression of several protein-encoding genes involved in transmembrane transport and mitochondrial β-oxidation of fatty acids [47]. As shown in Figure 5C, Low-ARNI/SGLT2i treatment increased the expression of PPAR target genes involved in fatty acid transport (Slc27a1, Slc27a6, Acsbg1, Acsl3, Acsl4, Fabp4, Fabp5) and oxidation (Cpt1a, Cpt1b, Acaa1a, Ehhadh). Consistent with our transcriptomic data, immunoblotting and quantitative RT-PCR ( Figure 5G,H) revealed that PPARα, CD36, and Cpt1a were increased in the Low-ARNI/SGLT2i group, which may contribute to the increased oxidation rates of myocardial fatty acid. This preservation of fatty acid oxidation suggests the promotion of catabolism for energy production, thereby improving myocardial energetics. Moreover, Low-ARNI/SGLT2i treatment induced myocardial expression of 3-hydroxy-3-methylglutaryl-CoA synthase 2 (Hmgcs2), a mitochondrial enzyme involved in the second rate-limiting step of ketogenic pathway. Ketone bodies are an alternative fuel source for failing hearts [48]. Our transcriptomic data of increased expression of Hmgsc2 and elevation of circulating β-hydroxybutyrate in the Low-ARNI/SGLT2i treatment group indicate that ketones are also potential contributors to improving myocardial energetics ( Figure 5F). Moreover, increased expression of GLUT4 and inactivation of GSK3β may influence the re-partitioning of glucose metabolism, which might have enhanced energy efficiency by generating higher ATP levels (Supplementary Figure S3). Altogether, combined treatment with SGLT2i and suitable dosage of ARNI potentiates myocardial energetics through the utilization of fatty acid, glucose, and ketone bodies.
We acknowledge the limitations of our study. Healthy animals were used in our study to yield the effect of ARNI and SGLT2i on doxorubicin-induced heart failure, but these drugs may interfere with chemotherapeutic efficacy. Furthermore, we could not measure diastolic function; therefore, it was not possible to evaluate whether diastolic function was changed by the combination of two drugs. Moreover, further investigations are needed to test the ability of ARNI and SGLT2i combination by metabolite profiling to identify the dynamic metabolic changes in the heart.

Conclusions
We report that the addition of low-dose ARNI with SGLT2i resulted in greater benefits than single treatment with either ARNI or SGLT2i in protecting cardiac function against doxorubicin-induced cardiotoxicity through PPAR signaling pathway. Our data may offer novel mechanistic insight into the benefits observed with Low-ARNI/SGLT2i combination. Even though future investigations are required to confirm the favorable role of ARNI and SGLT2i combined therapy in CTRCD, our findings would be applicable in developing optimized CTRCD management.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/pharmaceutics14122629/s1, Figure S1: Low-ARNI/SGLT2i treatment prevents body weight loss during acute doxorubicin administration; Figure S2: Transcriptomic analysis during ARNI and SGLT2i treatment in the chronic doxorubicin model of heart failure; Figure S3: ARNI and SGLT2i treatment effects on glucose metabolism re-partitioning in chronic doxorubicin-injected mice; Table S1: Primer sequences used in the present study.