Dual Angiotensin Receptor-Neprilysin Inhibition With Sacubitril/Valsartan Attenuates Systolic Dysfunction in Experimental Doxorubicin-Induced Cardiotoxicity

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  Figure 1. Schematic of Experimental Design A parallel group design was employed for 6 weeks using 4 groups of rats (n = 25 each group; N = 100). The groups included control (CTL) animals, doxorubicin (DOX)-treated rats (DOX), DOX-treated rats receiving valsartan (Val) (DOX + Val), and DOX-treated rats receiving sacubitril (Sac)/Val (DOX + Sac/Val). Animals were treated with either DOX (2.15 mg/kg/dose, 15 mg/kg total, n = 75) or saline (CTL group, n = 25) every 3 days for 21 days, denoted by red arrows. Following the first treatment, CTL animals were gavaged daily with water (n = 25), while DOX-treated animals were gavaged daily with water (n = 25), Val (31 mg/kg; n = 25), or Sac/Val (68 mg/kg; n = 25) for 4 to 6 weeks. Echocardiography was performed at baseline (prior to chemotherapy, n = 25 per group) and at 4 weeks (n = 25 per group) and 6 weeks (n = 10 per group) following initiation of chemotherapy to assess changes in cardiac function. In a subgroup of rats, myocardial matrix metalloproteinase (MMP) activity and cardiotoxicity severity were assessed ex vivo with 99mTc-RP805 and standard histological techniques (n = 7 to 10 per group), respectively, at 4 and 6 weeks following the…

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  Figure 2. Transthoracic Echocardiography–Derived Systolic Function and Ventricular Dimensions (A) Left ventricular (LV) ejection fraction, (B) fractional shortening, (C) LV end-diastolic volume, and (D) LV end-systolic volume at baseline (n = 25 per group), 4 weeks (n = 25 per group), and 6 weeks (CTL, n = 10; DOX, n = 10; DOX + Val, n = 9, DOX + Sac/Val, n = 10) following the initiation of chemotherapy. ∗p < 0.05 over horizontal bars indicates between-group difference. ∗p < 0.05 vs. baseline; ∗∗p < 0.01 vs. baseline; ∗∗∗∗p < 0.0001 vs. baseline; #p < 0.05 vs. 4 weeks. Abbreviations as in Figure 1.

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  Figure 3. LV 99mTc-RP805 Uptake (A) LV 99mTc-RP805 uptake between CTL animals (n = 10) and rats treated with DOX (n = 9), DOX + Val (n = 10), and DOX + Sac/Val (n = 9) at 4 weeks and between CTL animals (n = 10) and rats treated with DOX (n = 9), DOX + Val (n = 10), and DOX + Sac/Val (n = 9) at 6 weeks following chemotherapy initiation. Correlation between (B) LV ejection fraction and LV 99mTc-RP805 uptake and (C) LV end-systolic volume and LV 99mTc-RP805 uptake in all rats at 6 weeks. ∗p < 0.05 between group difference. Abbreviations as in Figures 1 and 2.

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  Figure 4. Representative Pathohistological Images Over Time Representative images (Hematoxylin and Eosin [H and E] and Masson's Trichrome [MT]) of myofiber variation (top panel) and myofiber vacuolation with cellularity (bottom panel) for each group at 4 and 6 weeks following initiation of chemotherapy. Doxorubicin toxicity was progressive and characterized by marked variation in myofiber diameter (blue arrows, B, F, J, N) and intracellular vacuolation (black arrows, F and N). Small foci of inflammatory cells (white arrows, G, H, l) or rare vacuolated myofibers (black arrow, P) are still seen in DOX rats treated with Val and Sac/Val, albeit less than that observed without these treatments. Abbreviations as in Figures 1 and 2.

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  Figure 5. Quantification of Histopathology Scoring Over Time Scoring was categorized as (A) myofiber vacuolation with diameter variation and (B) cellularity between CTL animals (n = 10) and rats treated with DOX (n = 10), DOX + Val (n = 10), and DOX + Sac/Val (n = 9) at 4 weeks and between CTL animals (n = 10) and rats treated with DOX (n = 9), DOX + Val (n = 10) and DOX + Sac/Val (n = 9) at 6 weeks following chemotherapy initiation. ∗p < 0.05, ∗∗p < 0.01 over horizontal over horizonal bars denotes between-group difference. Abbreviations as in Figure 1.

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  Figure 6. Representative PSR and SMA Staining Images and Quantification (A) Picrosirius red (PSR) and (B) smooth muscle actin (SMA) staining and (C, D) respective quantification of these variables between CTL animals (n = 9) and rats treated with DOX (n = 7), DOX + Val (n = 7) and DOX + Sac/Val (n = 7) at 4 weeks and between CTL animals (n = 8) and rats treated with DOX (n = 8), DOX + Val (n = 7) and DOX + Sac/Val (n = 7) at 6 weeks following chemotherapy initiation. ∗ p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 over horizontal bars denotes between-group difference. #p < 0.05 vs. 4 weeks. Abbreviations as in Figure 1.

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  Figure 7. Cardiomyocyte Cross-Sectional Area and Capillary Density Over Time (A) Representative images of LV sections co-stained with laminin (red) and isolectin B4 (green) at 4 and 6 weeks after the initiation of chemotherapy. (B) Quantification of myocyte cross-sectional area (μm2) and (C) capillary density (expressed as capillary number per cardiomyocyte area) for CTL animals (n = 10) and rats treated with DOX (n = 9), DOX + Val (n = 10), and DOX + Sac/Val (n = 9) at 4 weeks and for CTL animals (n = 10) and rats treated with DOX (n = 9), DOX + Val (n = 9–10), and DOX + Sac/Val (n = 8–10) at 6 weeks following chemotherapy initiation. Horizontal bars denote group difference at the significance levels of, ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. †p < 0.01 vs. 4 weeks. Abbreviations as in Figures 1 and 2.

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  Central Illustration. Sac/Val Is Superior to Val at Reducing Anthracycline-Induced Cardiotoxicity Echocardiography was performed at baseline in all rats (n = 100). Rats were then administered doxorubicin (DOX) (2.15 mg/kg) every 3 days for 21 days (15 mg/kg total) (n = 75) or an equal volume of 0.9% saline (control animals, n = 25) intraperitoneally. After the first dose, DOX rats remained untreated (DOX, n = 25), or were administered valsartan (Val) (31 mg/kg; DOX+ Val, n = 25) or sacubitril (Sac)/Val (68 mg/kg; DOX+ Sac/Val, n = 25) by gavage for the duration of study. Control and DOX-alone animals were gavaged with water. At 4 weeks post-chemotherapy, echocardiography-derived left ventricular (LV) function (LV ejection fraction), in vivo myocardial matrix metalloproteinase activity (following 99mTc-RP805 injection) or histological myocardial toxicity as assessed by hematoxylin and eosin and Masson's Trichrome staining was no different (blue horizontal double-sided arrow) in the DOX groups compared with control animals. At this time point, myocardial fibrosis (Picrosirius red) was only increased (red arrow) in the DOX-alone group but was prevented with Val and Sac/Val treatment compared with…