Hydrogen Sulfide Attenuates Renin Angiotensin and Aldosterone Pathological Signaling to Preserve Kidney Function and Improve Exercise Tolerance in Heart Failure

Figures

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  Figure 1. Experimental Compounds and Experimental Protocol (A) Chemical structures of JK-1 and control. Hydrolysis of JK-1 yields H2S and the chemical backbone of JK-1, which was selected as Control. The control compound is devoid of sulfur and does not release H2S. (B) Experimental design and protocol. At 1 week following baseline echocardiography, mice were subjected to TAC or sham TAC surgery. Mice were studied for a period of 18 weeks following TAC. The H2S donor, JK, was administered at either 3 or 10 weeks following TAC at a dose of 100 μg/kg b.i.d. through i.p. injection. This study involved 4 groups: sham TAC, HF plus Control compound, HF plus JK-1 administered at 3 weeks following TAC, or HF plus JK-1 administered starting at 10 weeks post TAC. b.i.d. = twice daily; BNP = B-type natriuretic peptide; HF = heart failure; LV = left ventricle; RAAS = renin-angiotensin-aldosterone system; TAC = transverse aortic constriction.

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  Figure 2. LV Structure, Function, and Circulating BNP Levels at 18 Weeks Post TAC (A) (B) LVEDD and LVESD (mm) throughout the experimental protocol. (C) Percent of LVEF. (D) Left ventricular end-diastolic pressure (mm Hg) at 18 weeks following TAC. (E) LV relaxation constant Tau (ms) at 18 weeks post TAC. (F) Circulating BNP levels (ng/ml) in mice from Sham, HF + Control, HF + 3-week-delayed JK-1 treatment, and HF + 10-week-delayed JK-1 treatment. *p < 0.05 between Sham vs. HF + Control; **p < 0.01 between Sham vs. HF + Control; ****p < 0.0001 between Sham vs. HF + Control; #p < 0.05 between HF + JK-1 3 weeks post TAC vs. HF + Control; ###p < 0.001 between HF + JK-1 3 weeks post TAC vs. HF + Control; ####p < 0.0001 between HF + JK-1 3 weeks post TAC vs. HF + Control; $p < 0.05 between HF + JK-1 10 weeks post TAC; $$$p < 0.001 between HF + JK-1 10 weeks post TAC; $$$$p < 0.0001 between HF + JK-1 10weeks post TAC. LVEDD, LVESD, and LVEF data were analyzed with 2-way-ANOVA, whereas LVEDP, Tau, and circulating BNP data were analyzed with 1-way ANOVA. Circles inside bars denote sample size. LVEDD = left ventricular end-diastolic dimensions; LVEF = left ventricular ejection fraction; LVESD =…

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  Figure 3. Cardiac Fibrosis at 18 Weeks Post TAC (A) Representative images (original magnification ×20) of Picrosirius red-stained cardiac tissue sections from HF + Control and HF + JK-1 (3 weeks and 10 weeks, respectively) delayed treatment. (B) Quantified cardiac fibrosis. (C) Relative fold changes in myocardial mRNA levels of a panel of fibrosis-related genes in mice from HF + Control, HF + JK-1 3 weeks post TAC, and HF + JK-1 10 weeks post TAC groups at 18 weeks post TAC. All data in Figure 3 were analyzed with 1-way ANOVA. Circles inside bars denote sample size. Abbreviations as in Figure 1.

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  Figure 4. Renal Fibrosis and Plasma Creatinine Levels at 18 Weeks Post TAC (A) Plasma creatinine levels. (B) Representative images (original magnification ×20) of Picrosirius red- stained renal tissue sections. (C) Quantified global renal fibrosis. (D) Relative fold changes in renal mRNA levels of fibrosis-related genes in mice from HF + Control, HF + JK-1 3 weeks post TAC, and HF + JK-1 10 weeks post TAC groups at 18 weeks post TAC. All data were analyzed with 1-way ANOVA. Circles inside bars denote the sample size. Abbreviations as in Figure 1.

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  Figure 5. Aortic Vasorelaxation at 18 Weeks Post TAC Freshly isolated thoracic aortic rings were allowed to equilibrate, and pre-contracted with 1 μM phenylephrine. (A) Concentration-response curve (% relaxation from 10−9 to 10−5). (B) EC50 (nM). (C) Maximal relaxation (%) achieved for acetylcholine (Ach)-mediated vasorelaxation in mice from Sham, HF + Control, HF + JK-1 3 weeks post TAC, and HF + JK-1 10 weeks post TAC groups at 18 weeks post TAC. (D) Concentration-response curve (% relaxation from 10−10.5 to 10−6.5) (E) EC50 (nM), and (F) maximal relaxation (%) achieved for SNP mediated vasorelaxation in mice from Sham, HF + Control, HF + JK-1 3 weeks post TAC, and HF + JK-1 10 weeks post TAC groups at 18 weeks post TAC. *p < 0.05 between Sham vs. HF + Control; ****p < 0.0001 between Sham vs. HF + Control; # p < 0.05 between HF + JK-1 3 weeks post TAC vs. HF + Control; ##p < 0.01 between HF + JK-1 3 weeks post TAC vs. HF + Control; ####p < 0.0001 between HF + JK-1 3 weeks post TAC vs. HF + Control; $$$p < 0.001 between HF + JK-1 10weeks post TAC; $$$$p < 0.0001 between HF + JK-1 10weeks post TAC. Concentration-response curve were generated using a model of nonlinear regression with…

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  Figure 6. Treadmill Exercise Capacity at 18 Weeks Post TAC Exercise capacity was evaluated in all study groups at 18 weeks following TAC surgery. (A) Duration (min) of the running before exhaustion. (B) Distance (m) of the running before exhaustion. (C) Shocks per minute in mice from Sham, HF + Control, HF + JK-1 3 weeks post TAC, and HF + JK-1 10 weeks post TAC groups at 18 weeks post TAC. All data were analyzed with 1-way ANOVA. Circles inside bars denote the sample size. Abbreviations as in Figure 1.

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  Figure 7. H2S and NO Bioavailability and 8-Isoprostane Levels at 18 Weeks Post TAC H2S levels (μM) in plasma (A), myocardial tissue (B), and renal tissue (C). Nitrite levels (μM) in plasma (D), myocardial tissue (E), and renal tissue (F). 8-Isoprostane (8-Iso) levels (pg/ml) in circulation (G), myocardial tissue (H), and renal tissue (I) in mice from Sham, HF + Control, HF + JK-1 3 weeks post TAC, and HF + JK-1 10 weeks post TAC groups at 18 weeks post TAC. All data were analyzed with 1-way ANOVA. Circles inside bars denote the sample size. Abbreviations as in Figure 1.

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  Figure 8. Renal Norepinephrine Content, Circulating Renin, Ang II, and Aldosterone Levels at 18 Weeks Post TAC (A) Renal tissue norepinephrine content (ng/ml). (B) Plasma renin (ng/ml). (C) Plasma Ang II (pg/ml). (D) Circulating Aldo levels (pg/ml). (E) Renal Aldo levels (pg/ml) in mice from Sham, HF + Control, HF + JK-1 3 weeks post TAC, and HF + JK-1 10 weeks post TAC groups at 18 weeks post TAC. All data were analyzed with 1-way ANOVA. Circles inside bars denote the sample size. Aldo = aldosterone; Ang II = angiotensin II; other abbreviations as in Figure 1.