Combination Sodium Nitrite and Hydralazine Therapy Attenuates Heart Failure With Preserved Ejection Fraction Severity in a “2‐Hit” Murine Model

Figures

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  Figure 1. NO bioavailability is reduced in the “2‐hit” mouse model of HFpEF. Study protocol for characterization of the “2‐hit” murine model of cardiometabolic HFpEF as compared with control mice fed a normal chow and drinking water (A). Echocardiographic measurements of left ventricular (LV) ejection fraction (%) (B) and LV E/E’ ratio (C); body weight (grams) (D); invasive hemodynamic catheter measurements of systolic blood pressure (mmHg) (E) diastolic blood pressure (mmHg) (F); LV relaxation time constant Tau (ms) (G); and LV end‐diastolic pressure (LVEDP) in mmHg (H). mRNA levels of endothelial nitric oxide synthase and inducible nitric oxide synthase in cardiac tissue (I). Cardiac tissue nitrite levels (nmole/g of tissue) (J) and plasma nitrite concentrations in μmol/L (K). All data were analyzed using an unpaired Student t‐test. *P<0.05, **P<0.01. Numbered circles inside bars indicate sample size for each group. Ach indicates acetylcholine; DBP, diastolic blood pressure; HFpEF, heart failure with preserved ejection fraction; L‐NAME, N(ω)‐nitro‐L‐arginine methyl ester; NS, not significant; SBP, systolic blood pressure; and SNP, sodium nitroprusside.

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  Figure 2. Altered mitochondrial respiration, vascular dysfunction and reduced exercise capacity in HFpEF. Mitochondrial respiration in different activity states was quantified using pyruvate as a substrate (A) and palmitoyl‐L‐carnitine as a substrate (B). Isolated aortic ring vascular relaxation (%) following precontraction with phenylephrine (1 μmol/L) to increasing concentrations of acetylcholine (10−9 to 10−5) (C) and sodium nitroprusside (10−10 to 10−7) (D). Quantification of treadmill exercise duration in minutes (E), exercise distance in meters (F), and work expenditure (kg×m) during exercise testing (G). All data were analyzed using an unpaired Student t‐test. *P<0.05, **P<0.01. Numbered circles inside bars indicate sample size. Ach indicates acetylcholine; HFpEF, heart failure with preserved ejection fraction; and NS, not significant.

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  Figure 3. Sodium nitrite + hydralazine therapy restored NO bioavailability and reduced oxidative and nitrosative stress in “2‐hit” mouse model of HFpEF. A, Experimental study protocol for 2‐hit murine model of HFpEF depicting the 2 study groups consisting of Vehicle and sodium nitrite+hydralazine therapy. Circulating plasma nitrite (μmol/L) (B) and cardiac nitrite (μmol/L) (C) in vehicle and sodium nitrite+hydralazine groups. Circulating levels of 8‐isoprostane (pg/mL) (D), 3‐nitrotyrosine (3‐NT) in μg/mL (E), and high sensitivity C‐reactive protein (hsCRP) in pg/mL (F). All data were analyzed using an unpaired Student t‐test. *P<0.05, **P<0.01. Numbered circles inside bars indicate sample size. HFpEF indicates heart failure with preserved ejection fraction; hsCRP, high‐sensitivity C‐reactive protein; L‐NAME, N(ω)‐nitro‐L‐arginine methyl ester; LV, left ventricular; NS, not significant; and SNP, sodium nitroprusside.

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  Figure 4. Combination sodium nitrite+hydralazine therapy attenuates diastolic dysfunction. A, Echocardiography measurements of left ventricular ejection fraction (LVEF); B, E/A ratio; and C, E/E’ ratio measurements accordingly. Invasive hemodynamic catheter measurements of left ventricular end‐diastolic pressure (LVEDP) in mm Hg (D); LV relaxation time constant (Tau) in milliseconds (E). F and G, systolic and diastolic blood pressures (mm Hg) respectively. All data were analyzed using an unpaired Student t‐test. *P<0.05, **P<0.01. Numbered circles inside bars indicate sample size. DBP indicates diastolic blood pressure; NS, not significant; and SBP, systolic blood pressure.

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  Figure 5. Sodium nitrite + hydralazine therapy improves exercise capacity, vascular reactivity, and mitochondrial respiration in HFpEF. Treadmill exercise tolerance testing with exercise duration in minutes (A); exercise distance in meters (B); and total work exerted while during the challenge (C). Ex‐vivo aortic ring vascular reactivity measurements following administration of acetylcholine (10−9 to 10−5) plotted as percentage (%) of maximal relaxation to phenylephrine (1 μmol/L) (D) and sodium nitroprusside (10−10 to 10−7) plotted as maximal relaxation (E). Respiration responses of isolated cardiac mitochondria measured at differing activity states with pyruvate as a substrate (F) and palmitoyl‐L‐carnitine as a substrate (G). All data were analyzed using an unpaired Student t‐test. *P<0.05, **P<0.01. Numbered circles inside bars indicate sample size. Ach indicates acetylcholine; NS, not significant; and SNP, sodium nitroprusside.

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  Figure 6. Combination therapy reduces cardiac perivascular fibrosis. A, Representative photomicrographs of cardiac tissue stained with Masson's trichrome to evaluate interstitial cardiac fibrosis. Images were taken and blindly evaluated to quantify both interstitial and perivascular cardiac fibrosis. B, Bar graph of % fibrosis in vehicle and sodium nitrite treated groups. Images are presented for Vehicle, sodium nitrite, hydralazine, and combination therapy groups. C, Representative photomicrographs of cardiac perivascular fibrosis following Masson's trichrome staining. D, Bar graph of cardiac perivascular fibrosis (%) in vehicle and sodium nitrite+hydralazine groups. E, Representative imaging of renal tissue fibrosis following Masson's trichrome staining. F, Bar graph of renal fibrosis quantification in vehicle and sodium nitrite+hydralazine groups. All data were analyzed using an unpaired Student t‐test. *P<0.05, **P<0.01. Numbered circles inside bars indicate group sample size. HFpEF indicates heart failure with preserved ejection fraction; and NS, not significant.