Uncoupled cardiac nitric oxide synthase mediates diastolic dysfunction

Abstract

Background: Heart failure with preserved ejection fraction is 1 consequence of hypertension and is caused by impaired cardiac diastolic relaxation. Nitric oxide (NO) is a known modulator of cardiac relaxation. Hypertension can lead to a reduction in vascular NO, in part because NO synthase (NOS) becomes uncoupled when oxidative depletion of its cofactor tetrahydrobiopterin (BH(4)) occurs. Similar events may occur in the heart that lead to uncoupled NOS and diastolic dysfunction.

Methods and results: In a hypertensive mouse model, diastolic dysfunction was accompanied by cardiac oxidation, a reduction in cardiac BH(4), and uncoupled NOS. Compared with sham-operated animals, male mice with unilateral nephrectomy, with subcutaneous implantation of a controlled-release deoxycorticosterone acetate pellet, and given 1% saline to drink were mildly hypertensive and had diastolic dysfunction in the absence of systolic dysfunction or cardiac hypertrophy. The hypertensive mouse hearts showed increased oxidized biopterins, NOS-dependent superoxide production, reduced NO production, and dephosphorylated phospholamban. Feeding hypertensive mice BH(4) (5 mg/d), but not treating with hydralazine or tetrahydroneopterin, improved cardiac BH(4) stores, phosphorylated phospholamban levels, and diastolic dysfunction. Isolated cardiomyocyte experiments revealed impaired relaxation that was normalized with short-term BH(4) treatment. Targeted cardiac overexpression of angiotensin-converting enzyme also resulted in cardiac oxidation, NOS uncoupling, and diastolic dysfunction in the absence of hypertension.

Conclusions: Cardiac oxidation, independently of vascular changes, can lead to uncoupled cardiac NOS and diastolic dysfunction. BH(4) may represent a possible treatment for diastolic dysfunction.

Figure 1

Representative echocardiographic assessments of LV diastolic function.Panels a and b: Left ventricular inflow propagation velocity (Vp) interrogated with color M-mode Doppler. A control mouse shows a steeper isovelocity line slope, corresponding to a higher Vp compared with a hypertensive DOCA mouse. Panels c and d: Septal mitral annulus velocities interrogated with tissue Doppler imaging (TDI). The control animal has a higher E’ (early diastolic velocity), and lower A’ (late diastolic velocity) than the hypertensive animal. Panels e and f: Conventional pulsed wave Doppler shows a normal E/A (early to late diastolic filling velocity ratio) of >1 and <2 for both the control and DOCA mice, a pseudonormal” pattern.

Figure 2

Invasive hemodynamic assessment of LV diastolic dysfunction. Panel a: Baseline pressure-volume loops for hypertensive and control animals. Panel b: Comparison of LVESP for hypertensive and control animals (p=0.002). Panel c: Comparison of LVEDP for hypertensive and control animals (7.2 ± 0.7 vs. 4.5 ± 0.4 mmHg, p=0.004).Panel d: The time constant for isovolemic relaxation (τ_Glantz) is increased in hypertensive mice compared to controls (p=0.03).Panel e: The end-diastolic pressure-volume relation (EDPVR) slope is steeper in hypertensive mice as compared to controls (p=0.0004).Panel f: Pearson correlation coefficients for linear fitting of the EDPVR.Panel g: LV contractility assessed by the end-systolic pressure-volume relation (ESPVR) slope (p=NS) and the volume axis intercept Vo (p=NS) are similar between DOCA and control groups. Panel h: Mean heart rate between groups (p=NS).Panel i: Arterial elastance (Ea) a measure of vascular stiffness is similar between hypertensive and control mice (p=NS).

Figure 3

Hypertensive mice show cardiac oxidation and reduced BH₄. Panel a: Typical HPLC spectra obtained from a control (top), DOCA (middle), BH₄ treated DOCA mice (bottom) show a larger oxyethidium (2HO-ET) peak in the DOCA animal.Panel b: Mean 2HO-ET levels are significantly higher in DOCA mice as compared to control and BH₄ treated mice (days 1–14). Panel c: The percent O₂^•− reduction between control and DOCA LV tissue treated with the non-selective NOS inhibitor, L-NAME, and the selective neuronal NOS inhibitor, 7-nitroinidazole (7N).Panel d: BH₄ and oxidized biopterins are quantified using the differential oxidation method and HPLC. The ratio of reduced BH₄ to oxidized pterins in the heart is lower in DOCA mice versus controls. Feeding BH₄ to hypertensive mice (days 1–14) increases the cardiac reduced BH₄ to oxidized pterins ratio. Panel e: Total NOS activity is reduced to a similar degree in DOCA and L-NAME treated control hearts.

Figure 4

Tetrahydrobiopterin (BH₄) prevents or reverses diastolic dysfunction. Panel a: DOCA mice treated with BH₄ or hydralazine (days 12–24) show a reduction in systolic blood pressure (SBP) to levels observed in control animals. Treatment with tetrahydroneopterin (H₄N; days 12–24), an enzymatically inactive analog of BH₄ with equivalent antioxidant properties, has no effect on blood pressure in hypertensive DOCA mice. Panels b, c, and d: Assessment of diastolic function using echocardiography: E’ (early septal mitral annulus velocity measured with tissue Doppler imaging), Vp (left ventricular inflow propagation velocity interrogated with color M-mode Doppler) and E/E’ ratio show BH₄, but not H₄N or hydralazine, prevents or reverses diastolic dysfunction in hypertensive DOCA mice. Panel e: Sample pressure-volume loops obtained during inferior vena cava occlusion demonstrate the EDPVR and ESPVR in control-, DOCA-, and BH₄-treated mice.Panel f: The mean EDPVR is steeper in DOCA mice versus control, BH₄ prevention, and BH₄ treatment groups.Panel g: Isolated myocytes from DOCA mice have a prolonged relaxation constant (τ) compared to control animals. The addition of exogenous BH₄ to isolated DOCA myocytes normalizes relaxation kinetics. Panel g: DOCA mice have a reduced phosphorylated to total PLB ratio compared to control, BH₄ prevention, and BH₄ treatment groups.

Figure 5

Cardiac-specific ACE overexpression results in increased oxidation and diastolic dysfunction.Panels a, b, and c: ACE 1/8 mice have reduced E’, lower Vp, and an increased E/E’ ratio when compared to wild-type (wt) controls.Panel d: Mean oxyethidium (2HO-ET) levels are significantly higher in ACE 1/8 mice versus controls.Panel e: L-NAME suppresses O₂^•− production in ACE 1/8 mice (p=0.004).