Ranolazine improves cardiac diastolic dysfunction through modulation of myofilament calcium sensitivity

Abstract

Rationale: Previously, we demonstrated that a deoxycorticosterone acetate (DOCA)-salt hypertensive mouse model produces cardiac oxidative stress and diastolic dysfunction with preserved systolic function. Oxidative stress has been shown to increase late inward sodium current (I(Na)), reducing the net cytosolic Ca(2+) efflux.

Objective: Oxidative stress in the DOCA-salt model may increase late I(Na), resulting in diastolic dysfunction amenable to treatment with ranolazine.

Methods and results: Echocardiography detected evidence of diastolic dysfunction in hypertensive mice that improved after treatment with ranolazine (E/E':sham, 31.9 ± 2.8, sham+ranolazine, 30.2 ± 1.9, DOCA-salt, 41.8 ± 2.6, and DOCA-salt+ranolazine, 31.9 ± 2.6; P=0.018). The end-diastolic pressure-volume relationship slope was elevated in DOCA-salt mice, improving to sham levels with treatment (sham, 0.16 ± 0.01 versus sham+ranolazine, 0.18 ± 0.01 versus DOCA-salt, 0.23 ± 0.2 versus DOCA-salt+ranolazine, 0.17 ± 0.0 1 mm Hg/L; P<0.005). DOCA-salt myocytes demonstrated impaired relaxation, τ, improving with ranolazine (DOCA-salt, 0.18 ± 0.02, DOCA-salt+ranolazine, 0.13 ± 0.01, sham, 0.11 ± 0.01, sham+ranolazine, 0.09 ± 0.02 seconds; P=0.0004). Neither late I(Na) nor the Ca(2+) transients were different from sham myocytes. Detergent extracted fiber bundles from DOCA-salt hearts demonstrated increased myofilament response to Ca(2+) with glutathionylation of myosin binding protein C. Treatment with ranolazine ameliorated the Ca(2+) response and cross-bridge kinetics.

Conclusions: Diastolic dysfunction could be reversed by ranolazine, probably resulting from a direct effect on myofilaments, indicating that cardiac oxidative stress may mediate diastolic dysfunction through altering the contractile apparatus.

Figure 1

Invasive hemodynamic assessment of LV diastolic dysfunction. A: Representative pressure-volume loops with vena cava occlusion for DOCA-salt and DOCA-salt + ranolazine groups. B: The EDPVR slope is greater in DOCA-salt vs. sham mice (P<0.05) and is improved with ranolazine treatment (P<0.05, vs. DOCA-salt). The mean EDPVR is significantly greater in the DOCA-salt mice compared to sham and ranolazine-treated mice. Additionally, treatment with ranolazine reduces the EDPVR in DOCA-salt mice to that of controls (n= 8, *p < 0.05 vs. all groups). C: LV contractility assessed by the end-systolic pressure-volume relation (ESPVR) slope (P=NS) and the volume axis intercept (Vo, P=NS) are similar in sham, DOCA-salt and DOCA-salt + ranolazine groups. D: Comparisions of LV end-diastolic pressure (LVEDP) for sham (3.2 ± 0.3 mmHg, P<0.05 vs. DOCA-salt), DOCA-salt (5.1 ± 0.9 mmHg), and DOCA-salt + ranolazine (3.5 ± 0.4 mmHg, P<0.05 vs. DOCA-salt) groups. E: Ranolazine shows an inverse concentration dependent effect on the EDPVR in DOCA-salt mice with a Pearson correlation coefficient of 0.70 (n=6, p < 0.05).

Figure 2

Functional analysis of isolated cardiomyocytes. A: Fractional shortening of isolated cardiomyocytes paced at 0.5 Hz at 37°C represented as the peak shortening divided by the baseline sarcomere length (n=12, p=NS). B: Time to 90% peak contraction in isolated cardiomyocytes (n=12, p=NS). C: Isolated cardiomyocytes from DOCA-salt mice have a prolonged relaxation constant (τ) compared to control animals. The addition of ranolazine to isolated DOCA-salt cardiomyocytes normalizes relaxation kinetics (n=12, *p < 0.0001 vs. all groups). D: The mean diastolic sarcomere length was significantly shorter in the DOCA-salt cardiomyocytes compared to the sham. The addition of ranolazine to the DOCA-salt cardiomyocytes significantly lengthened resting sarcomeres, but had no effect on sham cardiomyocytes (n=12, *p < 0.0001 vs. all groups).

Figure 3

DOCA-salt mice have no difference in intracellular calcium cycling when compared to sham mice. B: The peak Ca²⁺ fluorescence in isolated cardiomyocytes loaded with the ratiometric fluorescent dye, Fura-2AM, and paced at 0.5 Hz (n=12, p=NS). C: The time to 90% peak Ca²⁺ fluorescence representing the rate of calcium entry into the cytosol (n=12, p=NS). D: The rate of relaxation measured as the time constant τ did not differ among groups (n=12, p=NS).

Figure 4

DOCA-salt mice show no difference in late I_Na when compared to sham mice. A: Voltage-clamp studies show no increase in late I_Na in DOCA-salt myocytes with respect to sham (n=7, p=NS). Extracellular addition of ranolazine had little effect on late accumulated charge in both the DOCA-salt and sham groups (n=4, p=NS). B: Graph of the ratio of the mean accumulated late Na⁺ charge to the mean accumulated total Na⁺ charge during an activating voltage step.

Figure 5

Myofilament Ca²⁺ responsiveness in skinned fiber bundles isolated from DOCA-salt and sham hearts with and without ranolazine. A-D: The mean steady-state isometric tension of skinned fiber bundles is plotted as a function of pCa. DOCA-salt fibers demonstrate a significant (p<0.05) increase in maximum tension (44.51 ± 0.55 mN/mm²; n = 6) compared to shams (39.7 ± 0.79 mN/mm²; n = 6) and DOCA-salt fibers treated with ranolazine (36.03 ± 1.50 mN/mm²). DOCA-salt fibers demonstrate a significant (p<0.05) increase in mean Ca²⁺ sensitivity (pCa₅₀ = 6.09 ± 0.01; n = 6) compared to shams (pCa₅₀ = 6.02 ± 0.01; n = 6). Treatment with ranolazine normalized these changes. E: pCa-tension relations normalized to maximum tension. DOCA-salt fibers (pCa₅₀ = 6.1 ± 0.02; Hill n = 3.42 ± 0.30) demonstrate a significant increase in mean Ca²⁺ sensitivity (n=6, *p < 0.05) as compared to shams without DOCA treatment (pCa₅₀ = 6.0 ± 0.01; Hill n = 3.80 ± 0.61) shams with DOCA treatment (pCa₅₀ = 6.0 ± 0.01; Hill n = 3.91 ± 0.50) and fibers from DOCA mice and treated with ranalozine (pCa₅₀ = 6.0 ± 0.03; Hill n = 3.71 ± 1.0).

Figure 6

A comparison in the post-translational modifications of the myofilaments from sham and DOCA-salt hearts. A: Western blot analysis demonstrating detection of glutathionylated proteins when comparing myofilament samples from sham and DOCA-salt treated hearts. The blots were also analyzed for myosin binding protein C, indicating that this protein was the major modification. Note that one pair of comparisons was removed from the gel. B: Quantification of the difference between glutathionylated proteins (GSH) normalized to myosin binding protein C (MyBP-C) in sham and DOCA-salt myofilaments. (*p < 0.05, n=3).