Chronic inhibition of the Na+/H+ - exchanger causes regression of hypertrophy, heart failure, and ionic and electrophysiological remodelling

Abstract

Background and purpose: Increased activity of the Na+/H+ -exchanger (NHE-1) in heart failure underlies raised [Na+]i causing disturbances of calcium handling. Inhibition of NHE-1, initiated at the onset of pressure/volume overload, prevents development of hypertrophy, heart failure and remodelling. We hypothesized that chronic inhibition of NHE-1, initiated at a later stage, would induce regression of hypertrophy, heart failure, and ionic and electrophysiological remodelling.

Experimental approach: Development of heart failure in rabbits was monitored electrocardiographically and echocardiographically, after one or three months. Cardiac myocytes were also isolated. One group of animals were treated with cariporide (inhibitor of NHE-1) in the diet after one month. Cytoplasmic calcium, sodium and action potentials were measured with fluorescent markers and sarcoplasmic reticulum calcium content by rapid cooling. Calcium after-transients were elicited after rapid pacing. Sodium channel current (INa) was measured using patch-clamp techniques.

Key results: Hypertrophy and heart failure developed after one month and progressed during the next two months. After one month, dietary treatment with cariporide was initiated. Two months of treatment reduced hypertrophy and heart failure, duration of action potential QT-interval and QRS, and restored sodium and calcium handling and the incidence of calcium after-transients. In cardiac myocytes, parameters of INa were not changed by cariporide.

Conclusion and implications: In rabbit hearts with hypertrophy and signs of heart failure one month after induction of pressure/volume overload, two months of dietary treatment with the NHE-1 inhibitor cariporide caused regression of hypertrophy, heart failure and ionic and electrophysiological remodelling.

Figure 1

Time table of experimental protocol. ^†Termination of experiment.

Figure 2

Electrophysiological data. (a) Raw data of action potentials in 2 Hz stimulated myocytes from Ctrl, HF-1, HF-3 and HF-car groups of animals, measured using di-4-Anepps fluorescence ratio in dual-emission mode. (b) Mean±s.e.mean; P<0.05, ^*versus Ctrl, ^†versus HF-1, ^‡versus HF-3. APD₉₀, action potential duration at 90% repolarization in 2 Hz stimulated myocytes.

Figure 3

Activity of the Na⁺/H⁺ exchanger and cytoplasmic [Na⁺]_i. (a) Cytoplasmic [Na⁺]_i in 2 Hz stimulated myocytes of Ctrl (nine rabbits, 45 cells), HF-1 (nine rabbits, 45 cells), HF-3 (10 rabbits, 45 cells) and HF-car (10 rabbits, 50 cells). (b) Time course of increase of [Na⁺]_i in 2 Hz stimulated myocytes after inhibition of Na⁺/K⁺-ATPase with 100 μM ouabain in the absence of cariporide (solid lines) of Ctrl (nine rabbits, 45 cells), HF-1 (nine rabbits, 27 cells), HF-3 (10 rabbits, 45 cells) and HF-car (10 rabbits, 50 cells). Myocytes from all four groups were also pre-incubated for 5 min with 5 μM cariporide before assay. Only one set of data is shown, for HF-3 myocytes (nine rabbits, 45 cells: dotted line), as the increase of [Na⁺]_i was the same in all of the myocyte groups pre-incubated with cariporide. (c) The activity of NHE-1 was measured as the rate of cariporide-sensitive sodium influx (J_Na-NHE). J_Na-NHE was obtained by subtracting the initial rates of sodium influx immediately following sodium pump inhibition with 100 μM ouabain in the absence and presence of 5 μM cariporide (see also Baartscheer et al., 2003c). Data: mean±s.e.mean; P<0.05, ^*versus Ctrl, ^†versus HF-1 and ^‡versus HF-3. NHE-1, Na⁺/H⁺ exchanger-1.

Figure 4

Sodium current (I_Na) in Ctrl and failing myocytes. (a) Peak current density and current–voltage relation of I_Na in HF-3 (n=9) and Ctrl myocytes (n=9). (b) Peak current density and current–voltage relation of I_Na in HF-3 myocytes in the presence (n=8) and absence (n=8) of 5 μM cariporide. (c and d) The corresponding time constants (τ_fast and τ_slow) of a double-exponential fit of inactivation of I_Na. Data: mean±s.e.mean.

Figure 5

Calcium transient characteristics. (a) Typical example of steady-state calcium transients in stimulated myocytes (2 Hz; average of five successive calcium transients). (b) Average data of diastolic [Ca²⁺]_i, (c) average data of calcium transient amplitude and (d) calcium transient duration at 80% recovery. Ctrl (nine rabbits, 45 cells), HF-1 (nine rabbits, 45 cells), HF-3 (10 rabbits, 50 cells) and HF-car (10 rabbits, 50 cells). Data: mean±s.e.mean; P<0.05, ^*versus Ctrl, ^†versus HF-1 and ^‡versus HF-3.

Figure 6

SR calcium content and fractional release. (a) Typical examples of response to rapid cooling in stimulated (2 Hz) myocytes. Rapid cooling (indicated by arrows) was used to release all calcium from SR. (b) The increase of total calcium during an electrically stimulated calcium transient (Tr) and the increase of total calcium during rapid cooling (RC). (c) Fractional SR calcium release during a calcium transient (ratio of Tr/RC). Ctrl (nine rabbits, 45 cells), HF-1 (nine rabbits, 45 cells), HF-3 (10 rabbits, 50 cells) and HF-car (10 rabbits, 50 cells). Data: mean±s.e.mean; P<0.05, ^*versus Ctrl, ^†versus HF-1 and ^‡versus HF-3. SR, sarcoplasmic reticulum.

Figure 7

The stimulation-rate dependence of the calcium transient amplitude. The calcium transient amplitudes measured in myocytes stimulated from 0.5 to 3 Hz. Ctrl (nine rabbits, 45 cells), HF-1 (nine rabbits, 45 cells), HF-3 (10 rabbits, 50 cells), HF-car (10 rabbits, 50 cells). Data: mean±s.e.mean; P<0.05, ^*versus Ctrl, ^†versus HF-1 and ^‡versus HF-3.

Figure 8

Incidence of calcium after-transients. Upper panels: representative examples of calcium after-transients evoked by cessation of 10 s of rapid stimulation (3 Hz) in the presence of 100 nM noradrenaline, arrows mark the last three stimulated beats. (a) Single calcium after-transient, (b) double calcium after-transient and (c) train of calcium after-transient. Lower panels: corresponding incidence of calcium after-transients in myocytes of Ctrl (nine rabbits, 45 cells), HF-1 (nine rabbits, 45 cells), HF-3 (10 rabbits, 50 cells) and HF-car (10 rabbits, 50 cells). P<0.05, ^*versus Ctrl, ^†versus HF-1 and ^‡versus HF-3.